Cancer therapy

Abstract

The present disclosure provides agents, compositions of agents, for use in the treatment of cancer by contacting cancer cells with an agent that increases tension in the cell membrane of the cancer cells, thereby preventing or reducing cell migration and / or proliferation of the cancer cells and treating the cancer.

Description

[Technical Field]

[0001] Related applications This application claims priority under 119(e) of U.S. Patent Provisional Application No. 63 / 162,214, filed on 17 March 2021, and the disclosure of this application, by reference, constitutes an entire part of this specification.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which in whole constitutes part of this specification by reference. The name of the above ASCII copy, created on March 17, 2022, is 521_003WO1_SL.txt, and its size is 45,200 bytes. [Background technology]

[0003] Metastasis is a leading cause of cancer-related death, yet our understanding of the key determinants underlying tumor dissemination remains limited. From a biophysical perspective, given that cancer cells need to undergo significant deformation to migrate through tissues and enter blood vessels, it has long been proposed that changes in cellular mechanics are essential for metastatic dissemination. Recent advances in nanotechnology have revealed the "mechanical signature" of malignant cells, as evidenced by the strong correlation between decreased cellular stiffness and increased invasive and metastatic efficiency. This suggests that genetic changes in cellular mechanics, which should be caused by malignant progression, are crucial to the metastatic process. Conversely, epithelial cells may already possess strategies to maintain mechanical homeostasis, which may function as endogenous tumor suppressors. However, the cellular-specific physical parameters crucial for the transition to a malignant phenotype remain unclear, and our understanding of the relationship between oncogenic signaling and abnormal regulation of cellular mechanics, as well as how mechanical changes are transmitted (mechanotransduction) and regulate cancer cell motility, remains limited.

[0004] Cell motility is essential for metastatic dissemination. Recent studies using three-dimensional (3D) environments have shown that malignant cells exhibit two distinct modes of invasion and migration: mesenchymal migration and amoeboid migration. Mesenchymal migration, characterized by a spindle shape, relies on PM extrusion driven by Arp2 / 3 complex-dependent branched actin polymerization. In contrast, amoeboid migration, characterized by a rounded morphology, is highly heterogeneous and exhibits both actin-based extrusion and membrane blebing due to contractility. Importantly, these two migration modes are interconvertible; that is, cancer cells can actively switch between these migration modes and even exhibit mixed phenotypes of both. This flexibility and complexity are considered to be a major obstacle in developing therapeutic strategies to limit tumor invasion and dissemination.

[0005] Because PM reversibly associates with the actin cortex via linker proteins such as ezrin, radixin, and moesin (ERM) family proteins, the dynamics of the cell membrane are essentially dependent on the degree of membrane-cortical adhesion (MCA). In fact, it has been shown that PM tension, which is mainly determined by this complex structure, plays a crucial role in changes in cell shape and motility. Considering that a taut membrane inhibits cell membrane deformation, PM tension is thought to hinder the formation of arbitrary membrane protrusions and ultimately cell motility. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, there is a need for compounds and methods of use that increase membrane tension and thereby suppress the metastasis of malignant cells. This disclosure satisfies these needs. [Means for solving the problem]

[0007] In this disclosure, we identify constitutive PM tension as a mechanical inhibitor of cancer cell dissemination by counteracting mechanosensible BAR proteins, using optical tweezers, genetic interference, cancer genome data, mechanical perturbations, and in vivo studies. These data demonstrate that a decrease in PM tension is a mechanical characteristic of malignant cells, regardless of whether the cells exhibit mesenchymal or amoeboid movement. Maintaining high PM tension is sufficient to suppress such 3D migration, tumor invasion, and metastasis, while generally having no effect on non-tumorogenic cells. This research opens the door to new precision therapeutic strategies for treating metastatic cancer by targeting the cell membrane dynamics of cancer cells.

[0008] Accordingly, this disclosure provides a method for treating cancer, comprising contacting cancer cells with a substance that increases the tension of the cell membrane of the cancer cells, thereby treating the cancer cells.

[0009] In some embodiments, the active ingredient increases the cell membrane tension to approximately 100 pN / μm to 200 pN / μm or higher, and / or maintains it to approximately 100 pN / μm to 200 pN / μm or higher. This can be achieved in many ways, but is not limited to, manipulating the cell's permeability function (increasing or decreasing the permeability of the cell membrane) or increasing the intracellular pressure by increasing or decreasing the amount of cell membrane components. Such cell membrane components are one or more of lipids, phospholipids, glycolipids, proteins, glycoproteins, and cholesterol. In some embodiments, the phospholipid is phosphatidylinositol 4,5-bisphosphate (PIP2), and the amount of PIP2 in the cell membrane increases.

[0010] In some embodiments, the active ingredient used to increase cell membrane tension causes an increase in membrane-actin-cortical adhesion (MCA), and the increase in MCA is compared to cells not in contact with the active ingredient. In some embodiments, the active ingredient used to increase MCA is an expression vector or construct containing one or more phosphatidylinositol 4-phosphate 5-kinase (PIP5K) genes, the PIP5K genes encoding one or more PIP5K1A, PIP5K1B, and PIP5K1C which produce phosphatidylinositol 4,5-bisphosphate (PIP2).

[0011] In some embodiments, the active agent increases cell membrane tension by causing an increase in the expression of ezrin, radixin, and moesin (ERM) proteins, including one or more EZR, RDX, and MSN. In some embodiments, certain genes can be used to activate ERM proteins, such as upstream kinases of ERM proteins. In some embodiments, the kinase is one or more RHOA, ROCK1, ROCK2, SLK, and STK10 that phosphorylate ERM proteins directly or indirectly. In some embodiments, the active agent is one or more expression vectors containing one or more ERM proteins and / or one or more upstream kinases.

[0012] In other embodiments, the agent inhibits or reduces the expression of one or more proteins comprising a Bin, amphiphysin, and Rvs (BAR) domain. Preferably, the BAR domain protein comprises one or more gene products of MTSS1L / ABBA, FNBP1L / Toca-1, TRIP10 / CIP4, ARHGAP4, ARHGAP10 / GARF2, ARHGAP17 / RICH1, ARHGAP26 / GRAF1, ARHGAP29, ARHGAP42 / GRAF, ARHGAP44 / RICH2, ARHGAP45 / HMHA1, ARHGEF37, ARHGEF38, IRSp53 / BAIAP2, BAIAP2L1 / IRTKS, DNMBP / Tuba, FCHSD1, FCHSD2, FER; FES, FNBP1 / FBP17, GAS7, GMIP, MTSS1 / MIM, OPHN1, PACSIN1, PACSIN2, PACSIN3, SH3BP1, SRGAP1, SRGAP2, and SRGAP3.

[0013] In some embodiments, the agent is an antibody, aptamer, short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), nanobody, affimer, DNA, CRISPR / Cas9 system, or chemical compound.

[0014] In some embodiments, the agent is one or more of siRNA, miRNA, and shRNA that bind to one or more of the messenger ribonucleic acids of MTSS1L / ABBA, FNBP1 / FBP17, TRIP10 / CIP4, ARHGAP4, ARHGAP10 / GARF2, ARHGAP17 / RICH1, ARHGAP26 / GRAF1, ARHGAP29, ARHGAP42 / GRAF, ARHGAP44 / RICH2, ARHAGAP45 / HMHA1, ARHGEF37, ARHGEF38, IRSp53 / BAIAP2, BAIAP2L1 / IRTKS, DNMBP / Tuba, FCHSD1, FCHSD2, FER;FES, FNBP1L / Toca-1, GAS7, GMIP, MTSS1 / MIM, OPHN1, PACSIN1, PACSIN2, PACSIN3, SH3BP1, SRGAP1, SRGAP2, and SRGAP3. In some embodiments, one or more of the siRNA, miRNA, and shRNA bind to the messenger ribonucleic acids of MTSS1L / ABBA, FNBP1 / FBP17, and TRIP10 / CIP4.

[0015] In some embodiments, the agent is an expression vector or construct configured to express an ezrin fusion protein comprising a conserved myristoylation sequence of Lyn fused to ezrin, wherein ezrin comprises a phosphomimetic activating mutation (T567E). In other embodiments, the agent is an expression vector or construct configured to express one or more kinases selected from ROCK1, ROCK2, SLK, STK10, and RHOA that directly or indirectly phosphorylate an ERM protein, thereby activating the ERM protein.

[0016] In some embodiments, the agent is formulated as a composition comprising a pharmaceutically acceptable carrier.

[0017] This disclosure also provides methods for inhibiting cell migration and / or proliferation, comprising reducing the expression of one or more proteins containing a BAR domain or increasing the expression of ezrin, radixin, and moesin (ERM) proteins, thereby inhibiting cell migration and / or proliferation. In some embodiments, increased ERM phosphorylation leads to inhibition of cell migration and / or proliferation.

[0018] In another embodiment, the present disclosure provides a method for increasing or decreasing the rate of cell division of eukaryotic cells in cell culture, comprising contacting eukaryotic cells with an active substance that causes a change in the tension of the cell membrane of the cells, thereby increasing or decreasing the rate of cell division compared to the rate of cell division of eukaryotic cells that are not in contact with the active substance.

[0019] In some embodiments, the active ingredient that causes an increase in the rate of cell division reduces the expression of one or more ezrin, radixin, and moesin (ERM) proteins, or reduces the phosphorylation of one or more ERM proteins, thereby causing an increase in cell division. In another embodiment, the active ingredient is an expression vector or construct configured to express an ezrin fusion protein containing a conserved myristylated sequence of Lyn fused with ezrin, wherein the ezrin contains a phosphorylation-mimicking activating mutation (T567E).

[0020] In other embodiments, the active ingredient that causes an increase in the rate of cell division increases the expression of one or more BAR domain proteins. In other embodiments, the active ingredient that causes a decrease in cell division increases the expression of one or more ezrin, radixin, and moesin (ERM) proteins or phosphorylation of one or more ERM proteins, or decreases the expression of one or more BAR domain proteins. The active ingredient is preferably an antisense RNA, siRNA, shRNA, or miRNA, or an antibody.

[0021] These and other features and advantages of the present invention will be understood more fully from the following detailed description, together with the appended claims. Note that the claims are defined by the details in the claims and not by the specific descriptions of the features and advantages described herein.

[0022] The following drawings form part of this specification and are included to further illustrate certain specific embodiments or various aspects of the present invention. In some cases, embodiments of the present invention can be best understood by referring to the accompanying drawings in conjunction with the detailed description presented herein. This specification and the accompanying drawings may highlight certain specific examples or certain aspects of the present invention. However, those skilled in the art will understand that some examples or aspects may be used in combination with other examples or aspects of the present invention. [Brief explanation of the drawing]

[0023] [Figure 1]This figure shows that the cell membrane (PM) tension in non-invasive cells is higher than that of metastatic cells. a. Scatter plot comparing the tethering force of specified cells. n=35 (MCF10A), n=24 (MDCK II), n=31 (IAR-2), n=23 (AU565), n=23 (MCF7), n=24 (MDA-MB-231 with ruffling), n=14 (MDA-MB-231 with bleving), n=21 (Hs578T with ruffling), n=13 (Hs578T with bleving), n=19 (PC-3 with ruffling), n=14 (PC-3 with bleving), n=24 (PANC-1) cells pooled from three independent experiments. Mean ± SD. b. Quantification of confocal images of specified cells stained with anti-phosphorylated ERM (pERM) antibody and phalloidin. Membrane / cytoplasmic intensity ratios of pERM and F-actin in pERM and F-actin in cells pooled from three independent experiments: n=30 (MCF10A), n=26 (Au565), n=24 (MDA-MB-231 with ruffling), n=20 (MDA-MB-231 with bleving), n=22 (Hs578T with ruffling), and n=18 (Hs578T with bleving). Mean ± SD. **P=0.0073;***P=0.0014. c Quantification of confocal images of MCF10A or MDA-MB-231 cells stained with anti-pERM antibody, phalloidin, and wheat germ agglutinin (WGA) in a 3D collagen matrix (3D). Cell membrane (PM) was labeled with WGA. pERM and F-actin membrane / cytoplasmic intensity ratios of cells pooled from three independent experiments: n=24 (MCF10A), n=16 (AU565), n=15 (MDA-MB-231, elongated), n=12 (MDA-MB-231, rounded, with actin-based protrusions), n=17 (MDA-MB-231, rounded, with blebs), and n=17 (Hs578T). Mean ± SD. Significance tested using one-way ANOVA and Tukey's multiple comparison test (a) and two-sided Mann-Whitney test (b, c). ns, not significant; ****P<0.0001. All scale bars, 10 μm. [Figure 2]This figure shows that a decrease in PM tension converts epithelial cells to a mesenchymal migration phenotype in both 2D and 3D environments. a. Scatter plot comparing estimated PM tension of MCF10A cells treated with specified RNAi. n=60 (si-control), n=40 (si-RHOA), n=28 (si-ERM), and n=25 (si-SLK+STK10) cells pooled from three independent experiments. Mean ± SD. b. Quantification of confocal images of MCF10A cells treated with RNAi and stained with anti-pERM antibody and phalloidin. Membrane / cytoplasmic intensity ratios of pERM and F-actin in cells pooled from three independent experiments: n=29 (si-control), n=26 (si-RHOA), n=19 (si-ERM), and n=28 (si-SLK+STK10). Mean ± SD. c Phase-contrast images of MCF10A cells treated with specified RNAi and grown in 3D on-top culture. Images are representative of three independent experiments with similar results. Scale bar, 20 μm. d Quantification of siRNA-treated MCF10A cells migrating through an 8 μm pore. n=9 fields from three independent experiments. Mean ± SD. **P=0.0011. e Quantification of 3D migration phenotypes of n=155 (si-control), n=126 (si-RHOA), n=128 (si-ERM), and n=123 (si-SLK+STK10) cells from three independent experiments. f Quantification of confocal images of specified RNAi-treated MCF10A cells stained with anti-pERM antibody and phalloidin in 3D. Membrane / cytoplasmic intensity ratios of pERM and F-actin in n=20 (si-control), n=14 (si-RHOA), n=15 (si-ERM), and n=18 (si-SLK+STK10) cells pooled from three independent experiments. Mean ± SD. Significance was tested using two-sided Mann-Whitney tests (a, b, e), two-sided Student's t-test (c), and chi-squared test (d). ****P<0.0001. [Figure 3]This figure shows the correlation between decreased PM tension and malignant progression. a. Quantification of confocal images of MCF10A or Snail-expressing cells stained with anti-pERM antibody and phalloidin. Membrane / cytoplasmic intensity ratios of pERM and F-actin in n=23 (MCF10A) and n=25 (Snail-expressing cells) cells pooled from three independent experiments. Mean ± SD. b. Scatter plot comparing estimated PM tension of specified cells. N=38 (MCF10A), n=33 (Snail-expressing cells), and n=36 (Slug-expressing cells) cells pooled from three independent experiments. Mean ± SD. c. Phase-contrast images of MCF10A cells treated with specified RNAi and grown in 3D on-top culture. Images are representative of three independent experiments with similar results. Scale bar, 20 μm. d. Quantification of confocal images of MCF10A or Snail-expressing cells stained with anti-pERM antibody and phalloidin in 3D. Yellow arrows indicate actin-based protrusions. Scale bar, 10 μm. Membrane / cytoplasmic intensity ratios of pERM and F-actin in n=21 (MCF10A) and n=21 (Snail-expressing cells) cells pooled from three independent experiments. Mean ± SD. e Genetic changes in RHOA, SLK, and STK10 across 14 cancer types in The Cancer Genome Atlas (TCGA) data (6586 samples). f Kaplan-Meier plots showing overall survival of breast, lung, and gastric cancer patients stratified according to SLK+STK10 mRNA expression. Significance tested using two-sided Mann-Whitney tests (a, b, c) and two-sided log-rank tests (e). ****P<0.0001. [Figure 4]This figure shows that an increase in PM tension is sufficient to suppress 3D migration and translocation. a. Top: Schematic diagram of membrane-anchored ezrin. Bottom: Scatter plot comparing estimated PM tension of specified cells. n=26 (parent), n=29 (ezrin), and n=31 (MA-ezrin) cells pooled from three independent experiments. b. Quantification of protrusion of n=207 (parent), n=224 (ezrin), and n=214 (MA-ezrin) cells from three independent experiments. c. Quantification of migration or invasion rate of specified cells. n=9 fields from three independent experiments stained with anti-HA antibody and phalloidin. d. Quantification of 3D migration phenotype of n=176 (parent), n=187 (ezrin), and n=175 (MA-ezrin) cells from three independent experiments. e. Trajectory of the cell center of specified cells tracked in (d) over 8 hours. Right: Average velocity of one cell over 8 hours. n=34 (ezrin) and n=44 (MA-ezrin) cells pooled from three independent experiments. f Quantification of representative hematoxylin and eosin (H&E) stained sections of primary tumors and surrounding tissues from mice injected with specified cells. Tumor invasion area at the tumor margin was quantified. n=9 regions for 3 tumors per group. g Quantification of spontaneous lung metastases by quantitative PCR. n=6 mice (parent), n=3 (ezrin), and n=6 mice (MA-ezrin). **P=0.0152;***P=0.0119. h Quantification of H&E staining of whole lung images and lung sections after tail vein injection of specified cells (below). n=8 mice per group. ***P=0.0002. All data are expressed as mean ± SD. Significance was tested using the two-tailed Mann-Whitney test (a, c, g, h, i), the two-tailed Student's t-test (c), and the chi-squared test (b, d). ns, not significant; ****P<0.0001. [Figure 5]This figure shows that constitutive PM tension suppresses cancer cell migration by counteracting the BAR protein. a. Percentage of MCF10A spheroids with invasive structures grown in 3D on-top culture treated with specified RNAi. Control siRNA alone and siRNA targeting the BAR protein that reduces invasive structures induced by ERM deletion. Data are the mean of two independent experiments using at least 50 cells per experiment. b. Percentage of invasive structures of specified cells in 3D on-top culture in uptake images. Data are the mean ± SD of three independent experiments using at least 200 cells per experiment. Scale bar, 20 μm. **P=0.002;***P=0.0007. c. Quantification of phase-contrast images of MDA-MB-231 cells treated with specified RNAi for 3D migration phenotypes of n=153 (si-control), n=150 (si-MTSS1L), and n=155 (si-Toca protein) cells from three independent experiments. d. Trajectory of the cell center of a specified cell tracked in c over 8 hours. Right, average velocity of one cell over 8 hours. n=35 (si-control), n=43 (si-MTSS1L), and n=46 (si-Toca protein) cells pooled from three independent experiments. Mean ± SD. e. Quantification of the protrusion of a specified cell in 3D. n=151 (si-control), n=132 (si-MTSS1L), and n=138 (si-Toca protein) cells from three independent experiments. f. Quantification of confocal images of a specified cell expressing GFP-FBP17 spots in n=26 (si-control), n=22 (si-ERM), n=22 (si-SLK+STK10), and n=22 (Snail-expressing cells) cells pooled from three independent experiments. g. Quantification of confocal images of specified cells stained with phalloidin and WGA in 3D, showing GFP-FBP17 spots in n=20 (ezrin) and n=20 (MA-ezrin) cells pooled from three independent experiments. All data except a are expressed as mean ± SD. Significance was tested using two-sided Student's t-test (b, f, g), two-sided Mann-Whitney test (d), and chi-squared test (c, e). ****P<0.0001. [Figure 6]This figure shows a proposed model explaining how constitutive PM tension acts as a mechanical inhibitor of cancer cell dissemination. a Proposed model explaining how disruption of constitutive PM tension, which leads to BAR protein-mediated cancer cell dissemination, is related to cancer progression. b Constitutive PM tension, maintained by membrane-cortical adhesion (MCA), can maintain a non-motile state by suppressing the assembly of BAR proteins, which are key regulators of both actin-based and bleb-based protrusions. [Figure 7] This figure shows the analysis of tether force and PM tension. a Schematic diagram of the measurement of tether force (F-tether) using optical tweezers. k is the stiffness of the trap, and Δx is the displacement of the bead from the center of the trap. PM tension can be estimated using the formula shown. B is the bending stiffness of the membrane. See the Methods section for details. b Schematic diagram of PM tension regulation by membrane-cortical adhesion (MCA) mediated by ERM proteins. ERM proteins are activated by RHOA via ERM kinases including ROCK1 / 2, SLK, and STK10. c Mean fluorescence intensity of line scans across lateral PM from confocal images of specified cells stained with anti-pERM antibody, phalloidin, and wheat germ agglutinin (WGA). n=10 (MCF10A), n=10 (AU565), n=10 (MDA-MB-231), and n=10 (HS578T) cells from three independent experiments. d. Quantification of protrusion of specified cells in 3D collagen matrix (3D). n=144 (MCF10A), n=107 (AU565), n=175 (MDA-MB-231), and n=126 (Hs578T) cells from two independent experiments. Confocal images were taken of AU565 or Hs578T cells stained with anti-pERM antibody, phalloidin, and WGA in the 3D collagen matrix. Chi-square test. ns, not significant;****P<0.0001. [Figure 8]This figure shows that a decrease in PM tension induces a mesenchymal migration phenotype in epithelial cells. a Confirmation of downregulation of target protein expression by RNAi analysis using Western blotting. The image is representative of two independent experiments with similar results. b Scatter plot comparing tether forces of MCF10A cells treated with specified RNAi. n=60 (si-control), n=40 (si-RHOA), n=28 (si-ERM), and n=25 (si-SLK+STK10) cells pooled from three independent experiments. Mean ± SD. See also Figure 2a. c Western blot of endogenous phosphomyosin light chain (pS19MLC), MLC, E-cadherin, vimentin, and β-actin levels in specified cells. The image is representative of two independent experiments with similar results. d Orbital of the cell center of specified cells tracked over 6 hours in 2D. n=21 (si-control), n=22 (si-RHOA), n=17 (si-ERM), and n=16 (si-SLK+STK10) cells from three independent experiments. Right, scatter plot comparing aspect ratios of n=81 (si-control), n=98 (si-RHOA), n=81 (si-ERM), and n=85 (si-SLK+STK10) cells pooled from three independent experiments. Mean ± SD. e Quantification of AU565 or MCF7 cells infiltrated through Matrigel. n=6 fields from two independent experiments. Mean ± SD. f Growth rate of MCF10A cells treated with specified RNAi. Data are from the mean ± SD of three independent experiments. Statistical comparisons with appropriate controls were performed using two-sided Mann-Whitney tests (b, d) and two-sided Student's t-tests (e). ****P<0.0001. [Figure 9]This figure shows the downregulation of MCA regulators in cancer patients. a Western blot of endogenous E-cadherin, vimentin, and β-actin levels in specified cells. The image is representative of two independent experiments with similar results. b Scatter plot comparing estimated PM tension of specified cells. n=15 (MDCK II cells, Dox(-)) and n=16 (MDCK II cells expressing RasV12, Dox(+)) cells pooled from three independent experiments. Mean ± SD. Two-sided Mann-Whitney test. ****P<0.0001. c Genetic alterations of specified genes across 14 human tumor types in TCGA data (6586 samples). d Genetic alterations of specified genes across 961 cancer cells in Cancer Cell Line Encyclopedia (CCLE) data. [Figure 10] This figure shows that increased PM tension suppresses 3D migration. a Proliferation rate of MDA-MB-231 cells expressed as specified. Data are presented as mean ± SD from three independent experiments. b Quantification of drug-treated cells infiltrated through Matrigel. n=6 fields from two independent experiments. Mean ± SD. ns, not significant; ****P<0.0001. c Scatter plot comparing estimated PM tension of MDA-MB-231 cells treated with MβCD. n=20 (mock, water) and n=21 (MβCD) cells pooled from three independent experiments. Mean ± SD. ****P<0.0001. d Primary tumor growth after injection of specified cells into mammary fat body. n=12 mice (MDA-MB-231, parent), n=4 (Ezrin), and n=6 mice (MA-Ezrin). Mean ± SD. Average tumor volume is shown in the graph. **P=0.0095;***P=0.0001. Statistical analysis using two-tailed Student t-tests (b, c) and two-tailed Mann-Whitney test (d). [Figure 11]This figure shows that constitutive PM tension inhibits cancer cell migration by suppressing BAR protein assembly. a, b Quantification of specified RNAi-treated MDA-MB-231 cells (a) or MCF10A cells infiltrated through Matrigel (b) using phase-contrast imaging in 3D on-top culture. n=9 fields from 3 independent experiments. Mean ± SD. c Quantification of GFP-FBP17 or GFP-MTSS1L spots in PM. n=20 (GFP-FBP17) and n=20 (GFP-MTSS1L) cells pooled from 3 independent experiments. d Quantification of GFP-MTSS1L spots in PM of n=20 (si-control), n=20 (si-ERM), and n=20 (Snail-expressing cells) cells pooled from 3 independent experiments. Mean ± SD. e Quantification of GFP-FBP17 spots in PM of n=20 (mock, water) and n=20 (MβCD) cells pooled from three independent experiments. Mean ± SD. f Quantification of confocal images of GFP-FBP17 or GFP-MTSS1L in MDA-MB-231 cells before and after mechanical stretching (20%). n=20 (GFP-FBP17) or n=20 (GFP-MTSS1L) cells pooled from two independent experiments. Mean ± SD. Statistical analysis using two-sided Student's t-test (a, c, e), one-way ANOVA and Tukey's multiple comparison test (b), and two-sided Mann-Whitney test (d, f). ****P<0.0001. [Modes for carrying out the invention]

