Methods of Diagnosis and Treatment of Metastatic Cancer

Abstract

To provide compositions used in methods of diagnosing and treating metastatic cancer in a subject.SOLUTION: Provided is a composition comprising an effective amount of an inhibitor of at least one of ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122. The inhibitor is a gene silencing nucleic acid molecule that targets a gene having a nucleotide sequence encoding ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122.SELECTED DRAWING: None

Description

Technical Field

[0001] Generally, the present invention relates to the diagnosis and treatment of cancer. More specifically, the present invention relates to methods for diagnosing and treating metastatic cancer in a subject.

Background Art

[0002] Metastatic seeding is a major cause of cancer-related death (Mehlen and Puisieux, Nat Rev Cancer 6:449-458, 2006). Surgical resection of primary tumors in combination with systemic chemotherapy has been successful in the treatment of localized cancer, but metastatic disease is highly resistant even to modern targeted therapies, making these cancers refractory. In fact, many patients are undergoing highly morbid treatment regimens that negatively impact their quality of life in order to reduce the risk of future metastases (Lauer et al. Expert Opin Drug Discov 10:81-90, 2015). Clearly, therapies that target the rate-limiting steps of metastatic seeding of tumor cells can significantly improve cancer treatment by removing the threat of systemic disease and reducing dependence on systemic therapies with harmful side effects (Steeg, Nat Rev Cancer 16:201--218,2016; Li and Kang, Pharmacol Ther 161:79-96,2016; Zijlstra et al., Cancer Cell 13:221-234,2008; Mehlen and Puisieux, Nat Rev Cancer 6:449-458,2006).

[0003] The metastatic process depends on the tumor cell's ability to invade the bloodstream, disseminate to distant sites, evade immunodetection, survive, proliferate, and subsequently establish itself in a new microenvironment (Valastyan and Weinberg, Cell 147:275-292, 2011). Previously, it was shown that the rate of intravascular invasion is highly dependent on the motility of in vivo tumor cells, and that inhibiting motility using migration-blocking antibodies targeting the tetraspanin CD151 prevents both intravascular invasion and distant metastasis of cancer cells (Zijlstra et al. Cancer Cell 13:221-234, 2008; Palmer et al. Cancer Res 74:173-187, 2014). Given that the genes and signaling networks that promote in vivo motility and intravascular invasion differ from those required for efficient primary tumorigenesis, it may be possible to prevent intravascular invasion and metastasis by identifying and interfering with these genes. Furthermore, improved tests for detecting early metastatic disease could provide a timeframe for treatment opportunities prior to full signs of metastasis, potentially improving overall survival for people with advanced cancer. [Overview of the project]

[0004] According to one aspect of the present invention, a method for preventing cancer metastasis in a subject is provided. This method comprises administering an effective amount of at least one modulator from among Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 to the subject. In one embodiment, an effective amount of an inhibitor of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b is administered to the subject. In another embodiment, an effective amount of miRNA122, or a compound capable of upregulating the expression of miRNA122, is administered to the subject.

[0005] A further aspect of the present invention provides a method for detecting Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 in a patient. The method involves obtaining a biological sample from a human patient and contacting the sample with Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 antibodies, or nucleic acids complementary to Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 mRNA, thereby adding Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr The method includes detecting whether 2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 are present, and detecting the binding of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 to an antibody, or hybridization between nucleic acids complementary to Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 mRNA.

[0006] According to another aspect of the present invention, a method for diagnosing and treating cancer metastasis in a patient is provided. The method includes the steps of obtaining a biological sample from a human patient, and detecting whether at least one of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b or miRNA374b is present in the biological sample and / or whether miRNA122 is not present in the biological sample, and detecting whether Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, The method includes the steps of: diagnosing a patient with metastatic cancer or the onset of metastatic cancer when the presence of APBA2, miRNA130b, or miRNA374b is detected, and / or when miRNA122 is absent; and administering to the diagnosed patient an effective amount of at least one inhibitor of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b, and / or an effective amount of miRNA122 or a compound capable of upregulating miRNA122 expression.

[0007] A further aspect of the present invention provides the use of at least one of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA 374b, or miRNA122 for diagnosing metastatic cancer in a subject.

[0008] According to another aspect of the present invention, the use of at least one inhibitor of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b, and / or an effective amount of a compound capable of upregulating the expression of miRNA122, is provided for the prevention of cancer metastasis in a subject.

[0009] In one embodiment, the inhibitor is a gene-silencing nucleic acid molecule or a small molecule. Gene-silencing nucleic acid molecules are, for example, short interfering RNA, antisense oligonucleotides, short hairpin RNA, microRNA, ribozymes, or other RNA interfering molecules. Small molecules are peptides, peptoids, amino acids, amino acid analogs, organic or inorganic compounds.

[0010] In a further embodiment, the human patient has cancer.

[0011] In further embodiments, the biological sample is tumor biopsy material.

[0012] These and other features, aspects and advantages of the present invention will be better understood with reference to the following description and accompanying drawings. [Brief explanation of the drawing]

