Methods to control cell state transitions in cancer

Abstract

The present invention relates to a method of inducing mesenchymal-epithelial transition (MET) in basal-like (or mesenchymal-like) cancer cells by contacting the cancer cells with a mesenchymal-epithelial transition inducer for a time and under conditions sufficient to induce MET in the cells. In addition, a method of inhibiting epithelial-mesenchymal transition (EMT) of cancer in a subject is provided, the method comprising: administering a lipid metabolism inhibitor or regulator for a time and under conditions sufficient to inhibit epithelial-mesenchymal transition (EMT) of cancer in the subject.

Description

Technical Field

[0001] The present invention relates to the field of cancer biology and in particular provides a method for inducing mesenchymal-epithelial transition (MET) in cancer cells. Background Art

[0002] Breast cancer is a heterogeneous disease that is typically stratified by receptor expression status; this requires different treatment strategies to achieve optimal disease control. While estrogen receptor-positive (ER+) and ERRB2 / HER2-positive tumors generally respond well to targeted therapies, the triple-negative breast cancer (TNBC) subtype, defined by the absence of ER, progesterone receptor (PR), and HER2 expression, lacks targeted approaches and remains refractory to standard-of-care chemotherapy. Limited progress has been made in developing new adjuvant treatment options in the clinical management of TNBC, and phase II and III trials of therapeutic agents targeting TNBC have been lacking in promise. This emphasizes the need to explore more effective strategies for TNBC treatment. Activation of the epithelial-mesenchymal transition (EMT) program in cancer cells is often attributed to the acquisition of a drug-resistant phenotype, in part due to the acquisition of cancer stem cell-like properties. Several studies have conceptually suggested that reversal of the EMT program, i.e., mesenchymal-to-epithelial transition (MET), may result in cancer cells regaining sensitivity to chemotherapy.

[0003] While receptor status has been one of the mainstays of clinical classification, prognosis, and management decisions, gene expression profiling has been applied to classify breast cancer into several “intrinsic” molecular subtypes: luminal A, luminal B, HER2-enriched, basal-like, and claudin-low. Although there is still some debate, it has been observed that ER+ tumors tend to be luminal (luminal-like) and express “epithelial” marker genes, such as E-cadherin, whereas TNBCs carry a basal-like gene signature and express “mesenchymal” markers, including claudin and vimentin. Whether the acquisition of “epithelial” or “mesenchymal” features during cell state transitions is truly associated with luminal or basal-like carcinomas, respectively, is unclear. In addition to changes in cell state, MET may be able to transform basal-like breast cancers into more luminal breast cancers, in which ER or luminal cytokeratins are expressed.

[0004] The EMT program is well established and can be modulated by alterations in both extracellular and intracellular signaling pathways. For example, extrinsic signals such as TGFβ and PDGF lead to the activation of intracellular signaling cascades that ultimately lead to the activation of EMT-associated transcription factors such as TWIST, SNAIL, SLUG, and ZEB1. In contrast, the pathways that promote MET appear to be more diverse and poorly defined. For example, forskolin-induced MET acts via activation of the cAMP pathway leading to epigenetic reprogramming, whereas TGFβ inhibitors have been reported to mediate MET in germline stem cells. In both cases, the precise mechanisms remain elusive. Furthermore, although it is possible in principle that biochemical or genetic modulation of potential pathways such as cAMP activation or TGFβ receptor inhibition could contribute to the promotion of MET, their potential for clinically relevant applications may not emerge.

[0005] While the signaling pathways and transcriptional regulators that control cell states are well understood, other emerging aspects, especially metabolism that contributes to cancer cell function, remain poorly understood. Metabolic alterations have reemerged as a hallmark of neoplasia, emphasizing the role of metabolic pathways in regulating tumor cell behavior and their therapeutic implications. The vast majority of studies have focused on assessing metabolic differences between cancer cells and normal cells, particularly the requirement for glycolysis. Given the phenotypic diversity and plasticity among cancer cells, little is known about the relevance and contribution of their metabolic signatures to functional adaptation. Ostensibly, cancer cells transition between cell states with different energy requirements to support these functions. Summary of the invention

[0006] Provided herein is a method for inducing mesenchymal-epithelial transition (MET) in cancer cells. On the one hand, a method for inducing mesenchymal-epithelial transition (MET) in cancer cells is provided, the method comprising: contacting the cells with a mesenchymal-epithelial transition inducer at a time and under conditions sufficient to induce MET in the cells, wherein the cancer cells are basal-like (mesenchymal-like) cancer cells. Such cancer cells belong to the mesenchymal subtype of cancer.