[0024] definition The following definitions are included to provide a clear and consistent understanding of this specification and the claims. Where used herein, the terms listed have the following meanings. All other terms and expressions used herein have the ordinary meanings understood by those skilled in the art. Such ordinary meanings are as defined in Hawley's Condensed Chemical Dictionary 14. th This can be obtained by referring to specialized terminology dictionaries such as Edition, by RJ Lewis, John Wiley & Sons, New York, NY, 2001.

[0025] References to “an embodiment,” “an embodiment,” etc., in this specification indicate that the described embodiments may include certain aspects, features, structures, parts, or characteristics, but not all embodiments necessarily include such aspects, features, structures, parts, or characteristics. Furthermore, such expressions may, but not necessarily, refer to the same embodiments referred to in other parts of this specification. Moreover, if certain aspects, features, structures, parts, or characteristics are described in relation to a particular embodiment, whether explicitly stated or not, it is within the knowledge of those skilled in the art that such aspects, features, structures, parts, or characteristics affect or relate to other embodiments.

[0026] Unless otherwise clearly indicated by the context, the singular forms "a," "an," and "the" refer to multiple entities. For example, a reference to "a compound" includes multiple such compounds, and compound X includes multiple compounds X. Furthermore, it should be noted that claims may be drafted to exclude any arbitrary element. Thus, this statement is intended to be an antecedent of the use of exclusive terms such as "solely," "only," or "negative" limitation relating to any element described herein and / or elements of the claims.

[0027] The term "and / or" means any one of the items to which this term relates, any combination of items, or all of the items. The expressions "one or more" and "at least one" will be readily understood by those skilled in the art, especially when read in the context of their usage. For example, this expression may mean one, two, three, four, five, six, ten, one hundred, or any upper limit of approximately ten, one hundred, or one thousand times the lower limit described. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring.

[0028] As those skilled in the art will understand, all numerical values, including those representing the amount of components, molecular weight and other properties, and reaction conditions, are approximations and are in all cases optionally modified with the term "approximately." These values ​​may vary depending on the desired properties that those skilled in the art seek to obtain using the teachings described herein. It will also be understood that such values ​​inherently include variability that inevitably arises from the standard deviation observed in each test measurement. When values ​​are expressed as approximations using the antecedent "approximately," it will be understood that specific values ​​without the modifier "approximately" also form further aspects.

[0029] The terms “about” and “approximately” are used interchangeably. Both terms can refer to a variation of ±5%, ±10%, ±20%, or ±25% of a given value. For example, “about 50”% may have a variation of 45% to 55% in some embodiments, or may be defined otherwise by a particular claim. For integer ranges, the term “about” may include one or two integers greater than and / or less than the integers stated at both ends of the range. Unless otherwise specified herein, the terms “about” and “approximately” are intended to include values ​​close to the range described, such as weight percentages, that are equivalent in terms of the functionality of the individual components, compositions, or embodiments. The terms “about” and “approximately” may also modify the endpoints of the range described, as discussed above in this paragraph.

[0030] As will be understood by those skilled in the art, all scopes described herein, in order to provide all possible purposes, particularly written explanations, include all possible sub-scopes and combinations thereof, as well as the individual values, particularly integer values, that constitute the scope. It will therefore be understood that each unit between two specific units is also disclosed. For example, if 10–15 is disclosed, then 11, 12, 13, and 14 are also disclosed individually and as part of the scope. The scopes described (e.g., weight percentages or carbon groups) include each specific value, integer, decimal, or identity within that scope. Any scope enumerated can be readily recognized as fully explaining and enabling that the same scope can be divided into at least equal parts, one-third, one-quarter, one-fifth, or one-tenth. As a non-limiting example, each scope discussed herein can easily be divided into a lower third, a middle third, and an upper third, etc. Furthermore, as will be understood by those skilled in the art, all terms such as “maximum,” “at least,” “greater than,” “less than,” “more,” and “or more” include the numerical values ​​described, and such terms refer to ranges that may subsequently be divided into subranges, as discussed above. Similarly, all ratios described herein include all subranges that fall within broader ratios. Therefore, the specific values ​​described for radicals, substituents, and ranges are for illustrative purposes only and do not exclude other specified values ​​or other values ​​within the specified ranges for radicals and substituents. Moreover, it will be understood that each range endpoint is valid both in relation to the other endpoint and independently of the other endpoint.

[0031] This disclosure provides ranges, limits, and deviations for variables such as volume, mass, percentage, and ratio. Those skilled in the art will understand that ranges such as "Number 1" to "Number 2" mean a continuous range of numbers including integers and fractions. For example, 1 to 10 means 1, 2, 3, 4, 5, ..., 9, 10. This also means 1.0, 1.1, 1.2, 1.3, ..., 9.8, 9.9, 10.0, and 1.01, 1.02, 1.03, etc. If the disclosed variable is less than "Number 10", it means a continuous range including integers and fractions less than Number 10, as discussed above. Similarly, if the disclosed variable is greater than "Number 10", it means a continuous range including integers and fractions greater than Number 10. These ranges may be modified with the term "approximately," as explained above.

[0032] Furthermore, those skilled in the art will readily recognize that, when members are grouped in a common manner, such as a Markush group, the invention encompasses not only the entire group as a whole, but also each individual member of that group, and all possible subgroups of the principal group. Moreover, for all purposes, the invention encompasses not only the principal group, but also principal groups lacking one or more of their members. Thus, the invention assumes the explicit exclusion of one or more members of the described groups. Accordingly, conditions can be applied to either the categories or embodiments of the disclosure, thereby excluding one or more of the described elements, types, or embodiments from such categories or embodiments, for example, so that they are used in explicit negative limitations.

[0033] The term "to bring into contact" refers to the act of touching, bringing into contact with, or bringing very close to or in close proximity to, for example, in a solution, reaction mixture, in vitro, or in vivo, in order to bring about a physiological reaction, chemical reaction, or physical change, including at the cellular or molecular level.

[0034] "Effective dose" refers to the amount effective in treating a disease, disorder and / or condition, or in producing the described effect. For example, an effective dose may be the amount effective in reducing the progression or severity of the condition or symptom being treated. Determining the therapeutic effective dose is well within the capabilities of those skilled in the art. The term "effective dose" is intended to include, for example, the amount of a compound or combination of compounds described herein that is effective in treating or preventing a disease or disorder in a host, or in treating the symptoms of a disease or disorder. For this reason, "effective dose" generally means the amount that produces the desired effect.

[0035] Alternatively, the terms “effective dose” or “therapeutic effective dose,” as used herein, refer to a dose of an active substance, composition, or combination of compositions sufficient to alleviate, to some extent, one or more symptoms of the disease or condition being treated. The result may be a reduction and / or mitigation of the signs, symptoms, or causes of the disease, or any other desired change in the biological system. For example, an “effective dose” for therapeutic use is the amount of a composition containing the compounds disclosed herein required to produce a clinically significant reduction in the symptoms of the disease. The appropriate “effective” dose in any individual case may be determined using techniques such as dose escalation studies. The dose may be administered in one or more doses. However, the precise determination of what may be considered an effective dose may be based on individual patient factors, including, but not limited to, the patient’s age, physique, type or severity of the disease, stage of the disease, route of administration of the composition, type or extent of supplemental therapy used, ongoing disease process, and the type of treatment desired (e.g., aggressive treatment versus conventional treatment).

[0036] The terms “to treat,” “to treat,” and “treatment” include (i) preventing the onset of a disease, pathological condition, or medical condition (e.g., prevention), (ii) inhibiting or preventing the onset of a disease, pathological condition, or medical condition, (iii) alleviating a disease, pathological condition, or medical condition, and / or (iv) reducing symptoms associated with a disease, pathological condition, or medical condition. For this reason, the terms “to treat,” “treatment,” and “treatment” may extend to prevention and may include preventing, reducing, stopping, or reversing the progression or severity of the condition or symptoms being treated. Thus, the term “treatment” may include medical, therapeutic, and / or prophylactic administration as appropriate.

[0037] Where used herein, “subject” or “patient” means an individual having symptoms of or at risk of a disease or other malignant tumor. A patient may be human or non-human and may include, for example, a strain or species of animal used as a “model system” for research purposes, such as the mouse model described herein. Similarly, a patient may include either an adult or a minor (e.g., a child). Furthermore, a patient may mean any organism, preferably a mammal (e.g., human or non-human), that could potentially benefit from the administration of the composition intended herein. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates, e.g., chimpanzees, and other apes and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and experimental animals such as rodents, e.g., rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of the method provided herein, the mammal is human.

[0038] As used herein, the terms “provide,” “administer,” and “introduce” are used without distinction herein and refer to introducing the compounds of this disclosure into a subject by a method or route that results in at least partial localization of the compound to a desired site. The compounds may be administered by any suitable route that results in delivery to a desired site in the subject.

[0039] The compounds and compositions described herein may be administered together with additional compositions to maintain the stability and activity of the compositions, or in combination with other therapeutic agents.

[0040] The terms “inhibit,” “inhibit,” and “inhibition” refer to delaying, stopping, or reversing the growth or progression of a disease, infection, pathology, or group of cells. Inhibition may be about 20%, 40%, 60%, 80%, 90%, 95%, or more than 99% compared to the growth or progression that would occur, for example, in the absence of treatment or contact.

[0041] The term “substantially” as used herein is a broad and unrestricted term, but is used in its ordinary sense, including that the specified items are the majority but not necessarily all of them. For example, the term may refer to a number that may not be 100% complete. A complete number may be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% less.

[0042] Where the term “comprising” is used herein, the options of using the terms “consisting of” or “essentially consisting of” are contemplated. Where used herein, “comprising” is synonymous with “including,” “containing,” or “characterizing,” and is comprehensive or open-ended and does not exclude additional undisclosed elements or methods or processes. Where used herein, “consisting of” excludes any elements, processes, or components not designated as elements of the embodiment. Where used herein, “essentially consisting of” does not exclude materials or processes that do not substantially affect the fundamental novel characteristics of the embodiment. In each example herein, the terms “comprising,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms. Disclosures described exemplary herein may be adequately carried out in the absence of any element(s) or limitation(s) not specifically disclosed herein.

[0043] As used herein, “sequence identity” or “identity” in the context of two nucleic acid sequences or polypeptide sequences refers to the percentage of specified residues in the two sequences that are identical when aligned to the maximum extent possible across a specified comparison window, as measured by a sequence comparison algorithm or visual inspection. When the percentage of sequence identity is used in relation to proteins, it is recognized that the positions of non-identical residues often differ by conservative amino acid substitutions, where the amino acid residues are replaced by other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), and therefore the functional properties of the molecule do not change. When sequences differ by conservative substitutions, the percentage of sequence identity can be increased to compensate for the conservative nature of the substitutions. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this correction are known to those skilled in the art. Typically, this involves scoring the conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Therefore, for example, if the same amino acid is assigned a score of 1 and a non-conservative substitution is assigned a score of 0, a conservative substitution will be assigned a score between 0 and 1. The scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif.).

[0044] As used herein, “percentage of sequence identity” means a value determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide sequence within the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0045] In the context of peptides, the term "substantial identity" indicates that the peptide contains a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the reference sequence across a specified comparison window. In certain embodiments, optimal alignment is performed using the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch, JMB, 48, 443 (1970)). An indicator that two peptide sequences are substantially identical is that one peptide immunologically reacts with an antibody produced against the second peptide. Therefore, a peptide is substantially identical to the second peptide if, for example, the two peptides differ only by conservative substitutions. Accordingly, the present invention also provides nucleic acid molecules and peptides substantially identical to those presented herein.

[0046] For sequence comparison, typically one sequence functions as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence(s) to the reference sequence based on the specified program parameters.

[0047] A gene is “targeted” by the siRNA according to the present invention when, for example, the siRNA molecule selectively reduces or inhibits gene expression. The expression “selectively reduces or inhibits,” as used herein, encompasses siRNA that affects gene expression. Alternatively, a gene is targeted by siRNA when one strand of the siRNA hybridizes with the gene's transcript, i.e., its mRNA, under stringent conditions. Hybridizing under “stringent conditions” means annealing to the target mRNA region under standard conditions, such as high temperature and / or low salt content, which tend to hinder hybridization. A preferred protocol (including 0.1 × SSC, 68°C for 2 hours) is described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, at pages 387-389.

[0048] Nucleic acid sequences cited herein are written in the 5'→3' direction unless otherwise specified. The term “nucleic acid” refers to either DNA (adenine “A”, cytosine “C”, guanine “G”, thymine “T”) or RNA (adenine “A”, cytosine “C”, guanine “G”, uracil “U”) containing purine or pyrimidine bases, or any modified form thereof. Interfering RNA provided herein may contain a “T” base, for example, at the 3' end, even though the “T” base is not naturally present in the RNA. In some cases, these bases may be denoted as “dT” to distinguish them from deoxyribonucleotides present in the ribonucleotide chain.