[0013] [Figure 1]This graph shows that genes identified by screening are necessary for proliferative cancer cell invasion in vivo. a) This image shows metastatic colonies produced by HEp3 cells transduced with scrambled shRNA or shRNA targeting Kif3b, SRPK1, or Nr2f1. The inset shows the trajectory of a representative cell within the metastatic colony. b) The left panel shows the invading front of a primary tumor produced by HEp3 cells transduced with scrambled shRNA or shRNA targeting Kif3b, SRPK1, or Nr2f1. The inset shows the trajectory of a representative cell on the invading front. The right panel shows invading cells from the red dotted square in the left panel. The color-coded arrows point to cell protrusions formed by individually color-coded labeled cells (c1-c3). c) This graph shows the trajectory velocity of individual cells for the control cell line and mutant cell line from Figure 1a. d) A graph showing the displacement rate (productivity) of individual cell trajectories for the control cell line and mutant cell line from Figure 1a. e) A graph showing the trajectory speed of individual cell trajectories for the control cell line and mutant cell line from Figure 1b. d) A graph showing the displacement rate of individual cell trajectories for the control cell line and mutant cell line from Figure 1b. g) A graph showing the number of invasive cells per field of view that migrated from the primary tumor for the cell line from Figure 1b. h) A graph showing the number of cell protrusions per cell for the control cell line and mutant cell line from Figure 1b. [Figure 2]This graph shows that targeting genes identified through screening inhibits spontaneous cancer cell metastasis in vivo. a) Stereofluorescence images of nude mouse lungs subcutaneously injected with control (scrambled) shRNA-transduced HEp3 cells or HEp3 cells stably expressing shRNAs targeting Kif3b, SRPK1, and Nr2f1. b) A graph showing the accurate quantification of HEp3 cancer cells metastasized to the lungs, determined by human alu q-PCR. The data represents the relative metastasis amount as a percentage and the total number of cancer cells detected (colored number) when estimated using a standard curve. c) A graph showing the primary tumor weight of the control cell line and knockdown cell line used to induce the tumors in the experiment. [Figure 3] These graphs show the quantitative validation of clones identified by re-injection screening. a) Representative images of compact colony-forming clones isolated during screening. The inset shows the CI score of the composite material and the shRNA present in the clones, sorted by abundance. Representative colonies formed by HEp3 cells transduced with the original (wt) and scrambled shRNA are also shown. shRNAs selected for further analysis are highlighted in red. b) A graph showing the linear exponential distribution of clones identified by screening. c) A graph showing the density exponential distribution of clones identified by screening. d) A graph showing the area exponential distribution of clones identified by screening. [Figure 4]These graphs show the occurrence of mutant cell line knockdown due to the expression of genes identified in screening. a) A graph showing Western blotting analysis of Kif3b mutant cell lines and control cell lines (HEp3, MDA-MB-231, and PC3). b) A graph showing Western blotting analysis of SRPK1 mutant cell lines and control cell lines (HEp3, MDA-MB-231, and PC3). c) A graph showing Western blotting analysis of Nr2f1 mutant cell lines and control cell lines (HEp3 and MDA-MB-231). d) A graph showing q-PCR analysis of TMEM229b mutant cell lines and control cell lines (expression of HEp3 and wild-type HEp3 set to 100%). e) A graph showing q-PCR analysis of C14orf142 mutant cell lines and control cell lines (expression of HEp3 and wild-type HEp3 set to 100%). The insets in (d) and (e) show representative images of colonies induced by the second independent shRNAs TMEM229b and C14orf142. [Figure 5] These graphs show the effects of Kif3b and SRPK1 expression knockdown on in vitro cancer cell migration. A modified cell scratch assay using magnetically adhering stencils (Mat) was used. a) Graph showing the Mat (magnetically adhering stencil) in vitro migration assay for control cell lines and mutant Kif3b cell lines. b) Graph showing the Mat in vitro migration assay for control cell lines and mutant SRPK1 cell lines. For each cell line, the wild-type mean was set to 100%. [Figure 6]This graph shows that elevated expression of genes identified through screening correlates with the metastatic behavior of cancer cells in major types of human cancer. a) This figure shows the expression of selected screening hits in metastatic lesions to primary tumors in skin cancer, prostate cancer, head and neck cancer, lung cancer, ovarian cancer, and colon cancer (Oncomine). b) This image shows immunohistochemical analysis of Nr2f1, C14orf142, and Kif3b expression in skin (melanoma) cancer. c) This image shows immunohistochemical analysis of SRPK1 and Kif3b expression in prostate cancer. d) This image shows immunohistochemical analysis of Kif3b expression in head and neck (squamous cell carcinoma) cancer. e) This image shows immunohistochemical analysis of SRPK1 and TMEM229b expression in lung cancer. f) This image shows immunohistochemical analysis of Nr2f1 expression in ovarian cancer. g) This image shows immunohistochemical analysis of Nr2f1 expression in colon cancer. The red arrows point to the anterior surface of the invasive tumor. [Figure 7] This graph shows the composite compactness index (CI) distribution of screening hits relative to positive (anti-CD151) and negative (scrambled shRNA) controls. Screening hits that are significantly more compact than the negative controls are shown in green. Clones containing a single shRNA species are shown in bold. For clones containing multiple shRNAs, the two most dominant shRNAs are shown. Statistical significance was determined using one-way ANOVA with Fisher's LSD test (*p<0.05, **p, 0.01, ***p<0.001). [Figure 8]These graphs show that increased expression of miRNAs identified by screening inhibits the formation of invasive metastatic lesions. a) Images showing metastatic lesions formed by cancer cells expressing control, miR122, miR374b, or anti-miRNA-130b constructs. b) Graph showing quantification of the contact length between cancer cells and blood vessels for metastatic lesions formed by cancer cells expressing control, miR122, miR374b, or anti-miRNA-130b constructs. c) Graph showing quantification of the percentage of vascular cells in contact with cancer cells for metastatic lesions formed by cancer cells expressing control, miR122, miR374b, or anti-miRNA-130b constructs. [Figure 9] These graphs show that increased expression of miRNAs identified through screening inhibits cancer cell invasion. a) Image showing metastatic lesions formed by cancer cells expressing control (red) or miR122 (green) overexpression cells. b) Image showing the cell migration trajectory of cancer cells expressing control (red) or miR122 (green) overexpression cells. c) Graph showing quantification of cancer cell displacement rate for control (red) or miR122 o / e constructs. d) Graph showing quantification of metastatic amount (spontaneous metastasis) of control (red) or miR122 o / e cancer cells (green). [Figure 10] These graphs show that increased expression of miRNAs identified by screening inhibits cancer cell invasion along the vascular system. a) Image showing scrambled control (red) or miR122 (green) overexpressing cells next to the blood vessel wall. b) Graph showing contact between cancer cells and blood vessels for or miR122 o / e overexpressing cells (two independent constructs). c) Image showing color coding of control and miR122 o / e cancer cell protrusions along the vascular system. d) Image showing color coding of control and miR122 o / e cancer cell protrusions along the vascular system. e) Graph showing quantification of the angle between cancer cell protrusions and the blood vessel wall for control and miR122 o / e cancer cells. f) Image showing the interaction between control and miR122 o / e cancer cells and vascularly oriented collagen fibers (SHGs). [Figure 11]This graph shows that increased expression of miRNAs identified by screening inhibits the invasion of cancer cells into the collagen matrix. a) Image showing invasion of scrambled control, miR122 overexpressing cells, or MT1-MMP inhibitor (phenantrione, ph) treated cells into a 3D collagen matrix (rat tail collagen gel). b) Graph showing cancer cell invasion into the collagen matrix. c) Graph showing quantification of collagen degradation. d) Image showing representative optical section from (a) showing collagen degradation by control or miR122 o / e cells. e) Representative 2D and 3D images of control and miR122 o / e cancer cells in chicken CAM collagen matrix (SHG). Note that miR122 o / e cells cannot invade collagen and grow on the surface (bottom panel). f) Graph showing quantification of metastatic colony depth for control and miR122 o / e cells 1-5 days after cancer cell injection. g) This graph shows the quantification of aligned collagen bundles in control and miR122 o / e cells 1 to 5 days after cancer cell injection. [Figure 12]This graph shows that increased expression of miRNAs identified by screening inhibits the transport and localization of normal MT1-MMP. a) Representative images showing the localization and trajectory of MT1-MMP vesicles in control and miR122 o / e cells. b) Graph showing the quantification of MT1-MMP vesicle trajectory length in control and miR122 o / e cancer cells. c) Representative images of control and miR122 o / e cancer cells in chicken CAM collagen matrix (SHG). Note that miR122 o / e cells are unable to properly localize MT1-MMP to the collagen in contact with the protrusions. d) Representative images of control and miR122 o / e cancer blood vessels in contact with cells in chicken CAM. Note that miR122 o / e cells are unable to properly localize MT1-MMP to the contact area between cancer cells and the blood vessel wall. f) This image shows the signal intensity line scanning the image of (d) along the dashed line. The red arrow indicates contact between cancer cells and the blood vessel wall. g) This graph shows the quantification of MT1-MMP signal intensity in the protrusion for control and miR122 o / e cancer cells. [Figure 13] This graph shows that increased expression of miRNAs identified by screening inhibits extravasation of cancer cells. a) Representative image showing control (red) and miR122 o / e (green) cells extravasating from the chicken CAM vascular system. b) Graph showing quantification of extravasation of control and miR122 o / e cancer cells. c) Representative image of control and miR122 o / e cancer cells (MT1-MMP overexpression, red) extravasating from the chicken CAM vascular system. Note that miR122 o / e cells are unable to properly localize MT1-MMP to the vascular wall in contact with the protrusion. d) Graph showing quantification of MT1-MMP signal intensity at the protrusion for control and miR122 o / e cancer cells. [Modes for carrying out the invention]

[0014] The following description is merely an example of a specific embodiment, not limiting the combinations necessary to carry out the present invention.

[0015] According to one embodiment, a method for preventing metastasis in a subject having cancer is provided. The method includes regulating the expression of at least one of kinesin-like protein 3b (Kif3b), serine / threonine-protein kinase 1 (SRPK1), transmembrane protein 229b (TMEM229b), chromosome 14 open reading frame 142 (C14orf142), nuclear receptor subfamily 2, group F, member 1 (Nr2f1), miRNA130b, miRNA374b, or miRNA122 in a cancerous tumor. In some embodiments, the method includes reducing, preventing, or "silencing" the expression of at least one of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, and miRNA374b in a cancerous tumor. In other embodiments, the method includes increasing the expression of miRNA122 in a cancerous tumor.