[0007] In one aspect, a mesenchymal-epithelial transition (MET) inducing agent for inducing mesenchymal-epithelial transition (MET) in cells of a patient is provided.

[0008] In one aspect, there is provided a use of a mesenchymal-epithelial transition (MET) inducer in the preparation of a medicament for inducing mesenchymal-epithelial transition in cells of a patient.

[0009] In one aspect, a method for treating cancer in a patient in need thereof is provided, the method comprising administering to the patient a mesenchymal-epithelial transition (MET) inducer in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator, or a lipid metabolism inhibitor or regulator at a time and under conditions sufficient to treat the patient's cancer.

[0010] In one aspect, a mesenchymal-epithelial transition (MET) inducer for treating cancer in a patient in need thereof is provided, wherein the mesenchymal-epithelial transition (MET) inducer is administered to the patient in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator or a lipid metabolism inhibitor.

[0011] In one aspect, provided is a use of a mesenchymal-epithelial transition (MET) inducer in the preparation of a medicament for treating cancer in a subject, wherein the medicament is administered to the patient in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator or a lipid metabolism inhibitor.

[0012] In one aspect, a method of sensitizing a cancer patient to an anti-cancer therapy is provided, the method comprising administering a retinoid to the patient at a time and under conditions that sensitize the patient to the anti-cancer therapy.

[0013] In one aspect, a mesenchymal-epithelial transition (MET) inducer for sensitizing a cancer patient to an anti-cancer therapy is provided.

[0014] In one aspect, there is provided use of a mesenchymal-epithelial transition (MET) inducer in the preparation of a medicament for sensitizing a patient to an anti-cancer therapy.

[0015] In one aspect, a method for identifying an agent that induces mesenchymal-epithelial transition of cells is provided, the method comprising:

[0016] a) contacting the cells with a test agent to be screened; and

[0017] b) detecting the expression of the promoter of a gene selected from the group consisting of CD24, CD44, CD166, CDH1, ZEB1 and SEPINE1; wherein a change in the expression of one or more genes compared to the control indicates that the test agent is capable of inducing mesenchymal-epithelial transition in the cell.

[0018] In one aspect, a method is provided for identifying a cancer patient who responds to a combination therapy, the combination therapy comprising a compound that induces mesenchymal-epithelial transition (MET) in combination with a chemotherapeutic agent, an epigenetic modification compound, an immunomodulator, or a lipid metabolism inhibitor, the method comprising: detecting a mesenchymal subtype of cancer in a sample obtained from the patient, wherein the presence of basal-like carcinoma indicates that the patient responds to the combination therapy.

[0019] In one aspect, a method is provided for identifying and treating a cancer patient who is responsive to a combination therapy, the combination therapy comprising a compound that induces mesenchymal-epithelial transition (MET) in combination with a chemotherapeutic agent, an epigenetic modification compound, an immunomodulator, or a lipid metabolism inhibitor, the method comprising: detecting a mesenchymal subtype of the cancer in a sample obtained from the patient, wherein the presence of the mesenchymal subtype of the cancer indicates that the patient is responsive to the combination therapy; and treating the patient with the combination therapy.

[0020] In one aspect, a method of inhibiting epithelial-mesenchymal transition (EMT) of cancer in a subject is provided, the method comprising administering a lipid metabolism inhibitor or regulator for a time and under conditions sufficient to inhibit epithelial-mesenchymal transition (EMT) of cancer in the subject.

[0021] In one aspect, a lipid metabolism inhibitor or modulator for inhibiting epithelial-mesenchymal transition (EMT) in a cancer in a subject is provided.