[0049] As used herein, “expression vector” means a vector that enables the expression of polynucleotides within a cell. Polynucleotide expression includes transcriptional events and / or post-transcriptional events.

[0050] The term “gene,” as used herein, refers to any individual coding region of the host genome, or a region that codes only for functional RNA (e.g., tRNA, rRNA, regulatory RNA, e.g., ribozymes), as well as associated non-coding regions and optionally regulatory regions. In certain embodiments, the term “gene” includes, within its scope, open reading frames, introns, and adjacent 5' and 3' non-coding nucleotide sequences that code for a particular polypeptide, and that are involved in the regulation of expression. In this regard, a gene may further include regulatory signals such as promoters, enhancers, termination signals and / or polyadenylation signals that are naturally associated with a given gene, or heterologous regulatory signals. Gene sequences may be eDNA or genomic DNA or fragments thereof. Genes can be introduced into vectors suitable for extrachromosomal maintenance or integration into a host.

[0051] The term "mRNA" refers to messenger RNA, a "transcript" produced within cells using DNA as a template, which itself codes for proteins. mRNA typically consists of a 5'-UTR, which is the protein-coding region, and a 3'-UTR. mRNA has a limited half-life within cells, which is partly determined by stability elements, particularly within the 3'-UTR, but also found in the 5'-UTR and the protein-coding region.

[0052] "Operationally linked" or "operationally ligated" refers to the linking of functionally related polynucleotide elements. A nucleic acid is "operationally ligated" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operationally ligated to a coding sequence when it affects the transcription of that coding sequence. Operationally ligated means that the nucleic acid sequences being ligated are typically contiguous and within a reading frame when it is necessary to join two protein coding regions. A coding sequence is "operationally ligated" to another coding sequence when RNA polymerase transcribes two coding sequences into a single mRNA, which is subsequently translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences do not need to be contiguous with each other, as long as the expressed sequence is ultimately processed to produce the desired protein. "Operationally linking" a promoter to a transcribable polynucleotide means that the transcribable polynucleotide (e.g., a protein-coding polynucleotide or other transcript) is placed under the regulatory control of the promoter, which controls the transcription and optionally translation of that polynucleotide. In constructing heterologous promoter / structural gene combinations, it is generally preferable to position the promoter or its variant at approximately the same distance from the transcription start site of the transcriptable polynucleotide as the distance between the promoter and the gene it controls in its natural context, i.e., the gene from which the promoter originates. As is known in the art, some variation in this distance can be adapted without loss of function. Similarly, the preferred arrangement of regulatory sequence elements (e.g., operators, enhancers, etc.) for a transcriptable polynucleotide under control is determined by the arrangement of the elements in their natural context, i.e., the gene from which they originate.

[0053] The term "promoter" generally refers to a region of DNA located upstream (5') of the coding region that at least partially controls the initiation and level of transcription. References to "promoter" herein are interpreted in their broadest context and include transcriptional regulatory sequences of classical genomic genes, including TATA and CCAAT box sequences, as well as additional regulatory elements (i.e., activators, enhancers, and silencers) that alter gene expression in response to developmental and / or environmental stimuli, or in a tissue-specific or cell-type-specific manner. Promoters are typically located upstream or 5' of the structural gene whose expression they regulate, but not necessarily. Furthermore, regulatory elements, including promoters, are typically located within 2kb of the gene's transcription start site. Promoters according to the present invention may include additional specific regulatory elements located more distal to the start site to further enhance intracellular expression and / or alter the timing or inducibility of the expression of the structural gene to which they are manipulably attached. The term "promoter" includes inducible, repressive, and constitutive promoters, as well as minimal promoters. A minimal promoter typically refers to the smallest expression regulatory element capable of initiating transcription of a manipulably linked selected DNA sequence. In some cases, minimal promoters cannot initiate transcription beyond the basal level in the absence of additional regulatory elements (e.g., enhancers or other cis-acting regulators). Minimal promoters often consist of a TATA box or a TATA-like box. Numerous minimal promoter sequences are known in the literature. For example, minimal promoters can be selected from a wide variety of known sequences, including the promoter regions of fos, CMV, SV40, and IL-2. Examples of using a minimal CMV promoter or a minimal IL2 gene promoter (-72 to +45 relative to the start site; Siebenlist, 1986) are presented.

[0054] Embodiments of the present invention This disclosure provides a method for treating cancer, comprising increasing the tension of the cell membrane of cancer cells, thereby preventing the migration and proliferation of cancer cells. The cell membrane tension is preferably increased and / or maintained at approximately 100 pN / μm to 200 pN / μm.

[0055] This disclosure also provides methods for increasing or decreasing the rate of cell division, cell motility, and cell proliferation.

[0056] In some embodiments of this disclosure, increasing cell membrane tension may involve manipulating the permeability function of cancer cells. For example, certain active agents (e.g., drugs, proteins, nucleic acids) may come into contact with cancer cells and cause changes in membrane permeability, resulting in the influx or outflow of solutes from the cancer cells. The influx or outflow of solutes may increase the internal pressure, thereby causing an increase in cell membrane tension. In other embodiments, an active agent may come into contact with cancer cells and cause an increase or decrease in one or more cellular components that regulate internal pressure (e.g., ion transporter proteins, small molecule transporter proteins, water channel proteins, glycosynthesis proteins, and transport proteins (e.g., glucose transporters)).

[0057] In some embodiments, membrane permeability can be influenced to induce the influx of water molecules into cancer cells.

[0058] In other embodiments of the present disclosure, increasing the tension of the cell membrane includes, but is not limited to, increasing or decreasing the amounts of cell membrane components such as lipids, phospholipids, glycolipids, proteins, glycoproteins, and cholesterol.

[0059] In some embodiments, an increase or decrease in cell membrane tension can be regulated, modified, or achieved by strengthening or weakening membrane-actin-cortical adhesion (MCA). Therefore, the agents of this disclosure can target various genes and / or their protein products that regulate MCA.

[0060] In one embodiment, the active agent promotes the activation of various membrane-actin linker proteins, resulting in the enhancement or increase of MCA. The enhancement or increase of MCA can be compared to control cells that are not in contact with the active agent or that have defects in one or more MCA interactions. In some embodiments, the increase in MCA can be directly confirmed by using optical tweezers and measuring the force applied to the formed membrane tether. Since MCA is known to be dependent on linker proteins (particularly ERM proteins) that link the membrane to actin, the enhancement of MCA compared to control cells can be indirectly measured by analyzing ERM activity (in this case, phosphorylation state).

[0061] One molecule that can enhance or increase MCA is the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is a component of the cell membrane and can activate membrane-actin linker proteins such as ERM proteins. Therefore, in some embodiments, MCA is increased or enhanced by increasing the expression of one or more proteins known to synthesize PIP2. For example, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) increases PIP2 synthesis (see, e.g., Ben-Aissa et al., Cell Biology, Volume 287, Issue 20, pp. 16311-16323, May 2012). In some embodiments, increasing the expression of one or more PIP5k proteins, such as PIP5K1A (SEQ ID NO: 77), PIP5K1B (SEQ ID NO: 79), and PIP5K1C (SEQ ID NO: 81), leads to an increase in MCA and cell membrane tension by subsequently increasing the amount of PIP2, which activates the ERM protein. In other embodiments, an expression vector containing one or more PIP5K genes can be introduced into cells to increase MCA. These genes include PIP5K1A (SEQ ID NO: 76), PIP5K1B (SEQ ID NO: 78), and PIP5K1C (SEQ ID NO: 80).

[0062] In some embodiments, the expression level and / or activity of one or more ERM proteins is regulated to increase cell membrane tension. This can be achieved, for example, by increasing the expression of certain proteins known to activate ERM proteins directly or indirectly (e.g., by phosphorylation), thereby increasing the amount of active ERM protein and consequently increasing the amount of MCA and cell membrane tension. In some embodiments, certain upstream kinases of ERM proteins are used to increase the activity of ERM proteins by phosphorylation of ERM. In some embodiments, kinases that phosphorylate ERM proteins and increase their activity are one or more of ROCK1 (DNA sequence: SEQ ID NO: 210; amino acid sequence: SEQ ID NO: 211), ROCK2 (DNA sequence: SEQ ID NO: 212; amino acid sequence: SEQ ID NO: 213), SLK (DNA sequence: SEQ ID NO: 214; amino acid sequence: SEQ ID NO: 215), STK10 (DNA sequence: SEQ ID NO: 216; amino acid sequence: SEQ ID NO: 217), and RHOA (DNA sequence: SEQ ID NO: 7; amino acid sequence: SEQ ID NO: 8).

[0063] In some embodiments of this disclosure, the increase in cell membrane tension is affected by bringing cancer cells or cells suspected of being cancerous into contact with an active substance that is internalized within the cancer cells and causes an increase in cell membrane tension.

[0064] In some embodiments, the active ingredient may comprise one or more antibodies, antibody fragments, antibody mimetics, aptamers, siRNAs, microRNAs, shRNAs, nanobodies, DNA, or chemical compounds. The active ingredient can inhibit the expression of a particular gene(s), or inactivate, sequester, degrade, or otherwise reduce or inhibit the protein products of such genes.

[0065] The term "antibody," as used herein, refers to a polypeptide (or set of polypeptides) of the immunoglobulin family capable of non-covalent, reversible, and specific binding to an antigen. For example, naturally occurring IgG-type "antibodies" are tetramers comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (V as used herein). H It consists of a heavy chain constant region (abbreviated as CH1) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (V in this specification). L It consists of a (abbreviated as) and a light chain steady region. The light chain steady region consists of one domain, namely CL. H Region and V L The region can be further subdivided into a more conserved region called the framework region (FR), and a hyper-variable region called the complementarity determination region (CDR) which incorporates these conserved regions. H and V LIt consists of three CDRs and four FRs arranged in the following order from the amino terminus towards the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen, which may be referred to herein as the antigen-binding domain. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies described herein), single-chain variable fragments, and single-domain antibodies. The antibody can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light chain (V L ) and heavy chain (V H ) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as one moves away from the antigen-binding site or the amino terminus of the antibody. The N-terminus is the variable region, the constant region is at the C-terminus, and the CH3 domain and CL domain actually contain the carboxy termini of the heavy and light chains, respectively.

[0066] In certain embodiments, the antibody portion can include one or more of a single-chain variable fragment (scFv), a single-domain antibody, a bispecific antibody, or a multispecific antibody.

[0067] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked via a short, flexible polypeptide linker, and the scFv is expressible as a single-chain polypeptide, retaining the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, scFv means V L Variable region and V H The variable region may have, for example, relative to the N-terminus and C-terminus of the polypeptide in either order, and scFv is V L -Linker-V H Even if it includes, or V H -Linker-V L It may also contain ScFv molecules, which are known in the art and are prepared as described, for example, in U.S. Patent Nos. 4,946,778 and 5,641,870.

[0068] The term "bispecific antibody" refers to an antibody that exhibits specificity to two different types of antigens. The term "multispecific antibody," as used herein, refers to a molecule that binds to one antigen or two or more different epitopes on two or more different antigens. Recognition of each antigen is generally achieved by an "antigen-binding domain." A multispecific antibody may comprise a single polypeptide chain containing multiple, for example, two or more, antigen-binding domains. In some embodiments, a multispecific antibody may comprise two, three, four or more polypeptide chains that together constitute multiple, for example, two or more, antigen-binding domains. Examples of the preparation and isolation of bispecific and multispecific antibodies are described, for example, in International Publication No. 2014031174 and International Publication No. 2009080252.

[0069] The term "single-domain antibody" refers to a variable region in either the heavy chain (VH) or light chain (VL) of an antibody. A single-domain antibody is described, for example, in U.S. Patent Application Publication No. 20060002935.

[0070] In certain embodiments of this disclosure, the antibody mimetic comprises or consists of an affibody, affilin, affimer, affitin, alphabody, antikalin and avimer, DARPin, fynomer, Knitz domain peptide or monobody.

[0071] As used herein, the term “antibody mimetic” is intended to describe an organic compound that specifically binds to a target sequence and has a structure distinct from naturally occurring antibodies. Antibody mimetics may include proteins, nucleic acids, or small molecules. The target sequence to which an antibody mimetic specifically binds may be an antigen. Antibody mimetics can offer superior properties to antibodies, including, but are not limited to, excellent solubility, tissue permeability, thermal and enzymatic stability (e.g., resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimetics include, but are not limited to, afibodies, affilins, affimers, afitins, alphabodies, antikalins, and abimers (also known as avidity polymers), DARPin (Designed Ankyrin Repeat Protein), finomers, Knitz domain peptides, and monobodies.

[0072] The affibody molecules of this disclosure include a protein scaffold comprising one or more α-helices that do not have any disulfide crosslinks. Preferably, the affibody molecules of this disclosure include or consist of three α-helices. For example, the affibody molecules of this disclosure may include an immunoglobulin-binding domain. The affibody molecules of this disclosure may include, for example, the Z domain of protein A.

[0073] The affilin molecules of this disclosure include a protein scaffold constructed by modifying, for example, the exposed amino acids of γB crystallin or ubiquitin. The affilin molecules functionally mimic the affinity of an antibody to an antigen, but do not structurally mimic an antibody. In any protein scaffold used to construct an affilin, these amino acids in a precisely folded protein molecule that are accessible to the solvent or possible binding partners are considered exposed amino acids. One or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.

[0074] The affimer molecules of this disclosure include a protein scaffold containing a highly stable protein that has been engineered to present a peptide loop that yields a high-affinity binding site to a specific target sequence. Exemplary affimer molecules of this disclosure include a protein scaffold based on a cystatin protein or its tertiary structure. Exemplary affimer molecules of this disclosure may have a common tertiary structure that includes an α-helix located on an antiparallel β-sheet.

[0075] In some embodiments, the active agent can affect the stability of RNA. As used herein, RNA stability refers to any regulation of the stability of ERM protein RNA, Bar domain protein RNA, or other RNAs of genes disclosed herein by the active agents disclosed herein. More specifically, RNA modification is a change in the chemical composition of a post-synthesized ribonucleic acid (RNA) molecule that may alter its function or stability.

[0076] RNA modifications that increase stability may include capping, i.e., the addition of a methylated guanine nucleotide cap to the 5' end of mRNA, cleavage, and polyadenylation, i.e., the addition of approximately 250 adenine residues to form a poly(A) tail following cleavage of the 3' end of RNA. Therefore, in some embodiments, the active ingredient can modulate RNA stability by directly or indirectly regulating any of the processes involved in capping, cleavage, and / or polyadenylation.

[0077] In other embodiments, the active ingredient can regulate RNA stability by directly or indirectly regulating RNA degradation. More specifically, RNA degradation is mediated by three major classes of intracellular RNA-degrading enzymes (ribonucleases or RNases): endonucleases that cleave RNA internally, 5'-exonucleases that hydrolyze RNA from the 5' end, and 3'-exonucleases that degrade RNA from the 3' end. The specificity of the RNA degradation mechanism is often conferred by cofactors such as helicases, polymerases, and chaperones.

[0078] ATP-dependent RNA helicases are a large family of proteins involved in almost all pathways of RNA processing and degradation. The eukaryotic exosome complex exhibits both 3' exonuclease and endonuclease activity and functions in the RNA degradation process together with helicase family members Mtr4 and Ski2.

[0079] Furthermore, in several additional or alternative embodiments, the active ingredient can increase or decrease the translation of RNA of, for example, the ERM protein gene and / or BAR domain protein gene disclosed herein.

[0080] In some embodiments, the active substance may be a nucleic acid molecule. More specifically, the nucleic acid molecule may be a molecule comprising at least one of single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), a nucleic acid molecule having at least one modified nucleotide, and any combination thereof.

[0081] More specifically, in certain embodiments, the active substance may be a nucleic acid molecule that reduces the amount or level of ERM and / or Bar domain protein RNA (by either reduced stability, increased degradation, and / or decreased synthesis thereof), and / or inhibits or reduces the activity of Erm or BAR domain RNA, and may include at least one of small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), and other nucleic acid derivatives.

[0082] Interfering RNA (sometimes referred to as RNAi or interfering RNA sequence without distinction) refers to double-stranded RNA capable of silencing, reducing, or inhibiting the expression of a target gene by any currently known or yet-to-be-disclosed mechanism of action. For example, RNAi may act by mediating the degradation of mRNA complementary to the RNAi sequence when the RNAi is in the same cell as the target gene. As used herein, RNAi may refer to double-stranded RNA formed by two complementary RNA strands or one self-complementary strand. RNAi may be substantially or completely complementary to the target mRNA, or it may contain one or more mismatches after alignment with the target mRNA. The sequence of the interfering RNA may correspond to the full-length target mRNA or any subsequence thereof.

[0083] Generally, RNAi is a multi-step process. In the first step, a large dsRNA is cleaved into 21-23 ribonucleotide-length double-stranded effector molecules called "small interfering RNA" or "short interfering RNA" (siRNA). These siRNA double helixes then associate with a complex containing an endonuclease known as the RNA-induced silencing complex (RISC). RISC specifically recognizes and cleaves endogenous mRNA / RNA containing a sequence complementary to one of the siRNA strands. One strand of the double-stranded siRNA molecule ("guide" strand) contains a nucleotide sequence complementary to the nucleotide sequence of the target gene or a portion thereof, and the second strand of the double-stranded siRNA molecule ("passenger" strand) contains a nucleotide sequence substantially similar to the nucleotide sequence of the target gene or a portion thereof.

[0084] In more specific embodiments, siRNA comprises a double-stranded or bifurcated region approximately 18–25 nucleotides long. Often, siRNA contains approximately 2–4 unpaired nucleotides at the 3' end of each strand. At least a portion of one strand of the double-stranded or bifurcated region of siRNA is substantially homologous or substantially complementary to a target sequence in a gene product (i.e., RNA) molecule as defined herein. The strand complementary to the target RNA molecule is the “antisense guide strand,” and the strand homologous to the target RNA molecule is the “sense passenger strand” (which is also complementary to the siRNA antisense guide strand). siRNA may be contained within structures such as miRNA and shRNA, which have additional sequences such as loops, ligation sequences, and stems and other folding structures.

[0085] As described above, RNAi includes small interfering RNAs, which may be referred to herein without distinction as siRNA. siRNAs are described, for example, in U.S. Patents 9,328,347, 9,328,348, 9,289,514, 9,289,505 and 9,273,312 (each of which, in its entirety, constitutes part of this specification by reference). siRNAs can be any interfering RNA having a double-strand length of approximately 15–60, 15–50, or 15–40 nucleotides, more typically approximately 15–30, 15–25, or 18–23 nucleotides. Each complementary sequence of a double-stranded siRNA may be 15–60, 15–50, 15–40, 15–30, 15–25, or 18–23 nucleotides, but other non-complementary sequences may be present. For example, an siRNA double helix may contain a 3' overhang of 1 to 4 nucleotides or more, and / or a 5' phosphate terminus containing 1 to 4 nucleotides or more. siRNA can be synthesized in any of a number of conformations. The types of siRNA conformations used for specific purposes are recognized by those skilled in the art. Examples of siRNA conformations include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate strand molecules, one of which is a sense strand and the other a complementary antisense strand; double-stranded polynucleotide molecules assembled from single-stranded molecules, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure with complementary sense and antisense regions; or cyclic single-stranded polynucleotide molecules having two or more loop structures and a stem having self-complementary sense and antisense regions. In the case of cyclic polynucleotides, the polynucleotides can be processed either in vivo or in vitro to produce active double-stranded siRNA molecules.