[0016] Using the methods described herein, it has been found that the expression of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 is related to cancer motility, and regulation of the expression of these genes prevented the cancer from spreading from the lesion site.

[0017] For the purposes of this discussion, the term "regulate" can mean either upregulation of gene expression or downregulation of gene expression compared to the basal level of expression in a cell.

[0018] It will be understood that gene expression may refer to the production of polypeptides from the nucleic acid sequence of a gene. Gene expression can include both transcription and translation processes, and therefore gene expression may refer to the production of nucleic acid sequences such as mRNA (i.e., transcription), the production of proteins (i.e., translation), or both. As an example, a vector (viral, plasmid-based, or otherwise) containing one or more copies of a particular gene, each driven by an appropriate promoter sequence (e.g., constitutive or inductive promoter), can be introduced into cells by transfection, electroporation, viral infection, or another appropriate method known in the art. Appropriate expression vector techniques for introducing specific genes into cells are known in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th Ed.), 2012, Cold Spring Harbor Laboratory Press).

[0019] As is known to those skilled in the art, nucleotide sequences for expressing a particular gene may encode or contain one or more appropriate features as described, for example, in "Genes VII", Lewin, B. Oxford University Press (2000), or "Molecular Cloning: A Laboratory Manual", Sambrook et al. Cold Spring Harbor Laboratory, 3rd edition (2001). Nucleotide sequences encoding polypeptides or proteins can be incorporated into appropriate vectors or expression cassettes such as commercially available vectors or expression cassettes. For example, as outlined in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)), vectors can also be individually constructed or modified using standard molecular biology techniques. Those skilled in the art will recognize that a vector may contain a nucleotide sequence encoding desired elements that can be operably linked to a nucleotide sequence encoding a polypeptide or protein. Such nucleotide sequences encoding desired elements may include appropriate transcription promoters, transcription enhancers, transcription terminators, translation initiators, translation terminators, ribosome binding sites, 5'-untranslated regions, 3'-untranslated regions, cap structures, polyA tails, and / or origins of replication. The selection of an appropriate vector may depend on several factors, including but not limited to, the size of the nucleic acid to be incorporated into the vector, the type of desired transcription and translation control elements, the desired expression level, the desired copy number, whether chromosomal integration is desired, the type of desired selection process, or the host cell or host range intended for transformation.

[0020] Included as part of the present invention are nucleic acid vectors, often expression vectors, which contain nucleic acid sequences that are complementary, or at least partially complementary, to the nucleic acids corresponding to the miRNA122 gene (gene ID: 406906), or to the nucleic acids corresponding to the Kif3b (gene ID: 9371), SRPK1 (gene ID: 6732), TMEM229b (gene ID: 161145), C14orf142 (gene ID: 84520), KB-1460A1.5, ACTC1 (gene ID: 70), Nr2f1 (gene ID: 7025), KIAA0922 (gene ID: 422400), KDELR3 (gene ID: 11015), APBA2 (gene ID: 321), miRNA 130b (gene ID: 406920), or miRNA374b (gene ID: 100126317). A vector is a nucleic acid molecule capable of transporting another nucleic acid to which it is bound, and may include plasmids, cosmids, or viral vectors. Vectors may be capable of autonomous replication or may be incorporated into host DNA. Viral vectors may include, for example, replication-deficient retroviruses, adenoviruses, and adeno-associated viruses.

[0021] Those skilled in the art will recognize that the expression of a particular gene within a cell can be reduced, prevented, or “suppressed” using any of the various well-known methods. In non-limiting examples, gene expression can be suppressed using gene-silencing nucleic acids such as siRNA (short interfering RNA), antisense oligonucleotides (AONs), short hairpin RNA (shRNA), microRNA (miRNA), or other RNA interference (RNAi) or antisense gene-silencing triggers (see, e.g., Gaynor et al. Chem. Soc. Rev. 39:4196-4184, 2010; Bennett et al. Annual Review of Pharmacology and Toxicology 50:259-293, 2010). Gene expression can also be reduced by other pre-transcriptional or post-transcriptional gene-silencing techniques known in the art. Given a specific gene sequence, those skilled in the art can design gene-silencing oligonucleotides that can target that gene sequence and reduce gene expression. Various software-based tools are available for designing siRNAs or AONs to target specific genes, including those available from the Whitehead Institute or from siRNA vendors. For example, siRNA antisense strands or antisense oligonucleotides that are completely or substantially complementary to a region of a gene expression mRNA sequence can be prepared and used for targeted gene silencing by inducing RISC or RNase H-mediated mRNA degradation. Gene silencing nucleic acids can be prepared, for example, as described in Current Protocols in Nucleic Acids Chemistry published by Wiley.

[0022] siRNA or RNAi is a nucleic acid that forms double-stranded RNA and has the ability to reduce or inhibit the expression of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene. siRNA is a short double-stranded RNA formed by a complementary strand. The complementary portion of siRNA that hybridizes to form a double-stranded molecule often has substantial or complete identity with respect to the target molecular sequence. In one embodiment, siRNA is a nucleic acid that has substantial or complete identity with respect to the target gene and forms double-stranded siRNA.

[0023] When designing siRNA molecules, the target region is often selected from a given DNA sequence 50–100 nucleotides downstream of the start codon. Initially, regions near the 5' or 3' UTR and start codon were avoided, assuming that the UTR-binding protein and / or translation initiation complex might interfere with the binding of the siRNP or RISC endonuclease complex. Sometimes, a 23-nucleotide region matching the sequence motif AA(N19)TT (N, nucleotide) and with a G / C content of approximately 30% to 70% (often with approximately 50% GIC content) is frequently selected. If a suitable sequence is not found, the search is often extended using the motif NA(N2 1). The sequence of sense siRNA sometimes corresponds to (N19)TT or N21 (positions 3 to 23 of the 23-base motif), respectively. In the latter case, the 3' end of the sense siRNA is often converted to TT. The rationale for this sequence conversion is to generate a symmetrical double helix with respect to the sequence composition of the sense and antisense 3' overhangs. Antisense siRNAs are synthesized as complementary strands to positions 1-21 of a 23-base motif. Since position 1 of the 23-base motif is not sequence-specifically recognized by antisense siRNA, the most 3' nucleotide residue of the antisense siRNA can be carefully selected. However, the second-to-last nucleotide of the antisense siRNA (complementary to position 2 of the 23-base motif) is often complementary to the target sequence. To simplify chemosynthesis, TT is often used. siRNAs corresponding to the target motif NAR(N17)YNN, where R is a purine (A, G) and Y is a pyrimidine (C, U), are often selected. Each 21-nucleotide sense and antisense siRNA often begins with a purine nucleotide and can be expressed from a pol III expression vector without altering the target site. If the first transcribed nucleotide is a purine, RNA expression from the pol III promoter may be more efficient.

[0024] siRNA sequences can correspond to full-length target genes or partial sequences thereof. In most cases, siRNAs are approximately 15 to 50 nucleotides long (for example, each complementary sequence of a double-stranded siRNA is 15 to 50 nucleotides long, and a double-stranded siRNA is approximately 15 to 50 base pairs long, sometimes approximately 20 to 30 nucleotides, or approximately 20 to 25 nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long). siRNAs are sometimes approximately 21 nucleotides long. Methods of using siRNA are publicly known in the art, and specific siRNA molecules can be purchased from numerous companies, including Dharmacon Research, Inc.

[0025] Gene expression can be inhibited by the introduction of double-stranded RNA (dsRNA) that induces a potent and specific phenomenon called gene silencing, RNA interference, or RNAi. (See, for example, Fire et al., U.S. Patent No. 6,506,559; Tuschl et al., PCT International Publication No. WO01 / 75164; and M. Kay et al., PCT International Publication No. WO03 / 010180A1). This process has been improved by reducing the size of double-stranded RNA to 20-24 base pairs (to create short interfering RNA or siRNA), thereby switching off genes in mammalian cells without initiating an acute-phase response, i.e., a host defense mechanism that often leads to cell death. In human cells, there is growing evidence of post-transcriptional gene silencing by RNA interference (RNAi) to inhibit targeted expression in mammalian cells at the mRNA level. Further evidence is available for its effectiveness in inhibiting tumor cell proliferation and migration in human patients and in inhibiting metastatic cancer.

[0026] In another embodiment, the gene-silencing nucleic acid is a ribozyme. A ribozyme specific to a target nucleotide sequence may include one or more sequences complementary to such nucleotide sequence, and a sequence having a known catalytic region that is responsible for mRNA cleavage (see, for example, U.S. Patent No. 5,093,246). For example, derivatives of Tetrahymena L-19 IVS RNA are sometimes used in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the mRNA (see, for example, Cech et al., U.S. Patent No. 4,987,071 and Cech et al., U.S. Patent No. 5,116,742). Alternatively, a catalytic RNA having specific ribonuclease activity can be selected from a pool of RNA molecules using the target mRNA sequence.

[0027] Modified nucleic acid molecules can be formed by modifying gene-silencing nucleic acid molecules such as antisense, ribozyme, RNAi, and siRNA nucleic acids. Nucleic acids can be modified at the base, sugar, or phosphate backbone to improve molecular stability, hybridization, or solubility. For example, the deoxyribose phosphate backbone of a nucleic acid molecule can be modified to produce peptide nucleic acids (see Hyrup et al. Bioorganic & Medicinal Chemistry 4(1):5-23, 1996). Peptide nucleic acids, or PNAs, refer to nucleic acid mimes such as DNA mimes, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone, and only the four native nucleic acid bases are retained. The neutral backbone of PNAs can enable specific hybridization to DNA and RNA under low ionic strength conditions. The synthesis of PNA oligomers can be carried out, for example, using the standard solid-phase peptide synthesis protocol described in Hyrup et al.

[0028] PNA nucleic acids can be used for the prognostic, diagnostic, and therapeutic applications described herein. For example, PNA can be used as an antisense or antigene agent for sequence-specific regulation of gene expression, for example, by inducing arrest of transcription or translation, or by inhibiting replication. PNA nucleic acid molecules can also be used as an artificial restriction enzyme in combination with other enzymes in the analysis of SNPs in genes (e.g., by PNA-directed PCR clamping) (e.g., S1 nuclease (Hyrup et al., previously cited) or as a probe or primer for DNA sequencing or hybridization (Hyrup et al., previously cited).

[0029] In some embodiments, a target gene, such as miRNA122, is overexpressed compared to the baseline level in cancerous tumors to minimize the likelihood of cancer metastasis. Gene overexpression can be achieved in numerous different ways, including, but is not limited to, transfecting cells / tissues with gene constructs that overexpress the target gene. Furthermore, transfecting cells / tissues with gene constructs that affect the transcription or translation mechanisms of genes / cells can also be used to induce overexpression of the target gene. In addition, small molecules that increase the expression of the target gene in cancerous cells can be developed.

[0030] In relation to the insertion of nucleic acid sequences into cells, gene introduction refers to “transfection,” “transformation,” or “transduction,” and includes the integration or introduction of nucleic acid sequences into eukaryotic cells, the nucleic acid sequences may be integrated into the cell’s genome or transiently expressed (e.g., transfected mRNA). Proteins or enzymes can be introduced into cells by delivering the protein or enzyme itself into the cell, or by expressing mRNA encoding the protein or enzyme in the cell and resulting in its translation.

[0031] Gene silencing nucleic acid molecules can be introduced into cells using any number of known methods. An expression vector (viral, plasmid-based, or otherwise) can be introduced into cells by transfection, electroporation, or other means, after which the gene silencing nucleotide can be expressed. Alternatively, the gene silencing nucleotide itself may be introduced into cells directly, for example, by transfection or electroporation (i.e., using transfection reagents such as, but not limited to, Lipofectamine®, Oligofectamine, or other suitable delivery agents known in the art), or through a targeted gene or nucleic acid delivery vehicle known in the art. Many delivery vehicles and / or agents are well known in the art, some of which are commercially available. Delivery strategies for gene-silencing nucleic acids are described, for example, in Yuan et al. Expert Opin. Drug Deliv. 8:521~536, 2011; Juliano et al. Acc. Chem. Res. 45:1067-1076, 2012; and Rettig et al. Mol. Ther. 20:483~512, 2012. Examples of transfection methods are described, for example, in Ausubel et al. (1994) Current Protocols in Molecular Biology, John Wiley & Sons, New York. Examples of expression vectors are described, for example, in Cloning Vector: A Laboratory Manual (Pouwels et al. 1985, Supp. 1987).

[0032] Those skilled in the art will understand that antibodies or antibody fragments targeting one or more amino acids, nucleic acids, proteins, or enzymes as described herein, such as monoclonal or polyclonal antibodies or their Fab fragments, can be produced using standard laboratory techniques to target specific amino acids, nucleic acids, proteins, or enzyme targets and thus silence the gene. As a non-limiting example, monoclonal antibodies against specific targets can be prepared using hybridoma techniques (see, e.g., Harlow et al. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al. Monoclonal Antibodies and T-Cell Hybridomas pp. 563-681 (Elsevier, NY, 1981)). Those skilled in the art will know methods and techniques for preparing antibodies against specific amino acids, proteins, nucleic acids, or enzyme targets. Such antibodies conjugate the amino acid, protein, nucleic acid, or enzyme target, preventing it from performing its normal function, resulting in effects similar to those resulting from gene silencing of the same amino acid, nucleic acid, protein, or enzyme. Therefore, in certain embodiments, antibodies can be used instead of gene-silencing nucleic acids to target or "silence" specific genes.

[0033] Compounds that inhibit the activity of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b may be useful in the present invention and may include small molecules. Small molecules include, but are not limited to, peptides, peptide mimetics (e.g., peptoids), amino acids, amino acid analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (i.e., heteroorganic compounds or organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0034] It will be understood that compounds and / or compositions comprising one or more nucleic acids and / or polypeptides described herein, or composed of them, may be used. The compositions may further comprise one or more pharmaceutically acceptable diluents, carriers, excipients, or buffers. The compositions may be used to administer one or more nucleic acids and / or polypeptides to cells in vitro or in vivo.

[0035] When used as a therapeutic agent, gene-silencing nucleic acid molecules are typically administered to a target (e.g., by direct injection into a tissue site) or generated in situ to hybridize or bind to genomic DNA encoding intracellular mRNA and / or polypeptides such as Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b, thereby inhibiting polypeptide expression, for example, by inhibiting transcription and / or translation. Alternatively, the gene silencing nucleic acid molecule may be modified to target selected cells and then administered systemically. In the case of systemic administration, gene-silencing nucleic acid molecules can be modified to specifically bind to receptors or antigens expressed on the surface of selected cells, for example, by binding the gene-silencing nucleic acid molecule to a peptide or antibody that binds to the cell surface receptor or antigen. Gene-silencing nucleic acid molecules can also be delivered to cells using vectors. Sufficient intracellular concentrations of gene-silencing nucleic acid molecules are achieved by incorporating a strong promoter, such as a pol II or pol III promoter, into the vector construct.