[0086] siRNA can be chemically synthesized, encoded in plasmids, transcribed, or vectorized by viruses engineered to express siRNA. siRNA may also be a single-stranded molecule having a complementary sequence that self-hybridizes into a double helix with a hairpin loop. siRNA can also be produced by cleaving the parental dsRNA using a suitable enzyme such as E. coli RNase III or Dicer (Yang et al., Proc. Natl. Acad. Sci. USA 99, 9942-9947 (2002), Calegari et al., Proc. Natl. Acad. Sci. USA 99, 14236-14240 (2002), Byrom et al, Ambion TechNotes 10, 4-6 (2003), Kawasaki et al, Nucleic Acids Res 31, 981-987 (2003), and Knight et al., Science 293, 2269-2271 (2001)). The parental dsRNA can be any double-stranded RNA capable of producing siRNA, such as a complete or partial mRNA transcript.

[0087] A mismatch motif can be any portion of an siRNA sequence that is not 100% complementary to the target sequence. An siRNA may have zero, one, two, three, or more mismatch regions. The mismatch regions may be contiguous or separated by any number of complementary nucleotides. A mismatch motif or region may contain a single nucleotide or two or more contiguous nucleotides.

[0088] siRNA molecules may be provided in several forms, for example, as one or more isolated siRNA double helixes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid. siRNA sequences may have overhangs (as 3' or 5' overhangs, as described in Elbashir et al, Genes Dev 15, 188 (2001) (each of which is incorporated herein by reference)) or may not have overhangs (i.e., have blunt ends).

[0089] siRNA can be obtained using one or more DNA plasmids encoding one or more siRNA templates. siRNA can be transcribed as a sequence that automatically folds from a DNA template in a plasmid having an RNA polymerase III transcription unit, based on, for example, nuclear small RNA U6 or naturally occurring transcription units of human RNase P, RNase H1, into a double helix with a hairpin loop (Brummelkamp et al, Science 296, 550 (2002), Donze et al, Nucleic Acids Res 30, e46 (2002), Paddison et al, Genes Dev 16, 948 (2002)). Typically, the transcription unit or cassette includes an RNA transcription promoter sequence, such as an H1-RNA or U6 promoter, operably linked to a template for transcription of the desired siRNA sequence and a termination sequence consisting of two or three uridine residues and a polythymidine (T5) sequence (polyadenylation signal). The selected promoter can result in constitutive or inducible transcription. Compositions and methods for DNA-dependent transcription of RNA interference molecules are described in detail in U.S. Patent No. 6,573,099 (which in whole forms part of this specification). Transcription units are incorporated into plasmids or DNA vectors from which interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. Patents No. 5,962,428 and No. 5,910,488 (which in whole forms part of this specification). Selected plasmids can result in transient or stable delivery of nucleic acids to target cells. It will be apparent to those skilled in the art that plasmids originally designed to express a desired gene sequence can be modified to include a transcription unit cassette for siRNA transcription.

[0090] Similar to the PCR method (see U.S. Patent Nos. 4,683,195 and 4,683,202, PCR Protocols: A Guide to Methods and Applications, Innis et al, eds, (1990)), methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are known in the art (see, for example, Gubler and Hoffman, Gene 25, 263-269 (1983), Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, (2001) (the entire contents of each of these are incorporated herein by reference)).

[0091] siRNA molecules may be chemically synthesized. In one example of chemical synthesis, single-stranded nucleic acids containing siRNA double-stranded sequences can be synthesized using any of the various techniques known in the art, such as those described in Usman et al, J Am Chem Soc, 109, 7845 (1987), Scaringe et al, Nucl Acids Res, 18, 5433 (1990), Wincott et al, Nucl Acids Res, 23, 2677-2684 (1995), and Wincott et al, Methods Mol Bio 74, 59 (1997) (each of which is incorporated herein by reference in its entirety). Common nucleic acid protecting groups and coupling groups, such as dimethoxytrityl at the 5' end and phosphoramidite at the 3' end, are used for the synthesis of single-stranded nucleic acids. As a non-limiting example, small-scale synthesis can be performed using an Applied Biosystems synthesizer (Thermo Fisher Scientific, Waltham, Mass.) with a 0.2 micromolar-scale protocol that includes a 2.5-minute coupling step for 2'-O-methylated nucleotides. Alternatively, 0.2 micromolar-scale synthesis can be performed using a Thermo Fisher Scientific 96-well plate synthesizer. However, larger or smaller-scale synthesis, including any currently known or yet-disclosed synthesis methods, is also encompassed by the present invention. Reagents suitable for the synthesis of siRNA single-stranded molecules, methods for RNA deprotection, and methods for RNA purification are known to those skilled in the art.

[0092] In certain embodiments, siRNA can be synthesized by tandem synthesis techniques, where both strands are synthesized as a single continuous fragment or strand separated by a linker, which then cleaves and hybridizes to produce separate fragments or strands that form an siRNA double helix. The linker can be any linker, including polynucleotide linkers or non-nucleotide linkers. Tandem synthesis of siRNA can be readily adapted to both multi-well / multi-plate synthesis platforms and large-scale synthesis platforms using batch reactors, synthesis columns, etc. In some embodiments, siRNA can be assembled from two different single-stranded molecules, one strand containing the siRNA sense strand and the other containing the siRNA antisense strand. For example, each strand can be synthesized separately and joined by hybridization or ligation after synthesis and / or deprotection. Either the sense strand or the antisense strand may contain additional nucleotides that are not complementary to each other and do not form a double-stranded siRNA. In certain cases, siRNA molecules can be synthesized as a single, continuous fragment, with self-complementary sense and antisense regions hybridizing to form an siRNA double helix having a hairpin secondary structure.

[0093] An siRNA molecule may contain a double helix having two complementary strands forming a double-stranded region, and at least one modified nucleotide is present in the double-stranded region. The modified nucleotide may be present on one or both strands. If the modified nucleotide is present on both strands, it may be at the same position on each strand or at different positions. Modified siRNA may have lower immunostimulatory activity than the corresponding unmodified siRNA sequence, but it retains the ability to silence the expression of the target sequence.

[0094] Examples of modified nucleotides suitable for use in the present invention include, but are not limited to, ribonucleotides having a 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl group. Modified nucleotides having conformations such as those described in, for example, Sanger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984) (which in whole forms part of this specification by reference) are also suitable for use in siRNA molecules. Other modified nucleotides include, but are not limited to, locked nucleic acid (LNA) nucleotides, G-clamp nucleotides, or nucleotide base analogs. Examples of LNA nucleotides include, but are not limited to, 2'-O,4'-C-methylene-(D-ribofuranosyl)nucleotide, 2'-O-(2-methoxyethyl)(MOE) nucleotide, 2'-methyl-thio-ethyl nucleotide, 2'-deoxy-2'-fluoro(2'F) nucleotide, 2'-deoxy-2'-chloro(2Cl) nucleotide, and 2'-azido nucleotide. G-clamp nucleotides refer to modified cytosine analogs, which, through modification, are given the ability to hydrogen bond with both the Watson-Crick and Hoogsteen faces of complementary guanine nucleotides within the double helix (Lin et al, J Am Chem Soc, 120, 8531-8532 (1998), the entire work which is incorporated herein by reference). Examples of nucleotide base analogs include C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (Loakes, Nucl Acids Res, 29, 2437-2447 (2001), which in whole constitutes part of this specification by reference).

[0095] An siRNA molecule may contain one or more nonnucleotides on one or both of its strands. The nonnucleotides are any subunits, functional groups, or other molecular entities that are not, or do not contain, commonly recognized nucleotide bases such as adenosine, guanine, cytosine, uracil, or thymine, and can be incorporated into a nucleic acid chain in place of one or more nucleotide units, such as sugars or phosphates.

[0096] Chemical modification of siRNA may involve attaching a conjugate to the siRNA molecule. The conjugate may be attached to the 5' and / or 3' ends of the sense and / or antisense strands of siRNA via covalent bonds such as nucleic acid or non-nucleic acid linkers. The conjugate may be attached to siRNA via carbamate groups or other linking groups (see, for example, U.S. Patent Publications 2005 / 0074771, 2005 / 0043219, and 2005 / 0158727, each of which is incorporated herein by reference). The conjugate may be attached to siRNA for any of a number of purposes. For example, the conjugate may be a molecular entity that facilitates the delivery of siRNA into cells, or it may be a molecule containing a drug or label. Examples of conjugate molecules suitable for attachment to siRNA of the present invention include, but are not limited to, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folic acid analogs and their derivatives), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cell receptors that can mediate cell uptake, and combinations thereof (see, for example, U.S. Patent Applications Publication Nos. 2003 / 0130186, 2004 / 0110296, and 2004 / 0249178, and U.S. Patent No. 6,753,423 (each in its entirety forming part of this specification by reference)). Other examples include lipophilic portions, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, minor groove binders, cleavage agents, and crosslinking agent conjugate molecules described in U.S. Patent Application Publications 2005 / 0119470 and 2005 / 0107325 (the entire contents of each of these publications constitute part of this specification by reference).Other examples include 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cationically modified pyrimidines, cationic peptides, guanidinium groups, amidinium groups, and cationic amino acid conjugate molecules, as described in U.S. Patent Application Publication 2005 / 0153337 (which in whole forms part of this specification by reference). Additional examples of conjugate molecules include hydrophobic groups, membrane-active compounds, cell-permeable compounds, cell-targeting signals, interaction modifiers, or steric stabilizers, as described in U.S. Patent Application Publication 2004 / 0167090 (which in whole forms part of this specification by reference). Further examples include conjugate molecules, as described in U.S. Patent Application Publication 2005 / 0239739 (which in whole forms part of this specification by reference).

[0097] In other embodiments, the strands of double-stranded interfering RNA (e.g., siRNA) can be linked to form a hairpin or stem-loop structure (e.g., shRNA). Therefore, as described above, the active substance may be low-molecular-weight hairpin RNA (shRNA).

[0098] In other embodiments, the active ingredient may include microRNA (miRNA). miRNA is a small RNA produced from genes encoding primary transcripts of varying sizes. miRNA has been identified in both animals and plants. The primary transcript (referred to as "pri-miRNA") is processed through various nucleolysis steps into a shorter precursor miRNA, or "pre-miRNA." Since pre-miRNA exists in a folded form, the final (mature) miRNA exists as a double helix, with both strands referred to as miRNA. Pre-miRNA is a substrate for a type of dicer that removes the miRNA double helix from the precursor, after which, like siRNA, the double helix can be incorporated into the RISC complex. Unlike siRNA, miRNA binds to transcription sequences with only partial complementarity and represses translation, usually without affecting steady-state RNA levels. Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (RISC) (see, for example, Michaels et al., Nature Communications, 19:818.2019).

[0099] The active agent may include a nucleic acid active agent that may contain at least one shRNA molecule. In a more specific embodiment, such shRNA may contain a nucleic acid sequence that is at least partially complementary to an EMR protein RNA or a BAR domain protein RNA, or any fragment(s) or variant(s) thereof. When used herein, the term "shRNA" refers to an RNA active agent having a stem-loop structure comprising a first and a second region of a complementary sequence. The degree of complementarity and orientation of the regions is sufficient for base pairing to occur between the regions. The first and second regions are joined by a loop region, which arises from the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. Some of the nucleotides within the loop may be involved in base pairing interactions with other nucleotides within the loop.

[0100] In some specific embodiments, the shRNA contains a sequence complementary to the target ERM protein RNA and / or BAR domain protein RNA and may have a length of about 5 to 50 nucleotides, specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 45, 46, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides or more. In a more specific embodiment, the complementary sequence may have a length in the range of 9 to 29 nucleotides.

[0101] In some embodiments, target genes such as ERM proteins and BAR domain proteins can be edited to reduce or suppress the expression of these proteins using clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related (Cas) proteins, as described in, for example, U.S. Patent Nos. 10,266,850, 10,227,611, 10,000,772, 10,113,167 and U.S. Patent Application Publication No. 20190134227.

[0102] Generally, the term “CRISPR system” refers collectively to transcripts and other elements involved in the orientation of CRISPR-related ("Cas") genes into expression or activity, including the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr-mate sequence (in the context of the endogenous CRISPR system, this includes “direct repeats” and partial direct repeats processed by tracrRNA), the guide sequence (also referred to as “spacers” in the context of the endogenous CRISPR system), and / or other sequences and transcripts from the CRISPR locus.

[0103] The CRISPR system may include, for example, a CRISPR / Cas nuclease, or the CRISPR / Cas nuclease system may include a sequence-specific non-coding RNA molecule (guide) RNA that binds to DNA and a Cas protein (e.g., Cas9) that has nuclease functionality (e.g., two nuclease domains). In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a specific organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0104] In some embodiments, a Cas nuclease and guide RNA (including a fusion of a target sequence-specific crRNA and a fixed tracrRNA) are introduced into cells. Generally, the target site at the 5' end of the gRNA targets the Cas nuclease to the target site, e.g., a gene, using complementary base pairing. In some embodiments, the target site is selected based on the position immediately 5' of a protospacer-adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to the desired sequence by modifying the first 20 nucleotides of the guide RNA to correspond to the target DNA sequence.

[0105] In some embodiments, one or more vectors promoting the expression of one or more elements of the CRISPR system are introduced into cells such that the expression of the CRISPR system elements is directed toward the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined into a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. In some embodiments, the CRISPR system elements combined into a single vector can be positioned in any preferred orientation such that one element is located 5' ("upstream") or 3' ("downstream") of the second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of the second element and oriented in the same or opposite direction. In some embodiments, a single promoter promotes the expression of a transcript encoding a CRISPR enzyme and one or more guide sequences, tracr mate sequences (optionally operably ligated to the guide sequence), and tracr sequences, which are incorporated into one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequences, tracr mate sequences, and tracr sequences are operably ligated to the same promoter and expressed from there.

[0106] In some embodiments, the vector includes one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as “cloning sites”). In some embodiments, one or more insertion sites (e.g., about one, two, three, four, five, six, seven, eight, nine, ten or more, or more than about one, two, three, four, five, six, seven, eight, nine, ten or more) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, the vector includes insertion sites upstream of a tracr mate sequence and optionally downstream of a regulatory element manipulably linked to the tracr mate sequence, and upon expression after insertion of a guide sequence into the insertion site, the guide sequence induces sequence-specific binding of the CRISPR complex to a target sequence in eukaryotic cells. In some embodiments, the vector includes two or more insertion sites, each insertion site located between two tracr mate sequences so that a guide sequence can be inserted at each site. In such configurations, two or more guide sequences may include two or more copies of a single guide sequence, two or more different guide sequences, or a combination thereof. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within a cell. For example, a single vector may contain about one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more guide sequences, or more than about one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more guide sequences. In some embodiments, vectors containing about one, two, three, four, five, six, seven, eight, nine, ten or more guide sequences, or more than about one, two, three, four, five, six, seven, eight, nine, ten or more guide sequences, are provided and can optionally be delivered to cells.

[0107] In some embodiments, the vector includes a regulatory element that is operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs or variants thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, unmodified CRISPR enzymes such as Cas9 have DNA cleavage activity.

[0108] In some embodiments, the CRISPR enzyme is Cas9, which may be Cas9 derived from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme is directed to cleave one or both strands at a location within the target sequence, such as within and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme is directed to cleave one or both strands within a range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 base pairs or more from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a CRISPR enzyme that is mutated from the corresponding wild-type enzyme, and the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 derived from S. pyogenes (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, the Cas9 nickase can be used in combination with guide sequences (sometimes more than one), for example, two guide sequences targeting the sense and antisense strands of a DNA target, respectively. This combination allows for the introduction of nicks into both strands, which can be used to induce non-homologous end joining.

[0109] Generally, the guide sequence is any polynucleotide sequence that hybridizes with the target sequence and has sufficient complementarity with the target polynucleotide sequence to direct the CRISPR complex to sequence-specific binding to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, when optimally aligned using a suitable alignment algorithm, or exceeds about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

[0110] The optimal alignment can be determined using any suitable algorithm for sequence alignment, and non-restrictive examples include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer, or longer than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer. In some embodiments, the guide sequence is less than or equal to about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides. The ability of a guide sequence to direct the sequence-specific binding of the CRISPR complex to a target sequence can be assessed by any suitable assay. For example, sufficient components of the CRISPR system to form a CRISPR complex containing the guide sequence to be tested can be supplied to cells having the corresponding target sequence, for example, by transfection with a vector encoding the components of the CRISPR sequence, and then selective cleavage within the target sequence can be assessed. Similarly, cleavage of a target polynucleotide sequence can be evaluated in vitro by preparing the target sequence, components of the CRISPR complex containing the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rates at the target sequence between the reactions of the test guide sequence and the control guide sequence.

[0111] The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the cell's genome. Exemplary target sequences include sequences specific to the target genome. In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm.

[0112] Generally, a tracr mate sequence contains any sequence that is sufficiently complementary to the tracr sequence in order to facilitate one or more of the following: (1) excision of a guide sequence flanked by intracellular tracr mate sequences containing the corresponding tracr sequence, and (2) formation of a CRISPR complex at the target sequence. The CRISPR complex contains a tracr mate sequence hybridized to the tracr sequence. Generally, the degree of complementarity is based on the optimal alignment along the shorter of the two sequences, the tracr mate sequence and the tracr sequence.

[0113] The optimal alignment can be determined by any suitable alignment algorithm, which may further describe secondary structures such as self-complementarity in either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence along the shorter of the two lengths when optimally aligned is about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher, or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 nucleotides or longer, or longer than approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 nucleotides or longer. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript such that hybridization between them produces a transcript having a secondary structure such as a hairpin. In some embodiments, the loop-forming sequence used for the hairpin structure is 4 nucleotides long and has the sequence GAAA. However, alternative sequences may be used, as well as longer or shorter loop sequences. In some embodiments, the sequence includes a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In some embodiments, the transcript or transcription polynucleotide sequence has at least two hairpins. In some embodiments, the transcript has two, three, four, or five hairpins. In further embodiments, the transcript has at most five hairpins. In some embodiments, a single transcript further comprises a transcription termination sequence, such as a polyT sequence, for example, six T nucleotides.