[0036] As defined herein, a therapeutically effective dose of protein or polypeptide (i.e., an effective dose) is in the range of approximately 0.001–30 mg / kg body weight, sometimes approximately 0.01–25 mg / kg body weight, often approximately 0.1–20 mg / kg body weight, more frequently approximately 1–10 mg / kg, 2–9 mg / kg, 3–8 mg / kg, 4–7 mg / kg, or 5–6 mg / kg body weight. The protein or polypeptide may be administered once a week for approximately 1–10 weeks, sometimes 2–8 weeks, often approximately 3–7 weeks, more frequently approximately 4, 5, or 6 weeks. Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective dose of protein, polypeptide, or antibody may consist of a single treatment or a series of treatments.

[0037] For antibodies, a dosage of 0.1 mg / kg body weight (generally 10 mg / kg to 20 mg / kg) is often used. If the antibody acts in the brain, a dosage of 50 mg / kg to 100 mg / kg is often appropriate. Generally, partially human antibodies and fully human antibodies have longer half-lives in the human body than other antibodies. Therefore, lower doses and less frequent administration are often possible. Antibodies can be stabilized using modifications such as lipidization to enhance uptake and tissue penetration (e.g., into the brain). Methods for lipidization of antibodies have been described by Cruikshank et al. (1997).

[0038] Antibody conjugates can be used to modify a given biological response, and the drug portion should not be interpreted as being limited to classical chemotherapeutic agents. For example, the drug portion may be a protein or polypeptide having the desired biological activity. Such proteins may include toxins such as abrin, lysine A, Pseudomonas exotoxin or diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator or polypeptides, or biological response modifiers such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF") or other growth factors. Alternatively, an antibody can be conjugated with a second antibody to form an antibody heteroconjugate, as described by Segal in U.S. Patent No. 4,676,980.

[0039] For compounds, exemplary doses include milligrams or micrograms of the compound per kilogram of the subject or sample weight, e.g., about 1 microgram / kg to about 500 milligrams / kg, about 100 micrograms / kg to about 5 milligrams / kg, or about 1 microgram / kg to about 50 micrograms / kg. It is understood that the appropriate dose of a small molecule depends on the potency of the small molecule with respect to the expression or activity being regulated. When administering one or more of these small molecules to an animal (e.g., a human) to regulate the expression or activity of a polypeptide or nucleic acid described herein, a physician, veterinarian, or researcher should, for example, prescribe a relatively low dose initially and then increase the dose until an appropriate response is obtained. Furthermore, specific dose levels for any particular animal subject depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health status, sex, and diet, the time of administration, the route of administration, the rate of excretion, any combination of drugs, and the degree of expression or activity to be regulated.

[0040] With respect to nucleic acid formulations, gene therapy vectors may be delivered to a target, for example, by intravenous injection, topical administration (see, e.g., U.S. Patent No. 5,328,470), or stereotactic injection (Chen et al. 1994). A pharmaceutically acceptable preparation of a gene therapy vector may comprise the gene therapy vector in an acceptable diluent, or a sustained-release matrix in which the gene delivery vehicle is embedded. Alternatively, if the complete gene delivery vector can be produced intact from recombinant cells (e.g., retroviral vectors), the pharmaceutically acceptable preparation may comprise one or more cells that produce the gene delivery system. Examples of gene delivery vectors are described herein.

[0041] In another embodiment, the metastatic potential of cancer is detected using the expression of at least one of the following: Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122. In this case, a biological sample is taken from a patient with cancer and analyzed to detect whether the levels of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b are elevated above normal baseline levels in the biological sample, or, in the case of miRNA122, whether they are decreased above normal baseline levels. To determine mRNA levels, nucleic acids are isolated from the biological sample obtained from the subject. For example, nucleic acids can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissues. Nucleic acid samples can be isolated from biological samples using standard techniques. Nucleic acid samples may be isolated from the subject and then used directly in the method, or the sample may be isolated, stored for a period of time (e.g., frozen), and then subjected to analysis.

[0042] Diagnostic methods include polymerase chain reaction (PCR) (e.g., U.S. Patent Nos. 4,683,195; 4,683,202 and 6,040,166, "PCR Protocols: A Guide to Methods and Applications," Innis et al. (Eds.), 1990, Academic Press: New York), reverse transcriptase PCR (RT-PCT), anchored PCR, competitive PCR (e.g., U.S. Rapid amplification of cDNA ends (RACE) (e.g., Gene Cloning and Analysis: Current Innovations, 1997, pp. 99-115), ligase chain reaction (LCR) (e.g., EP 01 320308), one-sided PCR (e.g., Ohara et al. Proc. Natl. Acad. Sci. 1989, 86:5673-5677), in situ hybridization, and Taqman-based assays (Holland et al. al. Proc. Natl. Acad. Sci. 1991, 88: 7276~7280), differential display method (e.g., Liang et al.) It will be recognized that any suitable method, including but not limited to, the determination of the expression level of at least one of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122, may be used, including but not limited to, other RNA fingerprinting techniques, nucleic acid sequence-based amplification (NASBA), and other transcription-based amplification systems (see, for example, U.S. Patent Nos. 5,409,818 and 5,554,527), Q beta replicase, strand substitution amplification (SDA), repair chain reaction (RCR), nuclease protection assays, subtraction-based methods, Rapid-Scan®, etc.

[0043] In other cases, the expression of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 can be detected at the protein level by a variety of techniques, including but not limited to immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Therefore, it is possible to determine whether an individual has or is susceptible to metastatic cancer by contacting a polypeptide or protein encoded by a nucleotide sequence derived from the target with an antibody that specifically binds to an epitope associated with Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122. Cells suitable for diagnosis can be obtained from the patient's blood, urine, saliva, tissue biopsy material, and autopsy material.

[0044] In another embodiment, the components necessary to carry out the method are provided as part of a kit. In particular, the kit includes molecules that bind to Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122, and any buffers necessary to carry out the assay. These molecules are gene-silencing nucleic acid molecules, small molecules, or biologics that downregulate the expression or function of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b, and / or small molecules or gene constructs that can upregulate the expression of miRNA122. Optionally, the kit may include a set of instructions for the use of the molecules in the assay. However, the instructions do not need to be a set of paper documents; instead, they may be provided via a URL address or a QR code.

[0045] It will be understood that numerous modifications thereto will be obvious to those skilled in the art. Therefore, the above description and accompanying drawings should be interpreted as illustrative of the invention, not restrictively. Generally, in accordance with the principles of the invention, it is intended to cover any modifications, uses, or adaptations of the invention, including deviations from this disclosure, that fall within the scope of known or customary practices in the relevant art, and that may be applied to essential features described herein prior to their specification, and within the scope of the accompanying claims, as follows:

[0046] The following examples are provided for illustrative purposes only and are intended for those skilled in the art. These examples are not intended to be limiting, and it will be understood that numerous variations and modifications are possible, which would be known to those skilled in the art in light of the teachings herein. example

[0047] Traditionally, identifying genes necessary for in vivo cell motility has been hampered by the inherent difficulties in visualizing metastatic lesion formation in vivo (Sahai, Nat Rev Cancer 7:737-749, 2007; Kishimoto et al. Nat Med 12:1213-1219, 2006). To address this, we used an in vivo imaging approach in shell-less extraovo chicken embryos to perform shRNA screening for gene products that regulate tumor cell motility in vivo. After intravenous injection, cancer cells widely disseminate throughout the embryo's vascular system. A significant portion of these cancer cells arrest as single cells in the ceralis membrane (CAM), where they migrate to the extravascular matrix and proliferate into invasive metastatic colonies. These colonies, each derived from a single cancer cell, grow to approximately 1 mm over 4 days. 2 Reaching a size of 50-100 cells per colony, they can be easily visualized using a biological microscope. Since thousands of individual metastatic colonies can be visualized simultaneously in a single embryo CAM, this approach can be used to screen large libraries of genes. Identifying the motility phenotype is straightforward. When highly motile cancer cells, such as human head and neck HEp3 cell lines, are injected, the resulting colonies exhibit a "diffusion" migration phenotype, where proliferating cells migrate a considerable distance from the point of origin. When in vivo motility of tumor cells is reduced, as observed with CD151-specific migration-blocking antibodies, metastatic colonies exhibit a very compact morphology that is easily distinguishable from the highly motile phenotype. These compact metastatic lesions, composed of densely packed cancer cells, can be easily excised from surrounding tissue and subjected to further analysis. As previously seen with CD151 targeting, inhibiting genes required for in vivo cell motility leads to a compact colony phenotype, and this approach can be used to screen therapeutic targets for cell motility that affect intravascular invasion and metastasis.