[0114] In some embodiments, the active ingredient inhibits or reduces the expression of one or more proteins containing a BAR domain. BAR (Bin / amphiphasin / Rvs) domains are found in more than 35 proteins encoded in the human genome. These proteins function in diverse cellular processes such as endocytosis (i.e., endophyllin, sorting nexin, and amphiphasin) and actin rearrangement (i.e., RhoGAP and RhoGEF). Functional BAR dimers are banana-shaped bundles of six helices, positively charged at the tips and along their concave surfaces, and capable of mediating phospholipid binding. The curvature of the concave surface conforms to a rounded membrane with a diameter of approximately 220 Å. For example, the BAR domain of amphiphasin is approximately 210 amino acids long and consists of a coiled coil composed of three elongated α-helices. Two monomers dimerize to form a functional banana-shaped bundle of six helices. The positive charges cluster at the tips of the banana-like structure and along its concave surface. These positive charges are thought to mediate binding to phospholipids, and the concave curvature fits rounded membranes with a diameter of approximately 220 Å. While all BAR domains can bind to curved lipids, a subset of BAR domains may induce membrane curvature. Therefore, it is predicted that BAR domains may function either by targeting proteins to curved membranes during vesicle formation or by physically assisting in the induction of membrane curvature.

[0115] Examples of BAR domain genes and their proteins include MTSS1L / ABBA, FNBP1 / FBP17, TRIP10 / CIP4, ARHGAP10 / GARF2, ARHGAP17 / RICH1, ARHGAP26 / GRAF1, ARHGAP29, ARHGAP42 / GRAF, ARHGAP44 / RICH2, ARHGAP45 / HMHA1, ARHGEF37, ARHGEF38, IRSp53 / BAIAP2, BAIAP2L1 / IRTKS, DNMBP / Tuba, FCHSD2, FER;FES, FCHSD1, GAS7, GMIP, MTSS1 / MIM, OPHN1, PACSIN1, PACSIN2, PACSIN3, SH3BP1, SRGAP1, SRGAP2, SRGAP3, and ARHGAP4.

[0116] In some embodiments, the BAR domain gene is MTSS1L / ABBA (SEQ ID NO: 11), FNBP1 / FBP17 (SEQ ID NO: 13), TRIP10 / CIP4 (SEQ ID NO: 15), ARHGAP10 / GARF2 (SEQ ID NO: 17), ARHGAP17 / RICH1 (SEQ ID NO: 19), ARHGAP26 / GRAF1 (SEQ ID NO: 21), ARHGAP29 (SEQ ID NO: 23), ARHGAP42 / GRAF (SEQ ID NO: 25), ARHGAP44 / RICH2 (SEQ ID NO: 27), ARHGAP45 / HMHA1 (SEQ ID NO: 29), ARHGEF37 (SEQ ID NO: 31), ARHGEF38 (SEQ ID NO: 33), IRSp53 / BAIAP2 (SEQ ID NO: 35), BAIAP2L1 / IRTKS (SEQ ID NO: 11). It is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to (number 37), DNMBP / Tuba (sequence number 39), FCHSD2 (sequence number 41), FER (sequence number 43); FES (sequence number 45), FCHSD1 (sequence number 47), GAS7 (sequence number 49), GMIP (sequence number 51), MTSS1 / MIM (sequence number 53), OPHN1 (sequence number 55), PACSIN1 (sequence number 57), PACSIN2 (sequence number 59), PACSIN3 (sequence number 61), SH3BP1 (sequence number 63), SRGAP1 (sequence number 65), SRGAP2 (sequence number 67), SRGAP3 (sequence number 69), ARHGAP4 (sequence number 71), and FNBPL1 (sequence number 73).

[0117] In some embodiments, the BAR domain protein is MTSS1L / ABBA (SEQ ID NO: 12), FNBP1 / FBP17 (SEQ ID NO: 14), TRIP10 / CIP4 (SEQ ID NO: 16), ARHGAP10 / GARF2 (SEQ ID NO: 18), ARHGAP17 / RICH1 (SEQ ID NO: 20), ARHGAP26 / GRAF1 (SEQ ID NO: 22), ARHGAP29 (SEQ ID NO: 24), ARHGAP42 / GRAF (SEQ ID NO: 26), ARHGAP44 / RICH2 (SEQ ID NO: 28), ARHGAP45 / HMHA1 (SEQ ID NO: 30), ARHGEF37 (SEQ ID NO: 32), ARHGEF38 (SEQ ID NO: 34), IRSp53 / BAIAP2 (SEQ ID NO: 36), BAIAP2L1 / IRTKS (SEQ ID NO: Column number 38), DNMBP / Tuba (sequence number 40), FCHSD2 (sequence number 42), FER (sequence number 44); FES (sequence number 46), FCHSD1 (sequence number 48), GAS7 (sequence number 50), GMIP (sequence number 52), MTSS1 / MIM (sequence number 54), OPHN1 (sequence number 56), PACSIN1 (sequence number 58), PACSIN2 (sequence number 60), PACSIN3 (sequence number 62), SH3BP1 (sequence number 64), SRGAP1 (sequence number 66), SRGAP2 (sequence number 68), SRGAP3 (sequence number 70), ARHGAP4 (sequence number 72), and FNBP1L (sequence number 74) are 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0118] Therefore, in some embodiments, the active substance is an siRNA, shRNA, or miRNA that binds to one or more mRNAs of ARHGAP4, ARHGAP10 / GARF2, ARHGAP17 / RICH1, ARHGAP26 / GRAF1, ARHGAP29, ARHGAP42 / GRAF, ARHGAP44 / RICH2, ARHGAP45 / HMHA1, ARHGEF37, ARHGEF38, IRSp53 / BAIAP2, BAIAP2L1 / IRTKS, DNMBP / Tuba, FCHSD1, FCHSD2, FER;FES, FNBP1 / FBP17, FNBP1L / Toca-1, GAS7, GMIP, MTSS1 / MIM, MTSS1L / ABBA, OPHN1, PACSIN1, PACSIN2, PACSIN3, SH3BP1, SRGAP1, SRGAP2, SRGAP3, and TRIP10 / CIP4.

[0119] In some embodiments, the siRNAs that bind to one or more mRNAs of the BAR domain protein include one of SEQ ID NOs. 82-85 that binds to ARHGAP4 mRNA, one of SEQ ID NOs. 86-89 that binds to ARHGAP10 mRNA, one of SEQ ID NOs. 90-93 that binds to ARHGAP26 mRNA, one of SEQ ID NOs. 94-97 that binds to ARHGAP29 mRNA, one of SEQ ID NOs. 98-101 that binds to ARHGAP42 mRNA, and BA One of sequence numbers 102-105 that binds to IAP2 mRNA, one of sequence numbers 106-109 that binds to BAIAP2L1 mRNA, one of sequence numbers 110-113 that binds to DNMBP mRNA, one of sequence numbers 114-117 that binds to FCHSD1 mRNA, one of sequence numbers 118-121 that binds to FCHSD2 mRNA, one of sequence numbers 122-125 that binds to FES mRNA, and one that binds to GAS7 mRNA. One of sequence numbers 126 to 129, one of sequence numbers 130 to 133 that bind to GMIP mRNA, one of sequence numbers 134 to 137 that bind to HMHA1 mRNA, one of sequence numbers 138 to 141 that bind to MTSS1 mRNA, one of sequence numbers 142 to 145 that bind to MTSS1L mRNA, one of sequence numbers 146 to 149 that bind to OPHN1 mRNA, and one of sequence numbers 150 to 153 that bind to PACSIN1 mRNA. Any one of the following: one of sequence numbers 154-157 that binds to PACSIN2 mRNA, one of sequence numbers 158-161 that binds to PACSIN3 mRNA, one of sequence numbers 162-165 that binds to SRGAP1 mRNA, one of sequence numbers 166-169 that binds to SRGAP2 mRNA, one of sequence numbers 170-173 that binds to SRGAP3 mRNA, one of sequence numbers 174-177 that binds to ARHGAP17 mRNA,It includes one of the sequence numbers 178-181 that bind to ARHGAP44 mRNA, one of the sequence numbers 182-185 that bind to SH3BP1 mRNA, one of the sequence numbers 186-189 that bind to ARHGEF37 mRNA, one of the sequence numbers 190-193 that bind to ARHGEF38 mRNA, one of the sequence numbers 194-197 that bind to FER mRNA, one of the sequence numbers 198-201 that bind to FNBP1 mRNA, one of the sequence numbers 202-205 that bind to TRIP10 mRNA, or one of the sequence numbers 206-209 that bind to FNBP1L mRNA.

[0120] In some embodiments, the active ingredient reduces or knocks down the bar domain genes MTSS1L / ABBA, FNBP1 / FBP17, and TRIP10 / CIP4 or their protein products.

[0121] In other embodiments, the active ingredient can cause an increase in ERM protein expression, such as one or more of EZR, RDX, and MSN, or upstream activating proteins such as kinases or other activating proteins. In some embodiments, the ERM gene is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to EZR (SEQ ID NO: 1), RDX (SEQ ID NO: 3), and MSN (SEQ ID NO: 5). In some embodiments, the ERM gene is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to EZR (SEQ ID NO: 2), RDX (SEQ ID NO: 4), and MSN (SEQ ID NO: 6). In one embodiment, the activating protein is a transformed protein RhoA (SEQ ID NO: 8) having the DNA sequence according to SEQ ID NO: 7. In other embodiments, the activating protein is ROCK1 (SEQ ID NO: 211), ROCK2 (SEQ ID NO: 213), SKL (SEQ ID NO: 215), and STK10 (SEQ ID NO: 217), or one or more genes encoding the above proteins, namely ROCK1 (SEQ ID NO: 210), ROCK2 (SEQ ID NO: 212), SKL (SEQ ID NO: 214), and STK10 (SEQ ID NO: 216). In other embodiments, the activating protein is an expression vector containing one or more PIP5K genes, which can be introduced into cells to activate the ERM protein. Examples of these genes include PIP5K1A (SEQ ID NO: 76), PIP5K1B (SEQ ID NO: 78), and PIP5K1C (SEQ ID NO: 80), which encode the PIP5K1A protein (SEQ ID NO: 77), PIP5K1B protein (SEQ ID NO: 79), and PIP5K1C protein (SEQ ID NO: 81), respectively.

[0122] In some embodiments, DNA containing the coding region of a fusion protein, such as one that immobilizes an ERM protein to the cell membrane, can be introduced into cells, where the DNA is subsequently expressed, causing the fusion protein to increase the tension of the cell membrane. In one embodiment, the DNA is an expression vector or construct configured to express an MA-ezrin fusion protein containing a conserved myristylated sequence of Lyn fused with ezrin, where ezrin contains a phosphorylation-mimicking activating mutation (T567E). The DNA sequence of the MA-ezrin fusion protein gene is SEQ ID NO: 9, and the amino acid sequence of the MA-ezrin protein is SEQ ID NO: 10. In some embodiments, the active ingredient is an expression vector containing the DNA sequence of the MA-ezrin fusion protein of SEQ ID NO: 9.

[0123] In other embodiments of the Disclosure, a method for inhibiting cell migration and / or proliferation includes reducing the expression of one or more proteins containing a BAR domain, thereby inhibiting cell migration and / or proliferation. Methods for reducing BAR domain proteins are discussed throughout the Disclosure.

[0124] The active substance can be delivered to and internalized in cells using methods known in the art. For example, in some embodiments, the active substance, such as siRNA, shRNA, or miRNA, can be encapsulated, bound to, or adsorbed onto cationic lipids, liposomes, cochleates, viromosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsomes, polycationic peptides, cationic nanoemulsions, or combinations thereof.

[0125] In some embodiments, active substances, particularly siRNA, shRNA, miRNA, or other polynucleotide molecules, can enter cells via liposomes. Various amphiphilic lipids can form bilayers in an aqueous environment, encapsulating an RNA-containing aqueous core as liposomes. These lipids may have anionic, cationic, or zwitterionic hydrophilic head groups. Liposome formation from anionic phospholipids dates back to the 1960s, while cationic liposome-forming lipids have been studied since the 1990s. Some phospholipids are anionic, while others are zwitterionic. Preferred classes of phospholipids include, but are not limited to, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. Useful cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids are DPPC, DOPC, and dodecylphosphocholine. Lipids can be saturated or unsaturated.

[0126] Liposomes can be formed from a single lipid or a mixture of lipids. The mixture may include (i) a mixture of anionic lipids, (ii) a mixture of cationic lipids, (iii) a mixture of zwitterionic lipids, (iv) a mixture of anionic and cationic lipids, (v) a mixture of anionic and zwitterionic lipids, (vi) a mixture of zwitterionic and cationic lipids, or (vii) a mixture of anionic, cationic, and zwitterionic lipids. Similarly, the mixture may contain both saturated and unsaturated lipids. For example, the mixture may contain DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and / or DMPG (anionic, saturated). When using a mixture of lipids, not all lipid components in the mixture need to be amphiphilic; for example, one or more amphiphilic lipids can be mixed with cholesterol.

[0127] The hydrophilic portion of a lipid can be PEGylated (i.e., modified by covalent bonding of polyethylene glycol). This modification enhances stability and prevents nonspecific adsorption of liposomes. For example, lipids can be conjugated to PEG using techniques such as those disclosed in Heyes et al. (2005) J Controlled Release 107:276-87.

[0128] Liposomes are typically classified into three groups: multilayer vesicles (MLVs), small monolayer vesicles (SUVs), and large monolayer vesicles (LUVs). MLVs have multiple bilayers in each vesicle, forming several separate aqueous compartments. SUVs and LUVs have a single bilayer encapsulating an aqueous core; SUVs typically have a diameter of 50 nm or less, while LUVs have a diameter greater than 50 nm. Liposomes useful in this invention are ideally LUVs having a diameter in the range of 50 nm to 220 nm. For compositions containing a population of LUVs with different diameters, (i) at least 80% must numerically have a diameter in the range of 20 nm to 220 nm, (ii) the average diameter of the population (Zav, by intensity) is ideally in the range of 40 nm to 200 nm, and / or (iii) the diameter must have a polydispersity index of less than 0.2.

[0129] Techniques for preparing suitable liposomes are known in the art; see, for example, Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers: Methods and Protocols. (ed. Weissig). Humana Press, 2009. ISBN 160327359X, Liposome Technology, volumes I, II & III. (ed. Gregoriadis). Informa Healthcare, 2006, and Functional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes). (eds. Arshady & Guyot). Citus Books, 2002.

[0130] In other embodiments, active agents such as siRNA, shRNA, miRNA, or other polynucleotide molecules can enter cells as part of microparticles. Various polymers can form microparticles and encapsulate or adsorb active agents. The use of substantially non-toxic polymers means that recipients can safely receive the particles, and the use of biodegradable polymers means that the particles can be metabolized after delivery to avoid long-term persistence. Useful polymers are also sterilizable to aid in the preparation of pharmaceutical-grade formulations.

[0131] Suitable non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acids), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanone, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinylpyrrolidinone or polyesteramides, and combinations thereof.

[0132] In some embodiments, the microparticles are formed from poly(α-hydroxy acids) such as poly(lactide) ("PLA"), copolymers of lactide and glycoside such as poly(D,L-lactide-co-glycolide) ("PLG"), and copolymers of D,L-lactide and caprolactone. Useful PLG polymers include those having lactide / glycolide molar ratios in the range of 20:80 to 80:20, for example, 25:75, 40:60, 45:55, 55:45, 60:40, and 75:25. Useful PLG polymers include those having molecular weights of 5000Da to 200000Da, for example, 10000Da to 100000Da, 20000Da to 70000Da, and 40000Da to 50000Da. Ideally, microparticles have a diameter in the range of 0.02 μm to 8 μm. For compositions containing a population of microparticles with different diameters, numerically at least 80% must have a diameter in the range of 0.03 μm to 7 μm.

[0133] Techniques for preparing suitable microparticles are known in the art; see, for example, Functional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes). (eds. Arshady & Guyot). Citus Books, 2002, Polymers in Drug Delivery. (eds. Uchegbu & Schatzlein). CRC Press, 2006. (Chapter 7), and Microparticulate Systems for the Delivery of Proteins and Vaccines. (eds. Cohen & Bernstein). CRC Press, 1996. To promote the adsorption of active ingredients, the microparticles may contain cationic surfactants and / or lipids, as disclosed, for example, in O'Hagan et al. (2001) J Virology 75:9037-9043 and Singh et al. (2003) Pharmaceutical Research 20: 247-251. An alternative method for producing polymer microparticles is by molding and curing, as disclosed, for example, in International Publication No. 2009 / 132206.

[0134] In some embodiments, RNAi molecules, such as siRNA or other polynucleotide molecules, can be adsorbed onto microparticles, and the adsorption is enhanced by the inclusion of cationic materials (e.g., cationic lipids) in the microparticles.

[0135] In some embodiments, the liposomes or microparticles may include a targeting moiety such as an antibody, antibody fragment, antibody mimetic, aptamer, or ligand, and the liposomes or microparticles may be targeted to specific cells, for example by targeting a specific receptor on the cell surface, thereby causing the liposomes or microparticles to be internalized.

[0136] In some embodiments, the active substance (e.g., siRNA molecules, shRNA molecules, etc.) can be introduced into cells by receptor-mediated endocytosis, as described in U.S. Patent No. 6,090,619.

[0137] In some embodiments, transfection reagents (e.g., cationic lipids such as lipofectamine) are typically used to facilitate cell transfection with siRNA, shRNA, or miRNA in the form of dsRNA, for example.

[0138] In other embodiments, the active ingredient can be delivered to target cells using a specific vector. Examples of such vectors include bacteriophages, plasmids, phagemids, viruses, embeddable DNA fragments, episomal plasmids / viruses, and other vehicles or systems that enable the transfer of nucleic acid molecules into desired target host cells or the embedding of specific nucleic acid molecules at specific locations, and in some further embodiments, result in the expression of the transduced nucleic acid molecule in the target cells. Non-limiting examples of systems that may be used in the present invention for the specific targeted transfer of nucleic acid molecules include short palindromic repeats (CRISPR / Cas) systems that form clusters and have regular spacing, transcription activator effector nucleases (TALENs), zinc finger protein (ZEN) systems, and any equivalent systems.

[0139] A vector is typically a self-replicating DNA or RNA construct comprising a desired nucleic acid sequence and manipulably linked gene regulatory elements that are recognized in a suitable host cell and achieve transcription and translation of the desired gene. Generally, the gene regulatory elements may include a prokaryotic promoter system or a eukaryotic promoter expression regulatory system. Such systems typically include a transcription promoter and a transcription enhancer to increase the level of RNA expression. A vector usually contains an origin of replication that allows the vector to replicate independently of the host cell. However, there are vectors that are intentionally designed to result in replication defects, which can be delivered to cells and integrated into the host genome, whether at random or pre-designed sites.

[0140] Therefore, the terms regulatory and modulatory elements include promoters, terminators, and other expression regulatory elements. Such regulatory elements are described in Molecular Cell Biology Editors: H. Lodish et al., 7 th edition 2013 (or 8 th As described in edition 2016, for example, any of the broad range of expression regulatory sequences that control the expression of a DNA sequence when operably ligated to the DNA sequence can be used in these vectors to express a DNA sequence encoding any desired RNA or protein using the method of the present invention.