[0048] To perform screening, HEp3 cells were transduced with a human shRNA GIPZ microRNA-compatible shRNA lentivirus library (Open Biosystems) constructed using a natural miR-30 primary transcript to enable processing via the endogenous RNAi pathway. This library contains 79,805 sequence-validated shRNAs targeting 30,728 human genes in seven pools, along with TurboGFP to monitor successful transduction. Using each pool, HEp3 cells in culture were transduced at MOI (0.2) to support the incorporation of a single shRNA per cancer cell according to a Poisson distribution. When 25,000 tumor cells were intravenously injected into chicken embryos, approximately 10% of the cells quiescently migrated out of the blood vessels into easily accessible and visible CAM organs, forming isolated metastatic colonies. Screening was performed in 100 embryos to ensure 3x coverage of the 79,805 shRNA clones with 99% confidence. Transduced GFP-expressing cells were intravenously injected into embryos in extraoocyte culture on day 10 of development. On day 15 of development, more than 200,000 colonies in the CAM of these 100 embryos were examined using an in vivo microscope. Of these, 67 morphologically compact metastatic lesions were identified and excised. These colonies were dissociated and cultured under selective conditions, and 50 clones were successfully grown in culture.

[0049] To identify the incorporated shRNAs, inserts from each clone were amplified by PCR using common flanking primers, and the resulting cDNA sequences were determined by deep sequencing on an Illumina platform. Raw sequence reads were subjected to a stringent filtering algorithm to identify adjacent miRNA sequences and eliminate reads with conflicting loop sequences and stem base pair mismatches. The filtered sequences were then subjected to BLAST analysis against both a library and a human nucleotide (nt) database, and ranked according to their abundance. Of the 50 isolated clones, 17 contained a single shRNA, while the remaining 33 clones each contained two or more shRNAs.

[0050] Next, gene targets were prioritized based on their impact on productive cell migration in vivo, according to the degree of their compact colony phenotype. This was achieved by using an experimental metastasis approach, thereby verifying the phenotype of each clone after intravenous injection into ectocotyledon embryos, and capturing images of the resulting metastatic colonies using in vivo imaging. A custom Matlab-based program was developed to analyze images of each metastatic colony using three complementary algorithms. No significant differences in proliferation rates were detected among the hit clones in vitro, but some clones were observed to proliferate at different rates in vivo (Figure 3a). Therefore, to mitigate the effects of differences in proliferation between individual colonies and to obtain an accurate assessment of cancer cell motility in vivo, the algorithms were designed to analyze two different parameters: A) the distance of cancer cells from the colony's center of gravity (linear index), B) the density of cancer cells within the metastatic colony region (density index), and C) the total area occupied by each metastatic colony (area index, Figures 3b-3d). Simply put, the first algorithm uses the GFP signal to create a mask that outlines the cancer cells, and then passes through the centroid in a 360-degree arc. 0A mean line plot fitted to a Gaussian distribution is created using line scanning. A colony linear index is generated using the deviation of the Gaussian radial line scan intensity distribution between colonies formed by individual clones relative to a control shRNA colony. The second and third algorithms measure individual translocation colony regions (area index) using a fluorescence mask and calculate the fluorescence density within each region (density index). For each clone obtained from the original screening, 10 individual colonies were analyzed and then classified based on their linear index and area index values. Each index resulted in a similarity ranking of the colonies identified in the screening, but some visually compact clones could not be adequately identified by one or the other method alone (Figures 3b-3d). Therefore, the three algorithms were combined to create a combined colony compactness index (CI), which was used to stratify the phenotype of hit clones compared to positive controls (colonies from embryos injected with migration-inhibiting antibodies) and negative controls (scrambled shRNA-expressing HEp3 clones, Figures 3a-3d). The CI was calculated for each index from the Z-score (experiment-control / SD control), and the formula was CI = Z(density index) - Z(linear index) - Z(area index).

[0051] The morphology of positive control colonies generated after treatment with a migration-blocking antibody targeting CD151 (positive control) showed the most dramatic increase in CI (17.1 ± 1.68) compared to highly invasive metastatic colonies generated by scrambled shRNA-expressing cells (negative control, 8.515e-009 ± 1.68) (Figure 3a). Statistical analysis of the CI index revealed 27 clones with a metastatic colony phenotype whose CI was significantly different from that of the negative control (p ≤ 0.05) (Figure 7). Of these 27 clones, 11 contained a single shRNA (Kif3b, ACTB, SRPK1, TMEM229b, C140rf142, KB-1460A 1.5, ACTC1, KDELR3, APBA2, KIAA0922, and Nr2f1). Clones containing a single shRNA and having a CI of 5.0 or higher were selected for downstream analysis (see Table 1).

[0052] [Table 1]

[0053] To confirm that the observed inhibition of in vivo motility was due to shRNA-mediated depletion of the target gene and not to off-target effects, new HEp3 clones were generated for Kif3b (CI=12.4), SRPK1 (CI=11.2), TMEM229b (CI=9.7), Nr2f1 (CI=5.9), and C14orf142 (CI=8.8) using independent shRNA constructs (Figures 4a-4e). Analysis of gene and protein expression of each target protein in the original hit clones and the newly induced clones confirmed specific knockdown of the target proteins (Figures 4a-4e). Subsequently, clones with independent shRNAs were validated using an in vivo transmissible colony formation assay, and all candidate genes reproduced a compact colony phenotype with CI values ​​similar to those of their primary screening hit clones (Figure 4f).

[0054] Based on their therapeutic relevance and the potential to develop specific inhibitors, further research focused on the Kif3b, Nr2f1, and SRPK1 genes. To gain further insight into the migratory phenotypes produced by knockdown of these genes, high-resolution in vivo time-lapse imaging was performed on the invading front of primary tumors from each clone compared to individual metastatic colonies and control (scrambled) shRNA-transduced HEp3 cells. ShRNA-mediated inhibition of each of these targets was observed to reduce both the rate and directionality of cancer cell migration (Figures 1a–1f). Cancer cells from each of the shKif3b, shNr2f1, and shSRPK1 clones exhibited either a lack of motility or unproductive migration patterns both within metastatic lesions (Figures 1a, 1c–1d) and on the invading front of primary tumors (Figures 1b, 1e–1f). Despite the fact that the average velocity of cancer cells is faster in front of invasive tumors compared to metastases, the number of cancer cells that evaded the tumor was significantly reduced in shKif3b, shNr2f1, and shSRPK1 clones compared to controls (Figure 1g). In vivo imaging of control or hit clones in front of invasive tumors showed that control HEp3 cells tended to form a single dominant protrusion in the direction of motility, while shKif3b, shNr2f1, and shSRPK1 clones tended to form multiple protrusions extending in all directions in an uncoordinated manner (Figures 1b, 1h). In conclusion, this screening approach primarily identified genes required to modulate directional in vivo cell migration.