[0141] The vector or delivery vehicle may additionally include restriction sites, antibiotic resistance, fluorescent tags, or other markers suitable for positive selection (e.g., G418 resistance) or negative selection (e.g., herpesvirus TK) of vector-containing cells. Plasmids are the most commonly used form of vector, but other forms of vectors that perform equivalent functions and are known or to be known in the art are also suitable for use herein. See, for example, Ausubel et al., Current Protocols in Molecular Biology (2016), Wiley online library.

[0142] In some embodiments, the delivery vehicle according to this disclosure may be at least one viral vector, such as a lentivirus, adenovirus, or AAV. Examples of adenovirus vectors and gene transfer methods are described in International Publication Nos. 00 / 12738 and 00 / 09675, and U.S. Patents Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730 and 5,824,544 (each in whole forming part of this specification).

[0143] In some embodiments, the expression construct comprises one or more elements selected from the group consisting of: (i) a multiple cloning site (e.g., an Ec1HK1 site) for introducing a nucleotide sequence that is appropriately cleaved enzymatically or otherwise biochemically to yield a linearization vector from which the PCR amplification product can be directly cloned; (ii) a reporter gene; (iii) a promoter and / or enhancer for regulating the expression of a transcriptional polynucleotide (e.g., a polynucleotide encoding a polypeptide); (iv) a polyadenylated sequence; (v) a selectable marker gene; and (vi) an origin of replication.

[0144] In certain embodiments, the expression construct is a vector or set of vectors, in particular, in plasmid form, used for studying, measuring, or monitoring gene expression (e.g., promoter or enhancer activity). The vector is appropriately in the form of a prokaryotic vector or a eukaryotic vector. Many other vectors, such as viruses, artificial chromosomes, and other non-plasmid vectors, may also be used.

[0145] This disclosure also provides methods for regulating the rate of cell division of eukaryotic cells in culture. Certain cells may be difficult to divide when grown in culture, while others simply exhibit a slow rate of cell division. In other cases, it may be beneficial to slow the rate of cell division of certain types of cells. This disclosure provides methods for regulating the rate of cell division to increase or decrease the rate of cell division, depending on the needs of the user of the cell culture.

[0146] The disclosure also provides a method for increasing or decreasing the rate of cell division of eukaryotic cells in cell culture, comprising contacting eukaryotic cells with an active substance that causes a change in the tension of the cell membrane of the cells, thereby increasing or decreasing the rate of cell division compared to the rate of cell division of eukaryotic cells that are not in contact with the active substance.

[0147] In some embodiments, the activator that causes a decrease in the rate of cell division increases the expression of one or more ezrin, radixin, and moesin (ERM) proteins, or causes an increase in the phosphorylation of ERM proteins. In some embodiments, the activator that causes an increase in the expression of ERM proteins includes an expression vector containing one or more ezrin (SEQ ID NO: 1), radixin (SEQ ID NO: 3), and moesin genes (SEQ ID NO: 5). In some embodiments, the activator that causes an increase in the phosphorylation of ERM proteins includes an expression vector containing one or more ROCK1 (SEQ ID NO: 210), ROCK2 (SEQ ID NO: 212), SLK (SEQ ID NO: 214), STK10 (SEQ ID NO: 16), and RHOA (SEQ ID NO: 7) genes. The activator may include several expression vectors containing one or more ERM proteins and / or ERM kinases as described above.

[0148] In one embodiment, the reduction in cell division rate is caused by an expression vector configured to express an ezrin fusion protein containing a conserved myristylated sequence of Lyn fused with ezrin, the ezrin containing a phosphorylation-mimicking activating mutation (T567E). In some embodiments, the ezrin fusion protein contains the DNA sequence and amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0149] In another embodiment, the agent that causes an increase in the rate of cell division increases the expression of one or more BAR domain proteins.

[0150] In some embodiments, the activator that causes an alteration (e.g., increase) in the rate of cell division reduces the expression or phosphorylation of one or more ezrin, radixin, and moesin (ERM) proteins. For example, the activator may be a compound that inhibits one or more upstream kinases of ERM proteins. In one example, the activator is an inhibitor of ROCK1 and / or ROCK2, such as Y-27632 or ROCKi-IV, as discussed, for example, in Ohata et al., Cancer Res; 72(19) October 1, 2012. Other inhibitors of ERM kinases are known in the art. In other embodiments, the activator is an antisense RNA, siRNA, shRNA or miRNA, or an antibody.

[0151] The disclosure also provides a method for modulating cellular properties, comprising contacting a cell with a substance that alters the tension of the cell membrane, thereby modulating cellular properties by altering the tension of the cell membrane.

[0152] In some embodiments, cell properties are controlled by membrane-actin cortical adhesion (MCA), and the active agent increases or decreases MCA compared to control cells.

[0153] In some embodiments, the cellular properties are increased or decreased cell motility and invasion into surrounding tissues. In other embodiments, the cellular properties are increased or decreased cell proliferation and tumorigenesis. Agents that may be used to modulate cellular properties are discussed throughout this disclosure.

[0154] Results and Analysis Malignant tumors are associated with changes in cellular mechanics that contribute to the significant cellular deformation required for metastatic dissemination. We hypothesized that cell-specific physical factors maintaining epithelial cell mechanics could function as tumor suppressors. Here, using optical tweezers, genetic interference, mechanical perturbations, and in vivo studies, we demonstrate that epithelial cells maintain higher membrane (PM) tension than metastatic cells, and that high PM tension strongly inhibits cancer cell migration and invasion by counteracting BAR family proteins that sense / generate membrane curvature. This tension homeostasis is achieved by membrane-cortical adhesion (MCA) regulated by ERM proteins, and its disruption spontaneously converts epithelial cells into a mesenchymal migration phenotype driven by BAR proteins. Induction of epithelial-mesenchymal transition (EMT) by forced expression of EMT transcription factors consistently results in a decrease in PM tension. In metastatic cells, increasing PM tension by manipulating MCA is sufficient to suppress both mesenchymal and amoeboid 3D migration and tumor invasion by impairing membrane-mediated mechanotransduction by BAR proteins, thereby revealing a previously undescribed mechanical tumor suppression mechanism.

[0155] Epithelial cells have higher PM tension than malignant cells. To determine if there is a difference in PM tension between epithelial cells and metastatic cells, we analyzed the membrane tether force proportional to the PM tension using optical tweezers (Figure 7a). Human non-invasive mammary epithelial cells (MCF10A) and metastatic breast cancer cells (MDA-MB-231) were mainly used as they are commonly used models of malignant metastasis. To avoid extracellular influences such as cell-cell adhesion, tether force was measured at the single-cell level. The tether force of MCF10A cells was found to be almost equivalent to that of low-invasive human breast cancer cells (AU565 and MCF7; Figure 1a). Unlike these low-invasive cells, metastatic breast cancer cells such as MDA-MB-231 cells and Hs578T cells formed both prominent membrane ruffles and blebs when cultured on an uncoated glass substrate. Interestingly, no difference in tethering force was observed between Ruffling cells and Breving cells, and their tethering force was found to be significantly lower than that of low-motility cells (Figure 1a). Similar results were obtained in advanced prostate cancer cells (PC-3) and pancreatic cancer cells (PANC-1) (Figure 1a). PM tension was approximately half that of low-motility cells in metastatic cells (Table 1). Since the tethering force of normal epithelial cells such as canine kidney MDCK II cells and rat liver IAR-2 cells was similar to that of MCF10A cells, the ability to maintain higher PM tension than malignant cells appears to be a common characteristic of epithelial cells across species and tissues (Figure 1a, Table 1).

[0156] [Table 1]

[0157] PM tension is contributed to by both in-plane tension of the lipid bilayer and MCA (Figure 7b). Since PM tension is thought to be highly dependent on MCA, we focused on ERM proteins and F-actin under the PM. ERM proteins are activated by phosphorylation of conserved threonine residues. In MCA10A and AU565 cells, a strong phosphorylated ERM (pERM) signal was observed to decorate the PM overall and partially co-localize with F-actin (Figures 1b and 7c). In contrast, MDA-MB-231 and Hs578T cells had generally lower levels of membrane-bound pERM and F-actin, regardless of whether they exhibited membrane ruffling or blebing, and some pERM levels were limited to the posterior cell and occasionally contractile bleb (Figures 1b and 7c). Consistent with these 2D observations, confocal reconstructed 3D images showed that MCF10A and AU565 cells embedded in the collagen matrix (3D) consistently exhibited a rounded shape and possessed uniform and strong levels of pERM and F-actin under the PM (Figure 1c and Figure 7d). MDA-MB-231 cells showed both an actin-based elongated lobular migration phenotype and an actin- and bleb-based rounded amoeboid migration phenotype in 3D (Figure 7d). In both cases, pre-membrane pERM and F-actin showed generally reduced levels (Figure 1c). Similarly, Hs578T cells, which primarily showed an actin-based elongated phenotype in 3D (Figure 7d), yielded a consistent phenotype (Figure 1c). These results suggest that the difference in PM tension observed between non-motile and invasive cells is primarily due to ERM-mediated changes in the MCA, and that such mechanical disruption correlates with the invasive phenotype, regardless of the type of protrusion or mode of movement.

[0158] Epithelial cells spontaneously switch to a mesenchymal migration phenotype when PM tension is reduced. Based on these findings, it was hypothesized that a reduction in PM tension might be sufficient for epithelial cells to acquire invasive behavior. To test this, MCAs were manipulated by simultaneously knocking down three members of the ERM (ezrin, radixin, and moesin) or their specific kinases (SLK and STK10 (also known as LOK)) (Figure 8a). RHOA was also considered, as it is known to act as an upstream regulator of these ERM kinases (Figure 7b). Paradoxically, despite its important role in cell migration, RHOA has been reported to play an inhibitory role in invasion and metastasis. In particular, knockdown of these proteins in MCF10A cells resulted in a significant decrease in tether force (Figure 8b), which corresponded to a decrease in PM tension (Figure 2a). Notably, phosphorylated myosin light chain (pS19MLC), a marker of myosin II activation, was unaffected in ERM and SLK+STK10 depleted cells (Figure 8c), indicating that the observed difference in PM tension was specifically attributable to MCA changes rather than actomyosin contractility. These knockdown cells showed an overall decrease in membrane-bound pERM and F-actin levels, along with cell diffusion accompanied by marked actin-based protrusions such as membranous pseudopods and membrane ruffling in 2D (Figures 2b, 8d). Next, the effects of reduced PM tension were evaluated in a 3D on-top culture system using a collagen-Matrigel mixture commonly used in cancer research. As expected, control cells formed perfectly rounded spheroids (Figure 2c). Surprisingly, over 60% of the hypotension cells exhibited an elongated, invasive phenotype and showed cell seeding into the surrounding matrix (Figure 2c). These cells consistently showed a marked increase in motility, including invasion through narrow gaps and restricted migration (Figure d). The observed invasive phenotype appeared to be independent of the classical EMT program, as these hypotonic cells retained epithelial characteristics (Figure 8c), and E-cadherin depletion caused a slight but significant inhibition of invasion and migration (Figure d). Deficiency of ERM proteins in low-invasive breast cancer cells also resulted in a dramatic increase in invasion (Figure 8e).The reduction in MCA did not affect the proliferation of MCF10A cells (Figure 8f).

[0159] The effect of reduced PM tension on unicellular motility behavior in 3D was further investigated. Time-lapse videos showed that in the unicellular state when embedded in the collagen matrix, approximately 95% of control RNAi-treated cells exhibited a rounded shape with a non-motile phenotype (Figure 2e). In contrast, knockdown of MCA regulators resulted in conversion to a mesenchymal-like motility phenotype in all cases. Approximately 50% of cells exhibited an elongated motility phenotype with dynamic cell shape changes showing membrane protrusion and retraction (Figure 2e). Amoeboid motility characterized by a rounded shape was not observed in these knockdown cells. In 3D, this elongated phenotype was closely associated with lower levels of pERM and F-actin under PM, as observed in metastatic cells (Figure 2f). These data suggest that reduced PM tension can spontaneously convert non-motile epithelial cells to a mesenchymal-like motility phenotype.

[0160] Correlation between decreased PM tension and malignant progression. We noted the striking similarity between the elongated cell shape changes induced by decreased PM tension and those induced by EMT. Therefore, we hypothesized that disruption of tension homeostasis in PM may generally be associated with malignant transformation. To test this hypothesis, we established MCF10A cell lines that stably overexpress Snail or Slug, transcription factors that strongly induce EMT. As expected, these cells exhibited EMT characteristics, including the formation of actin-based protrusions (Figure 3a, arrows) and altered expression of E-cadherin and vimentin (Figure 9a). Interestingly, Snail expression resulted in a significant decrease in membrane-bound pERM and F-actin levels (Figure 3a). Furthermore, MCF10A cells overexpressing Snail or Slug showed lower PM tension than the parental cells (Figure 3b). Similar results were obtained in MDCK II cells inducibly expressing the K-Ras activating mutant (G12V), a known key driver of cancer progression and metastasis (Figure 9b). In 3D, the elongated morphology induced by EMT was closely associated with changes in pERM and actin staining patterns, similar to 2D (Figures 3c and 3d), suggesting a close correlation between changes in PM tension and the acquisition of an invasive phenotype.

[0161] To further investigate whether decreased PM tension is a common feature involved in cancer progression, we analyzed cancer genomes using pan-cancer data from the Cancer Genome Atlas (TCGA). Surprisingly, a comprehensive analysis of 14 major cancers across 6586 patients revealed that epithelial tumors frequently exhibit putative heterozygous deletions of RHOA, SLK, STK10, and ERM (Figures 3e and 9c). A similar trend was observed in 961 cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) (Figure 9d). Furthermore, a meta-analysis revealed a significant association between increased ERM kinase expression and increased patient survival in breast, lung, and gastric cancers (Figure 3f). These data suggest that disruption of constitutive PM tension is a common mechanical property of malignant cells and may correlate with cancer progression.

[0162] An increase in PM tension is sufficient to suppress 3D migration, tumor invasion, and metastasis. If a decrease in PM tension is key to acquiring migration and invasion, then we hypothesized that an increase in MCA might be sufficient to suppress cancer cell dissemination. Therefore, we attempted to increase PM tension by directly manipulating MCA. Since ERM proteins dissociate entirely from PM in advanced cancer cells, we inferred that membrane-targeted active ezrin (MA-ezrin) could restore the decrease in PM tension. To test this, we designed an MA-ezrin construct by fusing a conserved myristoylation sequence of Lyn with ezrin, and then introduced a phosphorylation-mimicking activating mutation (T567E) to maintain activity throughout the PM (Figure 4a). In particular, its expression in MDA-MB-231 cells resulted in a significant increase in PM tension (Figure 4a). MA-ezrin was generally localized to the PM, resulting in significant suppression of actin and bleb-based membrane outcroping (Figures 4b and 4b). These hypertonic cells showed normal cell proliferation in vitro (Figure 10a), but exhibited a significant decrease in invasion and migration (Figure 4c).

[0163] It has also been reported that ERM activity is associated with cortical stiffness, at least in the rounding of mitotic cells. Treatment with kallicrin A, commonly used to increase cortical stiffness or tension by increasing myosin II activity, was found to have no effect on invasiveness, reflecting the flexibility of cancer cell migration due to changes in contractility (Figure 10b). Furthermore, treatment of cells with methyl-β-cyclodextrin (MβCD), a cholesterol-scavenging compound that increases PM tension (Figure 10c) but decreases cortical stiffness, significantly reduced invasion (Figure 10b), ruling out the potential influence of cortical stiffness on inhibiting cancer cell migration.

[0164] Next, we tested whether increasing PM tension affected 3D migration. From 3D reconstructed confocal images, ezrin-expressing cells showed a different protrusion phenotype, similar to the parental cells, but MA-ezrin-expressing cells were shown to have a rounded shape without protrusion formation, reminiscent of epithelial cell characteristics. Indeed, time-lapse videos showed that parental and ezrin-expressing MDA-MB-231 cells exhibited slender, lobular motility (approximately 20%), rounded, amoeboid motility, and phenotypic switching between the two modes (mixed phenotype; 50%), as previously reported (Figures 4d and 4e). In contrast, the majority of MA-ezrin-expressing cells exhibited a rounded shape, along with non-migrating behavior (80%) and significantly reduced migration speed (more than 4 times slower) (Figures 4d and 4e). These data demonstrate that increasing PM tension is sufficient to suppress both of the two distinct modes of 3D migration.

[0165] To further investigate whether maintaining high PM tension plays an active role in suppressing cancer cell dissemination, we used an orthotopic mouse model of human breast tumor formation, local invasion, and spontaneous metastasis. MA-ezrin-expressing MDA-MB-231 cells, when injected into the mammary fat body, proliferated in vitro at a rate comparable to that of parental cells (Table 2; Figure 10a), but showed reduced tumorigenic potential and produced significantly smaller tumors (Figure 10d).

[0166] [Table 2]

[0167] In addition, in contrast to control tumors that exhibited significant invasive behavior with numerous cancer cells infiltrating surrounding adipose tissue, MA-ezrin-expressing tumors showed a clear boundary between the tumor cell area and adjacent tissue, demonstrating a significant decrease in invasiveness (Figure 4f). Furthermore, MA-ezrin cells showed a significant reduction in spontaneous lung metastases (Figure 4g) and experimental (tail vein injection) lung metastases (Figure h). Taken together, these data suggest that PM tension maintained by MCA acts as a potent tumor suppressor, inhibiting tumorigenesis, invasion, and metastasis.

[0168] Constitutive PM tension suppresses cancer cell motility by counteracting BAR proteins. Next, we investigated the mechanism by which PM tension inhibits cancer cell motility. We previously demonstrated that FBP17, a BAR domain-containing protein that regulates membrane curvature, acts as a sensor of PM tension involved in actin-based directed movement. Mathematical models consistently showed that the polymerization ability of BAR proteins is essentially dependent on membrane tension, suggesting a universal tension sensing mechanism mediated by BAR proteins. Therefore, we performed siRNA screening of BAR proteins after ERM knockdown to investigate whether BAR proteins play an important role in the invasive phenotype induced by low PM tension in 3D on-top culture (Figure 5a). Several BAR proteins, including MTSS1L (also known as ABBA), were identified (Figure 5a). We also noted that, as previously reported, knockdown of Toca proteins such as FBP17 and CIP4 slightly reduced the elongated invasive behavior induced by ERM depletion, suggesting their functional redundancy. Indeed, simultaneous depletion of these proteins increased the suppression of the invasive phenotype (FBP17+CIP4+Toca-1, triple KD) (Figure 5a). Among the characterized proteins, MTSS1L and Toca proteins were also required for invasion in MDA-MB-231 cells (Figure 11a) or MCF10A cells (Figure 11b) induced by depletion of ERM protein or RHOA. Therefore, these proteins were the focus of subsequent analyses. MTSS1L and Toca proteins are involved in membranous pseudopod formation or membrane ruffling via activation of actin nucleation dependent on the Arp2 / 3 complex, suggesting that these proteins may play a role in hypotension-mediated actin-based protrusion. Indeed, knockdown of these BAR proteins was found to suppress the elongated invasive phenotype driven by Snail overexpression (Figure 5b). The role of BAR proteins in 3D cancer cell motility was further investigated.Time-lapse videos showed that depletion of MTSS1L or Toca protein suppressed motility in both mesenchymal and amoeboid cells, with the majority of cells exhibiting a non-motile phenotype characterized by a rounded shape and a three-fold slower migration speed (Figures 5c and 5d). Deletion of MTSS1L or Toca protein consistently resulted not only in the inhibition of actin-based overhang but also in the formation of nonpolar membrane blebs (Figure 5e).

[0169] These data suggest that low-tension-mediated mechanosignaling by BAR proteins plays a crucial role in cancer cell motility, and that this mechanism is typically suppressed by constitutive PM tension in non-motile epithelial cells. Indeed, GFP-FBP17 primarily exhibited cytoplasmic distribution in MCF10A cells, but knockdown of ERM, their kinases, or Snail expression resulted in accumulation of GFP-FBP17 in PM (Figure 5f). In contrast, in ezrin-expressing MDA-MB-231 cells, GFP-FBP17 spontaneously accumulated in both actin-based and bleb-based membrane protrusions in both 2D (Figure 11c) and 3D (Figure 5g) environments. However, no significant accumulation of GFP-FBP17 was observed in high-tension MA-ezrin-expressing cells (Figures 5g and 11c). Similar results were obtained with GFP-MTSS1L (Figures 11c and 11d). Furthermore, increasing PM tension by MBCD treatment was also sufficient to prevent the recruitment of FBP17 to the PM (Figures 11f and 11e). To further investigate whether PM tension directly controls BAR protein assembly, tension was increased by adjusting in-plane membrane tension using a cell stretching device. Mechanical stretching of the PM induced rapid deassembly of FBP17 or MTSS1L, accompanied by the disappearance of the leading edges. These data suggest that constitutive PM tension acts as a mechanical tumor suppressor that inhibits cancer cell dissemination by counteracting membrane-mediated mechanotransduction by BAR proteins.

[0170] Cellular mechanics has long been considered to be intrinsically related to invasion and metastasis. However, how such mechanical changes affect tumor dissemination ability at the cellular and molecular levels has remained unclear due to a lack of understanding of the key physical parameters underlying malignant phenotypes. Here, we show that metastatic cells exhibit significantly lower PM tension than epithelial cells, and that this mechanical property is closely related not only to membrane protrusion but also to tumor invasion and metastasis. Our data further demonstrate that this decrease in MCA-based tension is converted into Arp2 / 3 complex-dependent actin polymerization via the self-assembly of BAR proteins such as MTSS1L and Toca proteins, promoting cancer cell migration and invasion (Figures 6a and 6b). Recent studies have shown that various BAR proteins play an active role in the invasion and metastasis of various cancers. An important aspect of our findings is that invasive behavior, regardless of the type and mode of protrusion, can be phenotypically normalized simply by increasing MCA-based PM tension. This is consistent with recent studies showing that membrane-actin detachment is key to both actin-based and bleb-based protrusions. In vivo studies have reported that the deficiency of moesin (the only ERM protein in Drosophila) alone is sufficient to induce invasion in Drosophila. Furthermore, analysis of clinical samples has shown that ERM proteins generally exhibit cytoplasmic distribution in malignancies, including breast cancer, lung cancer, and head and neck squamous cell carcinoma. Therefore, our study, along with these observations, supports the general role of MCA-based PM tension as a mechanical tumor suppressor.

[0171] A limitation of our research is that, since ERM proteins are known to be involved in the regulation of various signaling molecules, we cannot rule out the possibility that MA-ezrin expression may have effects other than PM tension. Recent studies have reported the development of synthetic molecular tools (iMC linkers) that manipulate MCA by simply linking the cell membrane to the actin cortex. Interestingly, two recent supplementary studies using this tool and constitutively active ezrin have shown that stem cell diffusion, which correlates with cell differentiation, is inhibited by an increase in MCA-based tension. Further research using this tool will be needed to support the importance of ERM-mediated MCA in inhibiting cancer cell motility.

[0172] Our data suggest that ERM-mediated MCA is involved in maintaining homeostatic PM tension in non-invasive cells. However, changes in actin structure itself are also thought to affect PM tension. Migratory cells are characterized by a significantly increased actin filament turnover rate, primarily mediated by cofilin, which contributes to dynamic protrusion formation by supplying less stable F-actin and G-actin monomers. Such accelerated depolymerization may cause changes in PM tension, thereby synergistically promoting cancer cell migration. This raises the question of how changes in PM tension led to two different modes of protrusion formation and subsequent migration. In epithelial cells, an overall decrease in MCA appeared to induce only slow mesenchymal-like motility. This suggests that a localized increase in MCA, particularly in the posterior cell, may be required for rapid migration modes such as amoeboid migration. Furthermore, experimental studies and mathematical considerations, consistent with our tether force data and previous cortical tension measurements, suggest that a decrease in PM tension and an increase in cortical tension are favorable for bleb formation and, consequently, bleb-based migration. Therefore, a decrease in PM tension may be a prerequisite for both types of protrusion formation, and cortical tension is important for their switching and subsequent migration modes, which reflects why maintaining high PM tension effectively suppresses both migration modes. Future studies investigating the mechanical relationship between these two forces in migration behavior, particularly in a 3D environment, will deepen our understanding of how cell mechanics controls cancer cell migration.

[0173] Recent studies have shown that disruption of intercellular adhesion in epithelial cells does not necessarily lead to single-cell seeding. Our research suggests that constitutive PM tension is a cell-autonomous property of non-motile cells, and that this may partially explain the above phenomenon. In addition, cancer cells are known to utilize collective movement characterized by multicellular coordination through intercellular adhesion for seeding. Such collective processes are mechanically mediated by the coordination of cell adhesion forces and actomyosin contractility. It is interesting to investigate how PM tension integrates with these forces to control collective movement, and whether this type of movement can be suppressed by manipulating PM tension.

[0174] It was an unexpected discovery that constant PM tension may play an active role in inhibiting tumor formation and growth. Interestingly, it has been suggested that changes in cell surface mechanics may correlate with the stem cell nature of cancer. Furthermore, recent studies suggest a direct link between membrane protrusion and cancer progression. Our data show that such membrane fluctuations caused by EMT or oncogenic transformation can be explained by a decrease in PM tension, suggesting that maintaining high PM tension may also function as an effective mechanism to suppress tumorigenicity. In particular, it is interesting to further explore the relationship between PM tension and cancer progression in relation to the stem cell nature of cancer.

[0175] Our data showed that the PM tension of epithelial cells was approximately 100 pN / μm, which is equivalent to the membrane tension (100 pN / μm to 200 pN / μm) at which self-assembly of BAR domains is inhibited, as determined by reconstruction experiments and mathematical models, suggesting a threshold tension for BAR protein assembly. We propose that PM tension exceeding a critical threshold inhibits the self-assembly of BAR proteins, thereby suppressing the assembly of branched actin and subsequent actin-base migration. Since several BAR proteins have been reported to play an active role in tumorigenesis, this inhibition mechanism may also partially explain why increased PM tension suppresses tumor growth. Our unexpected discovery was that BAR proteins are also required for bleb-base movement. Our knockdown experiments suggested that BAR proteins are not required for membrane bleb formation but are involved in their polarization. This may relate to recent studies reporting that localized membrane invagination induced by BAR proteins enables localized membrane protrusions essential for directional bleb-based migration. Importantly, since PM tension, regulated by MCA, should act as a local parameter, localized membrane undulations upon its shedding may recruit curvature-sensing BAR proteins, driving motility in both polarized actin-based and bleb-based cells. Overall, these observations suggest that the low PM tension-BAR protein axis may act as a common form of membrane-mediated mechano-signaling driving cancer cell migration, and that PM tension is once again highlighted as a promising target for limiting tumor dissemination.

[0176] Abnormal changes in cell membrane shape, including the formation of microvesicles / exosomes and micropharmaceutical activity, are characteristic of cancer. It is tempting to hypothesize that these functions can be directly controlled by PM tension. Our findings provide a basis for future investigations into whether MCA manipulation can be used as a therapeutic intervention aimed at normalizing cell membrane dynamics.

[0177] Pharmaceutical formulations Using the compounds described herein, therapeutic pharmaceutical compositions can be prepared, for example, by combining the compounds with pharmaceutically acceptable diluents, excipients, or carriers. The compounds may be added to the carrier in the form of salts or solvates. For example, if the compound is sufficiently basic or acidic to form a salt of a stable, non-toxic acid or base, it may be appropriate to administer the compound as a salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as tosylates, methanesulfonates, acetates, citrates, malons, tartrates, succinates, benzoates, ascorbicates, α-ketoglutarates, and β-glycerophosphates. Suitable inorganic salts, including hydrochlorides, halides, sulfates, nitrates, bicarbonates, and carbonates, can also be formed.

[0178] Pharmaceutically acceptable salts can be obtained using standard procedures known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to obtain a physiologically acceptable ionic compound. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared by similar methods.

[0179] The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to mammalian hosts such as human patients in various forms. These forms can be particularly adapted to selected routes of administration, such as oral administration, or parenteral administration via intravenous, intramuscular, topical, or subcutaneous routes.

[0180] The compounds described herein can be administered systemically in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilated food carrier. For oral administration, the compounds can be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into the patient's diet. The compounds may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and formulations typically contain at least 0.1% of the active compound. The proportion of the compositions and formulations can be varied and may, conveniently, be about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10% of the weight of a given unit dosage form. The amount of the active compound in such therapeutically useful compositions may be such that an effective dose level can be obtained.

[0181] Tablets, lozenges, pills, capsules, etc. may contain one or more of the following: binders such as tragacanth gum, acacia gum, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; and lubricants such as magnesium stearate. Sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavoring may be added. If the unit dosage form is a capsule, in addition to the above types of materials, a liquid carrier such as vegetable oil or polyethylene glycol may be included. Various other materials may be present as coatings or otherwise to change the physical form of the solid unit dosage form. For example, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar. Syrups or elixirs may contain active compounds, sucrose or fructose as sweeteners, methylparaben and propylparaben as preservatives, colorants, and flavorings such as cherry or orange flavoring. Any materials used in the preparation of any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts used. In addition, active compounds can be incorporated into sustained-release formulations and devices.

[0182] The active compound may be administered intravenously or intraperitoneally by injection or infusion. Solutions of the active compound or its salts may be prepared in water and optionally mixed with a non-toxic surfactant. Dispersions may be prepared in glycerol, liquid polyethylene glycol, triacetin or mixtures thereof, or pharmaceutically acceptable oils. Under normal storage and use conditions, the formulation may contain preservatives to prevent microbial growth.

[0183] Pharmaceutical dosage forms suitable for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing active ingredients suitable for immediate preparation of sterile, injectable or injectable solutions or dispersions, and can optionally be encapsulated in liposomes. The final dosage form should be sterile, fluid, and stable under manufacturing and storage conditions. The liquid carrier or vehicle may be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by liposome formation, maintenance of the particle size required in the case of dispersions, or by the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. The sustained absorption of the injectable composition may be achieved by substances that slow down absorption, such as aluminum monostearate and / or gelatin.

[0184] Sterile injectable solutions can be prepared by incorporating the required amount of active compound, along with various other components listed above as needed, into a suitable solvent, and optionally following with filtration sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods include vacuum drying and freeze-drying techniques, which can yield powders of the active ingredient and any additional desired components present in the solution.

[0185] For topical administration, compounds can be applied in their pure form, for example, if they are liquids. However, it is generally preferable to administer the active substance to the skin as a composition or formulation, combined with a dermatologically acceptable carrier, which may be a solid, liquid, gel, etc.

[0186] Useful solid carriers include pulverized solids such as talc, clay, microcrystalline cellulose, silica, and alumina. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohol, glycol, or water-alcohol / glycol blends, to which the compound can be dissolved or dispersed at an effective level using optionally non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given application. The resulting liquid composition can be applied from an absorbent pad, used for impregnation into bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.

[0187] By using thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials together with a liquid carrier, it is also possible to form pastes, gels, ointments, soaps, etc., that can be applied directly to the user's skin.

[0188] The effective doses of the compounds described herein can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art. See, for example, U.S. Patent No. 4,938,949 (Borch et al.). The amount of compound or its active salt or derivative required for therapeutic use depends not only on the specific compound or salt selected, but also on the route of administration, the nature of the condition being treated, and the patient's age and condition, and is ultimately at the discretion of the attending physician or clinician.

[0189] However, generally speaking, a suitable dose is approximately 0.5 mg / kg to approximately 100 mg / kg per day, for example, in the range of approximately 10 mg / kg to approximately 75 mg / kg (body weight), for example, 3 mg to approximately 50 mg per kg of recipient's body weight per day, preferably in the range of 6 mg / kg / day to 90 mg / kg / day, and most preferably in the range of 15 mg / kg / day to 60 mg / kg / day.

[0190] The compound is preferably formulated into a unit dosage form containing, for example, 5 mg to 1000 mg of the active ingredient per unit dosage form, preferably 10 mg to 750 mg, and most preferably 50 mg to 500 mg. In one embodiment, the present invention provides a composition containing the compound of the present invention formulated into such a unit dosage form.

[0191] The compound may be, for example, 5 mg / m² per unit dosage form. 2 ~1000mg / m 2 Conveniently, 10 mg / m² 2 ~750mg / m 2 Most conveniently, 50 mg / m² 2 ~500mg / m 2 The active ingredient can be conveniently administered in unit dosage forms. The desired dose can be conveniently given as a single dose or as divided doses administered at appropriate intervals, for example, two, three, four or more lower doses per day. The lower doses themselves can be further divided, for example, into a number of individual doses at sparse intervals.

[0192] The desired dose can be conveniently administered as a single dose or as divided doses given at appropriate intervals, for example, two, three, four or more lower doses per day. The lower doses themselves can be further divided into, for example, a number of individual, sparsely spaced doses, such as multiple inhalations from an inhaler or multiple drops applied to the eyes.

[0193] The compounds described herein are effective antitumor agents and may have higher potency and / or reduced toxicity compared to BHPIs. Preferably, the compounds of the present invention are potent and less toxic than BHPIs and / or avoid potential sites of catabolism seen in BHPIs, i.e., have a different metabolic profile than BHPIs. Furthermore, the compounds described herein do not cause more severe ataxia than BHPIs and other known compounds.

[0194] The present invention provides a therapeutic method for treating cancer in vertebrates such as mammals, comprising administering an effective amount of a compound or composition described herein to a mammal having cancer. Mammals include primates, humans, rodents, dogs, cats, cattle, sheep, horses, pigs, goats, and the like. Cancer refers to any of the various types of malignant neoplasms generally characterized by undesirable cell proliferation, such as uncontrolled growth, lack of differentiation, local tissue invasion, and metastasis. Cancers that can be treated with the compounds described herein include, for example, breast cancer, cervical cancer, colon cancer, endometrial cancer, leukemia, lung cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, or uterine cancer, and in particular any ERα-positive cancer.

[0195] The ability of the compounds of the present invention to treat cancer can be determined using assays known in the art. For example, their biological importance in the design of treatment protocols, toxicity assessment, data analysis, quantification of tumor cell death, and use in transplantable tumor screening is known. In addition, the ability of the compounds to treat cancer can be determined using the following tests.

[0196] The following embodiments are intended to illustrate the above invention and should not be construed as limiting its scope. Those skilled in the art will readily recognize that the embodiments suggest many other ways in which the invention can be carried out. It should be understood that numerous variations and modifications can be made while remaining within the scope of the invention. [Examples]

[0197] Example 1. Materials and Method Cell culture. Human non-tumorogenic mammary epithelial cells (MCF10A), human breast cancer cells (MCF7, AU565, MDA-MB-231, and Hs578T), and human pancreatic cancer cells (PANC-1) were purchased from the American Type Culture Collection (ATCC). Normal rat liver (IAR-2) epithelial cells and human prostate (PC-3) cancer cells were obtained from the Japan Research Bioresource Collection (JCRB) cell bank. Normal canine kidney (MDCK II) epithelial cells were kindly provided by M. Murata (University of Tokyo). MDCK II cells with doxycycline-inducible RasV12 have been previously described. MCF10A cells were cultured in DMEM / F12 (Invitrogen) supplemented with 5% horse serum (Gibco), EGF (20 ng / ml; R&D Systems), insulin (10 μg / ml; Sigma-Aldrich), cholera toxin (80 μg / ml; Fujifilm Wako Pure Chemical Corporation), and hydrocortisone (0.5 μg / ml; Sigma-Aldrich). AU565 cells were cultured in RPMI-1640 (Nacalai Tesque Co., Ltd.) supplemented with 10% FBS (Sigma-Aldrich). Other cell lines were cultured in DMEM (Nacalai Tesque Co., Ltd.) supplemented with 10% FBS (Sigma-Aldrich). All cell lines were cultured at 37°C in 5% CO2. All cell lines were periodically tested for mycoplasma contamination using a PCR-based mycoplasma detection kit (Venor GeM Classic; MB minerva biolabs).

[0198] For 3D culture, cells were incorporated into a 3D collagen grid (Type I bovine collagen; final concentration, 1.67 mg / ml; Advanced BioMatrix). For 3D on-top culture, cells were grown in a Matrigel:collagen mixture as previously described (https: / / brugge.med.harvard.edu / Protocols). Matrigel with reduced growth factors (BD Biosciences) was used.

[0199] Measurement of tether force and estimation of PM tension using optical tweezers. Tether force measurements were performed using an optical tweezers system (NanoTracker® 2, JPK Instruments) equipped with an infrared (IR) laser light source (3W, 1064nm) and an Olympus IX-73 inverted microscope with a 60x (numerical aperture = 1.2) water immersion objective lens (Olympus). Silica beads (1.5 μm in diameter, Polysciences) were incubated with concanavalin A (Sigma-Aldrich) at 1 mg / ml for 1 hour. Cells were plated in a glass-bottom dish (WPI). The coated beads were added to cell culture medium supplemented with 25 mM HEPES (pH 7.5), and the experiment was conducted at 37°C. The tether force (F) can be calculated using Hooke's Law: F = kΔx (where k is the stiffness of the trap and Δx is the displacement of the bead from the center of the trap). For each experiment, trap stiffness (k, typically around 0.15 pN / nm) was calibrated by power spectral analysis. A single bead was captured by an optical trap and kept in contact with the cell membrane for 500 milliseconds. It was then separated by moving a piezo stage at 1 μm / second under computer control, forming a membrane tether (5 μm in length). The bead displacement (Δx) at the trap center was determined by a 4-segment photodiode detector with less than 1 nm resolution. Data analysis was performed using JPK data processing software. To compare PM tension between non-motile epithelial cells and malignant cells, the tether force on the cell sides was measured. It was noted that cells with low PM tension, such as malignant cells, often formed double tethers, showing a twofold increase in tether force. This may be due to their low tension. Therefore, careful verification was performed to determine if only one tether was formed, and data for double tethers were excluded. In contrast to epithelial cells, where tethers are easily broken, we also noted that metastatic cells tend to have longer membrane tethers (over 15 μm).