[0055] To investigate whether genes required for in vivo cell motility and directional cell migration are also required for intravascular invasion and metastasis, hit clones were evaluated in a mouse model of spontaneous lung metastasis. For this purpose, subcutaneous HEp3 tumors were established in the flanks of nude mice using parental scrambled shRNA controls or tumor cells expressing shKif3b, shNr2f1, and shSRPK1. The primary tumor was 1.5 cm. 3Once this was reached, the presence of lung metastases was quantitatively examined using a full-scale fluorescence stereomicroscope and then human alu-specific q-PCR (Figures 2a and 2b). Significant lung metastases were detected by fluorescence imaging in animals with shRNA scrambled control HEp3 tumors (n=23) (Figure 2a). In contrast, metastatic lesions were rarely observed in the lungs of animals with KIF3Bsh / sh2, SRPK 1sh / sh2, and NR2F 1sh / sh2 tumors, and the metastatic lesions were very small in size (Figure 2a). To accurately quantify the amount of metastatic HEp3 cancer cells in mouse lungs, we extracted genomic DNA and performed human-specific alu q-PCR. Then, by comparing this data with a standard curve created from HEp3 cells, we determined the accurate count of metastatic cells in the lungs. The scrambled shRNA control had an average of 2.4 million disseminated cancer cells per lung. In contrast, animals with KIF3Bsh / sh2, SRPK 1sh / sh2, and NR2F1sh / sh2 tumors showed dramatic suppression of metastatic dissemination, with reductions in lung metastasis being 99.55% and 99.67% for KIF3Bsh / sh2, 99.98% and 99.66% for SRPK 1sh / sh2, and 99.71% and 99.81% for NR2F1sh / sh2, respectively (Figure 2b). At sacrifice, there was no significant difference in primary tumor weight between controls and hit shRNA clone tumors (Figure 2c). These results confirm that Kif3b, Nr2f1, and shSRPK1 are necessary for both in vivo cancer cell motility and successful spontaneous metastasis, and therefore represent very promising therapeutic targets for metastasis.

[0056] Considering the possibility that the observed motility phenotype may be specific to the highly metastatic human epidermal carcinoma cell line HEp3, hits with Kif3b, SRPK1, and Nr2f1 were suppressed in two additional cell lines representing two different types of human epithelial carcinoma: MDA-MB-231 (breast cancer) and PC3 (prostate cancer). Silencing Kif3b expression efficiently inhibited in vitro cell migration in all cancer cell lines (Figure 5a). Interestingly, silencing SRPK1 significantly inhibited the motility of HEp3 and PC3 cells in vitro, but did not affect the in vitro motility of MDA-MB-231 (Figure 5b). Finally, silencing Nr2f1 inhibited the migration of HEp3 in vitro, but had no effect on MDA-MB-231. Nr2f1 expression was not detected in PC3 cells (Figure 5c). This may explain why these genes were not detected in conventional in vitro screening. In fact, SRPK1 and Nr2f1 would likely not be detected if screening were performed using MDA-MB-231 cells.

[0057] Validation of miRNA130b and miRNA122 clones using independent miRNA constructs confirmed their non-invasive phenotypes (Figure 8A). Importantly, HT1080 cells engineered to overexpress miRNA-122 or miRNA-130b inhibitors formed compact, metastatic colonies that showed little contact with the chicken CAM vascular system (Figures 8B, 8C).

[0058] To gain further insight into the mechanism by which miRNAs identified in screening inhibit the invasive migration of cancer cells in vivo, in vivo imaging experiments were conducted focusing on the effects of miRNA122 overexpression on cancer cell invasion and metastasis. Control (RFP) cells were found to strongly invade CAM tissue, preferentially traversing along existing blood vessels (Figures 9A-9C and 2A-2C). Furthermore, control and scrambled vector-transduced cells actively metastasized to chicken CAM in an intraocular metastasis model, while miRNA122-overexpressing cells were unable to do so (Figure 9F). Co-injection of differentially labeled control, scrambled vector-expressing cells (RFP), and miRNA122-overexpressing cells (GFP), followed by high-resolution in vivo imaging, revealed that control cells preferentially protruded and invaded along the vascular system, forming clear contact with perivascular collagen fibers, while miRNA122-overexpressing cells protruded and invaded independently of the vascular system and did not form contact with perivascular collagen. (Figures 10A-10F). Chicken CAM represents a collagen-rich membrane through which the vascular system permeates. Metastatic cancer cells invade the collagen-rich matrix by a) migrating along existing collagen fiber bundles and b) locally degrading and rearranging the collagen matrix to create aligned collagen bundles that are later used for directional cancer cell invasion. Indeed, miR122-overexpressing cells were unable to invade the artificial 3D collagen matrix. 3D collagen invasion was also blocked by the MMP inhibitor phenanthroline, confirming that this process is protease-dependent (Figures 11A, 11B). miRNA122-overexpressing cells invaded and degraded the collagen matrix significantly less than control cells, as indicated by the almost complete absence of collagen degradation regions within the 3D collage matrix (Figures 11C, 11D). When scrambled transdextrins (CAM) were injected intravascularly into chickens, the transdextrins remained in the collagen matrix and actively formed directional collagen bundles in its vicinity (Figures 11E-11G).In contrast, miRNA122 cells were unable to penetrate deeply into the collagen matrix substantially growing on the CAM surface and showed little to no collagen rearrangement (Figures 11E–11G). Invasion and rearrangement of the collagen-rich matrix require local proteolysis of these by cancer cell-associated proteases. MT1-MMP is a key matrix-degrading enzyme whose activity and localization have been shown to be important for efficient cancer cell invasion. Therefore, we investigated MT1-MMP transport and localization in control, scrambled, and miRNA122-overexpressing HT1080 cells. First, in in vitro culture, cells overexpressing miRNA122 were found to exhibit impaired MT1-MMP transport, along with enlarged MT1-MMP-positive vesicles showing significantly shorter trajectories (Figures 12A, 12B). High-resolution in vivo imaging revealed that MT1-MMP was localized to the contact site between cancer cell protrusions and collagen fibers in control HT1080 cells, while in miRNA122-overexpressing cells, MT1-MMP was mainly localized in the cytoplasm (Figure 12C). Furthermore, in perivascular control cells, MT1-MMP was localized to the contact site between cancer cells and the blood vessel wall, but in miRNA122-overexpressing cells, MT1-MMP did not show specific localization (Figures 12D-12G). Next, we investigated miRNA122-mediated MT1-MMP transport to determine whether it is necessary for the extravasation of cancer cells. miRNA122-overexpressing cells were found to migrate out of blood vessels significantly less efficiently than control scramble-infected HT1080 cells (Figures 13A, 13B). Importantly, in control cells, MT1-MMP showed clear localization from the protrusions to the invadopodia in miRNA122-overexpressing cells depleted of MT1-MMP (Figure 13C, Figure 13D).

[0059] Having identified several promising metastatic therapeutic targets in cancer cell lines, we investigated the potential relevance of these genes to human cancer progression and metastasis. To do this, we examined the Oncomine collection of human oncogene expression databases (Rhodes et al. Neoplasia 9:166-180, 2007) to determine whether their expression was associated with metastasis or poor clinical outcomes. Indeed, the analysis showed that the top-hit genes identified in the screening were significantly upregulated in metastatic lesions of several solid tumor types, including melanoma (Nr2f1, C14orf142, and Kif3b), prostate (SRPK1 and Kif3b), head and neck (Kif3b), lung (SRPK1 and TMEM229b), ovarian (Nr2f1), and colon (Nr2f1) (Figure 6a). Furthermore, a detailed investigation of immunohistochemical staining of human cancers in the Human Protein Atlas Database showed that SRPK1, Kif3b, Nr2f1, C14orf142, and TMEM229b all exhibited significantly increased expression in the invasive areas of the primary tumors of these cancers, as described by cancer pathologists (Figures 6b-6g).