[0200] PM tension is given by the following formula: T=F テザー 2 / 8Bπ 2 (In the formula, T is the apparent cell membrane tension (PM tension), and F テザー(where B is the tether force measured by optical tweezers, and B is the bending stiffness of the membrane) can be estimated by the fact that B is known to be relatively invariant among the cell types tested (1 × 10⁻¹⁰ -19 N / m ~ 3 × 10 -19 N / m), B(1.4×10 -19 PM tension was calculated using N / m.

[0201] Materials. Methyl-β-cyclodextrin (MβCD) (Sigma-Aldrich) and kallikrin A (Cell Signaling Technology) were used at final concentrations of 4 mM and 0.5 nM, respectively. The following antibodies were used: anti-ERM (rabbit polyclonal, 1:1000, for immunoblotting; #3142, Cell Signaling Technology (CST)); anti-phospho-ERM (rabbit monoclonal, 1:100, for immunostaining; #3726, CST); anti-RHOA (mouse monoclonal, 1:1000, for immunoblotting; #sc-418, Santa Cruz Biotechnology); anti-pS19 MLC (Phosphomyosin light chain 2 (Ser19)) (mouse monoclonal, 1:1000, for immunoblotting; #3675, CST); Anti-MLC (rabbit polyclonal, 1:1000, for immunoblotting; #3672, CST); Anti-E-cadherin (rabbit monoclonal, 1:1000, for immunoblotting; #3915, CST); Anti-vimentin (rabbit monoclonal, 1:1000, for immunoblotting; #5741, CST); Anti-MTSS1L (rabbit polyclonal, 1:200, for immunoblotting; #NBP2-57037, Novus) Biologicals); anti-FBP17 (FNBP1) (rabbit polyclonal, 1:1000, for immunoblotting); anti-CIP4 (TRIP10) (mouse monoclonal, 1:1000, for immunoblotting; #612556, BD Transduction Laboratories); anti-HA-Tag (rabbit monoclonal (C29F4), 1:100, for immunostaining; #3724, CST) and anti-β-actin (rabbit polyclonal, 1:2000, for immunoblotting; #PM053, MBL). Alexa-Fluor-488 conjugate secondary antibody (1:500, for immunostaining; rabbit, #A11034; mouse, #A11029) was obtained from Thermo Scientific.

[0202] Human Snail and Slug were subcloned into the pMXs-IRES-Puro retroviral vector (Cell Biolabs, Inc; modified by introducing an HA tag at the C-terminus). 10 -Ezrin T567E (MA-Ezrin) was constructed by fusing a PM target signal (MGCIKSKRKD (SEQ ID NO: 75), a myristoylation motif derived from Lyn tyrosine kinase) to the N-terminus of human ezrin, and then generating the T567E mutation using PCR primers, and its sequence was confirmed. Ezrin and MA-Ezrin constructs were subcloned into a pQCXIN-HA retroviral vector (Clontech; modified by introducing an HA tag at the C-terminus) containing a neomycin resistance gene. Human MTSS1L was subcloned into a pEGFP C-1 vector. For retroviral infection, cells were plated in 6-well plates and incubated with the virus in the presence of 4 μg / ml polyblen (Sigma-Aldrich). Infected cells were selected with G418 (0.8 mg / ml) or puromycin (1.5 μg / ml). Transgene expression was assessed by Western dysmetology and confocal microscopy.

[0203] Short-chain interfering RNA (siRNA) and transfection. For knockdown experiments, Dharmacon SMARTpool-ON-TARGETplus siRNA (a mixture of four different siRNAs; Thermo Scientific) was used against human genes: EZR / Ezrin (L-017370-00); RDX / Radixin (L-011762-00); MSN / Moesin (L-011732-00); RHOA (L-003860-00); SLK (L-003850-00); STK10 (L-004168-00); ARHGAP4 (L-003628-00); ARHGAP10 / GARF2 (L-009382-01); ARHGAP17 / RICH1 (L-008335-02); ARH GAP26 / GRAF1(L-008426-00);ARHGAP29(L-008277-00);ARHGAP42 / GRAF3(L-026507-01);ARHGAP44 / RICH2(L-009238-01);ARHAGP4 5 / HMHA1(L-023893-00);ARHGEF37(L-032927-01);ARHGEF38(L-020676-00);IRSp53 / BAIAP2(L-012206-02);BAIAP2L1 / IRTKS(L-0 18664-02);DNMBP / Tuba(L-026304-01);FCHSD1(L-015107-02);FCHSD2(L-021240-01);FER(L-003129-00);FES(L-003130-00) BP1 / FBP17(L-026214-02);FNBP1L / Toca-1(L-020718-01);GAS7(L-011492-00);GIMP(L-021160-01);MTSS1 / MIM(L-018506-00);M TSS1L / ABBA(L-022582-01);OPHN1 / oligophrenin1(L-009444-00);PACSIN1(L-007735-00);PACSIN2(L-019666-02);PACSIN3(L-015343 -00);SH3BP1(L-009546-01);SRGAP1(L-026974-00);SRGAP2(L-021531-02);SRGAP3(L-014175-00);TRIP10 / CIP4(L-012685-00).ON-TARGETplus Non-Targeting siRNA Pool (D-001810-10) was used as the control siRNA. RNA (25 nM) was transfected into cells using Lipofectamine RNAi MAX (Invitrogen). Analysis was performed 72 hours after transfection. Knockdown of major proteins (ERM protein, RHOA, SLK, STK10, MTSS1L, FBP17, and CIP4) was confirmed by Western blotting. Plasmid transfection was performed using FuGENE HD (Roche) according to the manufacturer's protocol. Transfected cells were tested 24 hours later.

[0204] In vitro invasion and migration assays. For the invasion and migration assays, a BioCoat Matrigel Invasion Chamber (Corning) and an 8.0 μm PET migration insert (Corning) were used, respectively. In the invasion assay, 1 × 10⁻¹⁶ samples were taken. 5 Cells were suspended in serum-free medium and seeded onto the membrane of a chamber with serum-containing medium at the bottom. For MβCD or kallikrin A treatment, the drug was added to both sides. Low-invasive epithelial cells (MCF10A, AU565, and MCF7) and MDA-MB-231 cells were incubated for 36 hours and 24 hours, respectively, and then fixed with 4% formaldehyde. Infiltrating cells were imaged at 10x magnification using a BZ-X700 microscope (Keyence Corporation) and counted. In the migration assay, 5 × 10⁶ cells were counted. 4 Cells were seeded onto a PET membrane, incubated for 24 hours, and analyzed as described in the invasion assay. For the quantification of invasive structures grown in 3D on-top culture (Figures 5a and 5b), elongated lobe-like morphology and rounded morphology were defined and quantified as invasive and non-invasive phenotypes, respectively. Amoeboid movement characterized by rounded shapes was not observed in ERM knockdown cells or Snail-expressing cells.

[0205] Confocal microscopy, live-cell imaging, and image analysis were performed. For immunofluorescence analysis, cells were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 10 minutes and permeabilized in PBS with 0.2% Triton X-100 for 10 minutes. Cells were blocked in 5% goat serum (Sigma-Aldrich) in PBS for 1 hour and incubated with primary antibody for at least 3 hours. Subsequently, cells were incubated with secondary antibody for 1 hour. For membrane visualization, Alexa Fluor® 350 conjugate wheat germ agglutinin (WGA) (Thermo Scientific) was incubated with fixed cells for 30 minutes before permeabilization. For F-actin visualization, Alexa Fluor® 568 phalloidin (Thermo Scientific) was incubated with fixed cells for 30 minutes. Fluorescence images were acquired using a confocal microscope system (FluoView 1000-D; Olympus) equipped with diode lasers at 405 nm, 473 nm, and 568 nm, through an objective lens (60x oil immersion objective lens, NA=1.35). For 3D imaging, Z-stack images of continuous optical planes spaced 0.5 μm apart were acquired for the entire cell. Maximum intensity z projections were reconstructed using Image J and Imaris 8.0.2. All other confocal images were displayed as single planes. In the 3D data shown in Figures 1-3, a planar image near the center of the 3D stack, where the membrane region is clearly identifiable, was selected as a representative image. For live imaging using phase-contrast microscopy, cells were mixed with collagen matrix and plated in an 8-well plate (IWAKI). Images were taken using a BZ-X700 microscope (KEYENCE Corporation) at 20x magnification, 37°C, and 5% CO2. Single cells were manually tracked using the Manual Tracking Tool ImageJ software plugin. Migration plots and cell velocities were obtained using the Chemotaxis and Migration Tool (Ibidi).To evaluate cell morphological dynamics in 3D, cells were classified into either a mesenchymal phenotype (elongated; aspect ratio greater than 4) or an amoeboid phenotype (rounded, with actin or bleb-based protrusions; aspect ratio less than 4), as previously described. The aspect ratio was determined as the ratio of the cell's long axis to its short axis and was automatically calculated using ImageJ. While elongated cells typically have an aspect ratio of 5-8, amoeboid cells were observed to have an aspect ratio of 2-3. To calculate the membrane / cytoplasmic intensity ratio, the entire membrane region was segmented using threshold images of the WGA channel, and the membrane region was manually selected using the brush selection tool to a size of 5 pixels (approximately 500 nm wide). The average intensity of pERM or F-actin along all membrane regions was then calculated and divided by the average intensity of the entire cytoplasm (avoiding the nucleus). To quantify the 3D images, a single planar image (corresponding to the image near the center of the 3D stack) with a membrane region clearly identifiable by WGA staining, as shown in Figure 1d, was used for quantitative analysis, similar to the 2D images. Furthermore, to account for the variation in fluorescence intensity due to the thickness of the 3D stack, two images shifted vertically from the selected central image in the z direction by approximately 1.5 μm (elongated cells) to 4 μm (rounded cells) were quantified. The average membrane / cytoplasmic intensity ratio of all three images was used as a single sample. To quantify BAR protein accumulation in PM, the number of BAR protein spots merged with membrane regions defined by membrane markers was quantified.

[0206] Western blotting. Cell lysates were extracted using Laemmli buffer. Samples were electrophoresed on an SDS-PAGE gel, transferred to a polyvinylidene fluoride membrane, blocked with 5% BSA or skim milk powder in TBS containing 0.1% Tween 20, incubated with primary antibody, and then incubated with secondary antibody.

[0207] Growth assay. 2 × 10 4The cells were seeded in pairs in a 6-well plate at each time point, and counted using a Countess Automated Cell Counter (Thermo Fisher) at 2 and 4 days later.

[0208] Analysis of clinical datasets. The TCGA and CCLE datasets were analyzed using the 2020 version of cBioPortal (www.cbioportal.org / ). Clinical datasets of cancer patients from the 2020 version of KMplotter (www.kmplot.com) were analyzed using each probe (SLK:206875_s_at and STK10:228394_at) and a self-selected best cutoff. The significance of survival differences between groups was assessed by the log-rank test.

[0209] Animal studies. For tumorigenesis, local invasion, and spontaneous metastasis assays, 1 × 10⁶ 7 The cells were resuspended in PBS (0.1 mL) and injected into the mammary fat body of 6-week-old female BALB / c nu / nu mice. The mice were maintained at a temperature of 23±1°C and humidity of 55±5% in a 12 / 12-hour light / dark cycle. The mice were sacrificed 7 weeks after injection, and the tumors and lungs were collected. The tumor volume was expressed using the formula V = 1 / 2 (A × B). 2 The tumor was calculated according to the formula (wherein A and B represent the maximum and minimum dimensions of the tumor, respectively). The excised tumor was fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E). For invasiveness analysis, the boundary between the tumor and adipose tissue was manually defined at 37829 μm. 2A quantification box was set up. In each box, the tumor invasion area was calculated using Image J. To quantify spontaneous metastasis, the human / mouse DNA ratio was assessed using human-specific quantitative PCR (qPCR) as described above. Briefly, qPCR was performed using PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific) and 100 ng of lung genomic DNA along with human and mouse-specific PTGER2 primer pairs. The primers used for qPCR are listed in Table 1. A standard curve was created using genomic DNA extracted from MDA-MB-231 cells and xenografted naive mouse lungs. qPCR was performed using the StepOnePlus Real-Time PCR System. For the experimental lung metastasis assay, 2 × 10⁻⁶ 6 The cells were resuspended in 0.1 ml of PBS and intravenously injected into 6-week-old female BALB / c nu / nu mice. Lung metastases were assessed after 8 weeks. The lungs were fixed and stained with H&E. All sections were examined under a BZ-X700 or BZ-8000 microscope (Keyence Corporation). All animal experiments were reviewed by the institutional ethics committee and conducted in accordance with the guidelines for experimental animal research of Tokyo University of Pharmacy and Life Sciences (Tokyo, Japan).

[0210] [Table 3]

[0211] Mechanical cell stretching. Cells were grown for 24 hours on a silicon chamber (STB-CH-04, Strex Co., Ltd.) coated with fibronectin (0.05 mg / ml; Sigma-Aldrich). The chamber was placed in a stretching device (STB-100, Strex Co., Ltd.) and stretched uniaxially for 5 minutes (20% stretching).

[0212] Statistics. Statistical analysis was performed using GraphPad Prism 6 and Excel. The dataset was tested for a Gaussian distribution using the D'Agostino-Pearson omnibus test and the Kolmogorov-Smirnov test (Dallal-Wilkinson-Lillie p-values ​​were used). Statistical significance was determined for two groups using a two-tailed Student t-test or a non-parametric Mann-Whitney U test, and for multiple comparisons, one-way ANOVA and Tukey's test were used. Phenotypic distributions were compared using the chi-squared test. Sample size, number of repetitions for each experiment, and specific tests are indicated in the legend of the figures.

[0213] Data Availability. The source data is presented in this book. The TCGA and CCLE datasets are available from cBioPortal (www.cbioportal.org / ). The clinical dataset for cancer patients is available from KMplot (www.kmplot.com).

[0214] Example 2. Pharmaceutical Dosage Forms The following formulations exemplify typical pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of compounds of the formulas described herein, compounds particularly disclosed herein, or pharmaceutically acceptable salts or solvates thereof (hereinafter referred to as "Compound X"). (i) Tablets 1 mg / tablet "Compound X" 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesium stearate 3.0 300.0 (ii) Tablets 2 mg / tablet "Compound X" 20.0 Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0 (iii) Capsule mg / capsule "Compound X" 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0 (iv) Injection 1 (1 mg / mL) mg / mL "Compound X" (free acid form) 1.0 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 Appropriate amount of 1.0N sodium hydroxide solution (Adjust pH to 7.0-7.5) Water for injection, up to 1 mL (appropriate amount) (v) Injection 2 (10mg / mL) mg / mL "Compound X" (free acid form) 10.0 Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.0 Appropriate amount of 0.1N sodium hydroxide solution (Adjust pH to 7.0-7.5) Water for injection, up to 1 mL (appropriate amount) (vi) Aerosol mg / can "Compound X" 20 Oleic acid 10 Trichloromonofluoromethane 5000 Dichlorodifluoromethane 10000 Dichlorotetrafluoroethane 5000

[0215] These formulations can be prepared by conventional procedures known in the pharmaceutical technology field. It will be understood that the above pharmaceutical compositions can be modified according to known pharmaceutical techniques to accommodate different amounts and types of active ingredient "compound X". Aerosol formulation (vi) can be used with a standard measuring aerosol dispenser. Furthermore, specific components and proportions are for illustrative purposes only. Components can be substituted with suitable equivalents, and proportions can be varied according to the desired properties of the dosage form in question.

[0216] Specific embodiments have been described above with reference to the embodiments and examples of the disclosure, but such embodiments are illustrative and do not limit the scope of the present invention. Modifications and changes can be made in accordance with the ordinary skill in the art without departing from the broader embodiments of the invention as defined in the following claims.

[0217] All publications, patents, and patent documents are part of this specification by reference, as each individually does by particular reference Tsujita et al., Nat Commun 12(1), 5930 (2021). No limitations that would conflict with this disclosure should be inferred from them. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many changes and modifications can be made while remaining within the spirit and scope of the invention.

Claims

1. A cancer treatment composition comprising a substance that increases the tension of the cell membrane of cancer cells, The active substance is, At least one selected from the group consisting of siRNA, miRNA, and shRNA that bind to the messenger ribonucleic acid of MTSS1L / ABBA; and / or An expression vector configured to express an ezrin fusion protein containing a conserved myristylated sequence of ezrin fused with ezrin, wherein the ezrin contains a phosphorylation-mimicking activating mutation (T567E).

2. The composition according to claim 1, wherein the tension of the cell membrane is increased and / or maintained at approximately 100 pN / μm to 200 pN / μm or more.

3. The composition according to claim 1, wherein increasing the tension of the cell membrane increases the internal pressure of the cancer cell.

4. The composition according to claim 3, wherein increasing the internal pressure of the cancer cells is a means of manipulating the permeability function of the cancer cells.

5. The composition according to claim 1, wherein increasing the tension of the cell membrane includes increasing or decreasing the amount of components of the cell membrane.

6. The composition according to claim 5, wherein the components of the cell membrane are one or more of lipids, phospholipids, glycolipids, proteins, glycoproteins, and cholesterol.

7. The composition according to claim 1, wherein the active substance is internalized in the cancer cells.

8. The composition according to claim 1, wherein the substance that increases the tension of the cell membrane causes an increase in membrane-actin cortical adhesion (MCA), and the increase in MCA is compared to cells that are not in contact with the substance.

9. The composition according to claim 1, wherein the active substance causes an increase in the phosphorylation of ezrin, radixin, and moesin (ERM) proteins, which include one or more of EZR, RDX, and MSN.

10. The composition according to claim 1, wherein the active substance inhibits or reduces the expression of the MTSS1L / ABBA protein.

11. The composition according to claim 1, further comprising a pharmaceutically acceptable carrier.

12. In vitro, a cell and, At least one selected from the group consisting of siRNA, miRNA, and shRNA that bind to the messenger ribonucleic acid of MTSS1L / ABBA; and / or An expression vector configured to express an ezrin fusion protein containing a conserved myristylated sequence of Lyn fused with ezrin, wherein the ezrin contains a phosphorylation-mimicking activating mutation (T567E). A method for inhibiting cell migration, including bringing the two into contact.

13. A method for regulating the properties of a cell, comprising bringing a cell into contact with a substance that alters the tension of the cell membrane of the cell in vitro, wherein the properties of the cell are regulated by altering the tension of the cell membrane. A substance that alters the tension of the cell membrane of the cell is At least one selected from the group consisting of siRNA, miRNA, and shRNA that bind to the messenger ribonucleic acid of MTSS1L / ABBA; and / or An expression vector configured to express an ezrin fusion protein containing a conserved myristylated sequence of Lyn fused with ezrin, wherein the ezrin contains a phosphorylation-mimicking activating mutation (T567E), A method wherein the modification of the cell characteristics is at least one selected from the group consisting of reduced cell motility, reduced invasion into surrounding tissue, and reduced tumor formation.

14. The method according to claim 13, wherein the characteristics of the cells are controlled by membrane-actin cortical adhesion (MCA), and the active substance increases the MCA compared to control cells.

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