[0060] In summary, we used a quantitative in vivo approach that enabled the discovery of anti-metastatic therapeutic targets. The rapid and quantitative nature of this assay allowed for efficient filtering through a vast number of initial candidate genes, leading to the discovery of several novel anti-metastatic targets. The anti-metastatic targets identified using this screening approach had little to no effect on the ability of cancer cells to migrate in vitro. Claims [Claim 1] A method for preventing cancer metastasis in a subject, comprising administering an effective amount of an inhibitor of at least one of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, or miRNA374b, or a compound that increases the expression of miRNA122 and / or miRNA122, to the subject. [Claim 2] The method according to claim 1, wherein the inhibitor is a gene-silencing nucleic acid molecule or a small molecule. [Claim 3] The method according to claim 2, wherein the gene silencing nucleic acid molecule is a short interfering RNA, an antisense oligonucleotide, a short hairpin RNA, a microRNA, a ribozyme, or another RNA interfering molecule. [Claim 4] The method according to claim 2, wherein the small molecule is a peptide, peptoid, amino acid, amino acid analog, organic or inorganic compound. [Claim 5] The method according to claim 1, wherein the inhibitor is an inhibitor of Kif3b. [Claim 6] The method according to claim 1, wherein the inhibitor is an inhibitor of SRPK1. [Claim 7] The method according to claim 1, wherein the inhibitor is an inhibitor of TMEM229b. [Claim 8] The method according to claim 1, wherein the inhibitor is an inhibitor of C14orf142. [Claim 9] The method according to claim 1, wherein the inhibitor is an inhibitor of Nr2f1. [Claim 10] A method for detecting Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 in patients, including the following: Steps to obtain biological samples from human patients, The step of detecting whether Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 are present in the sample, the step of which is Contacting the sample with an anti-Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KI or miRNA122 antibody, or a nucleic acid complementary to Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b or miRNA122 mRNA, and Kif3b, ACTB, SRPK1, TMEM229b, C1 This is done by detecting the binding of 4orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 to the antibody, or by detecting the hybridization of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 mRNA with nucleic acids complementary to these mRNAs. [Claim 11] The method according to claim 10, wherein a human patient is suffering from cancer. [Claim 12] The method according to claim 10, wherein the biological sample is a tumor biopsy material. [Claim 13] The method according to claim 10, wherein Kif3b is detected. [Claim 14] The method according to claim 10, wherein SRPK1 is detected. [Claim 15] The method according to claim 10, wherein TMEM229b is detected. [Claim 16] The method according to claim 10, wherein C14orf142 is detected. [Claim 17] The method according to claim 10, wherein Nr2f1 is detected. [Claim 18] The method according to claim 10, wherein miRNA122 is detected. [Claim 19] Methods for diagnosing and treating cancer metastasis in patients, including the following: Steps to obtain biological samples from human patients, A step of detecting whether at least one of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 is present in the biological sample. A step of diagnosing a patient with metastatic cancer or the development of metastatic cancer, wherein the diagnosis is made when the presence of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122 is detected in a biological sample, and The step of administering an effective amount of an inhibitor of at least one of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122, and / or a compound that increases the expression of miRNA122, to the diagnosed patient. [Claim 20] The method according to claim 19, wherein the biological sample is a tumor biopsy material. [Claim 21] The method according to claim 19, wherein the inhibitor is a gene-silencing nucleic acid molecule or a small molecule. [Claim 22] The method according to claim 21, wherein the gene silencing nucleic acid molecule is a short interfering RNA, an antisense oligonucleotide, a short hairpin RNA, a microRNA, a ribozyme, or another RNA interfering molecule. [Claim 23] The method according to claim 21, wherein the small molecule is a peptide, peptoid, amino acid, amino acid analog, organic or inorganic compound. [Claim 24] The method according to claim 19, wherein the inhibitor is an inhibitor of Kif3b. [Claim 25] The method according to claim 19, wherein the inhibitor is an inhibitor of SRPK1. [Claim 26] The method according to claim 19, wherein the inhibitor is an inhibitor of TMEM229b. [Claim 27] The method according to claim 19, wherein the inhibitor is an inhibitor of C14orf142. [Claim 28] The method according to claim 19, wherein the inhibitor is an inhibitor of Nr2f1. [Claim 29] Use to prevent cancer metastasis in the subject, with at least one of the following: Inhibitors of Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122, and / or compounds that increase the expression of miRNA122. [Claim 30] The use according to claim 29, wherein a human patient is suffering from cancer. [Claim 31] The use according to claim 29, wherein the inhibitor is a gene-silencing nucleic acid molecule or small molecule. [Claim 32] The use according to claim 31, wherein the gene silencing nucleic acid molecule is a short interfering RNA, an antisense oligonucleotide, a short hairpin RNA, a microRNA, a ribozyme, or another RNA interfering molecule. [Claim 33] The use according to claim 31, wherein the small molecule is a peptide, peptoid, amino acid, amino acid analog, organic or inorganic compound. [Claim 34] The use according to claim 29, wherein the inhibitor is an inhibitor of Kif3b. [Claim 35] The use according to claim 29, wherein the inhibitor is an inhibitor of SRPK1. [Claim 36] The use according to claim 29, wherein the inhibitor is an inhibitor of TMEM229b. [Claim 37] The use according to claim 29, wherein the inhibitor is an inhibitor of C14orf142. [Claim 38] The use according to claim 29, wherein the inhibitor is an inhibitor of Nr2f1. [Claim 39] Use for diagnosing metastatic cancer in the subject, at least one of the following: Kif3b, ACTB, SRPK1, TMEM229b, C14orf142, KB-1460A1.5, ACTC1, Nr2f1, KIAA0922, KDELR3, APBA2, miRNA130b, miRNA374b, or miRNA122. [Claim 40] The use according to claim 39, wherein a human patient is suffering from cancer. [Claim 41] The use according to claim 39, wherein Kif3b is detected. [Claim 42] The use according to claim 39, wherein SRPK1 is detected. [Claim 43] The use according to claim 39, wherein TMEM229b is detected. [Claim 44] The use according to claim 39, in which C14 or f142 is detected. [Claim 45] The use according to claim 39, in which Nr2f1 is detected. [Claim 46] The use according to claim 39, wherein miRNA122 is detected.

Claims

1. A composition for use in a method to prevent cancer metastasis in a target, comprising an effective amount of an inhibitor against C14orf142 as an inhibitor, The inhibitor is a gene-silencing nucleic acid molecule that targets genes having a nucleotide sequence encoding C14orf142. The aforementioned cancer metastases are related to melanoma. composition.

2. The composition according to claim 1, wherein the gene silencing nucleic acid molecule is an RNA interference molecule selected from short interfering RNA, antisense oligonucleotide, small hairpin RNA, microRNA, or ribozyme.

3. A composition for use in preventing cancer metastasis in a subject, The inhibitor includes an inhibitor of C14orf142, The inhibitor is a gene-silencing nucleic acid molecule that targets genes having a nucleotide sequence encoding C14orf142. The aforementioned cancer metastases are related to melanoma. composition.

4. The composition according to claim 3, wherein the human patient is suffering from cancer.

5. The composition according to claim 3, wherein the gene silencing nucleic acid molecule is an RNA interference molecule selected from short interfering RNA, antisense oligonucleotide, low molecular weight hairpin RNA, microRNA, or ribozyme.

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