[0022] In one aspect, there is provided a use of a lipid metabolism inhibitor or regulator in the preparation of a medicament for inhibiting epithelial-mesenchymal transition (EMT) of cancer in a subject.

[0023] In one aspect, a method of blocking or preventing cancer metastasis in a subject is provided, the method comprising administering a lipid metabolism inhibitor or regulator for a time and under conditions sufficient to block or prevent cancer metastasis in the subject.

[0024] In one aspect, a lipid metabolism inhibitor or regulator for blocking or preventing cancer metastasis in a subject is provided.

[0025] In one aspect, there is provided a use of a lipid metabolism inhibitor or regulator in the preparation of a medicament for blocking or preventing cancer metastasis in a subject.

[0026] Brief Description of the Figures

[0027] Certain embodiments are illustrated by the following figures. It should be understood that the following description is only for the purpose of describing specific embodiments and is not intended to be limiting of the description.

[0028] Figure 1: High-throughput chemical library screening identifies retinoids as promoters of mesenchymal-epithelial transition (A) Reporter cell lines for MET screening were generated using a small molecule compound library. (B) Dual-glo luciferase assay of NAMEC8-SERPINElprom-Luc cells treated with A83-01. Data are expressed as mean FF / Ren+ / -SEM, n=3, **P<0.01 by Student's t-test. (C) Dual-glo luciferase assay of NAMEC8-SERPINE1prom-Luc cells screened using LOPAC, anticancer drugs, kinase inhibitors, and bioactive lipid libraries. Data are expressed as mean FF / Ren, n=3. Hits are circled in red. (D) Scatter plot of primary and secondary screens with 61 compounds. FF / Ren values ​​from two screens were plotted and linear trend lines were calculated. (E) Dual-glo luciferase assays of 41 compounds were performed with a 5-point dose response. Compounds were divided into 9 categories based on physiological function. Data are presented as mean FF / Ren, n = 3. Grey boxes represent doses resulting in <50% cell viability by DMSO.

[0029] Figure 2: Retinoids induce basal-like breast cancer cell transformation into luminal and enhance their sensitivity to chemotherapy (A) Cell morphology images of NAMEC8 treated with DMSO, bexarotene (1 μM) or TTNPB (1 μM). Images were taken at 50× magnification. Scale represents 100 μm. (B) Expression of EMT and lineage marker genes in bexarotene-treated NAMEC8 cells relative to DMSO. Data are expressed as mean + / - SEM by Student's t-test, n=3, *P<0.05, **P<0.01, ***P<0.001. (C) Protein expression of EMT and lineage markers in DMSO-treated NAMEC cells. Bexarotene was treated with increasing concentrations of 0.1 μM, 0.5 μM and 1 μM. β-Actin was used as a loading control. (D) Immunofluorescence staining of EMT and lineage markers in DMSO-treated NAMEC8 cells relative to bexarotene. Images were taken at 100× magnification. Scale bar represents 50 μm. (E) Flow cytometry histogram of CD44 staining in DMSO vs. bexarotene-treated NAMEC8 cells. (F) Mammosphere formation assay of DMSO vs. bexarotene or TTNPB-treated NAMEC8 cells. Images were taken at 50× magnification. Scale bar represents 100 μm. Bottom right: Bar graph shows the number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, **P<0.01. (G) Protein expression of EMT and lineage markers in BC2.2, BC22.1, BC29.1, MetBr-007 DR, BC24.1, BC33.1: DMSO vs. bexarotene-treated cells. β-Actin was used as a loading control. (H) Immunofluorescence staining of EMT and lineage markers in BC2.2: DMSO vs. bexarotene-treated cells. Images were taken at 100× magnification. Scale bar represents 50 μm. (I) Mammosphere formation assay of BC2.2 cells treated with DMSO versus bexarotene. Images were taken at 50× magnification. Scale bar represents 100 μm. Right: Bar graph shows the number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, **P<0.01. (J) Cell morphology images of NAMEC8 treated with bexarotene in the presence or absence of 10 μM 5-FU. Images were taken at 50× magnification. (K) Dose response graph of NAMEC8 cells treated with DMSO versus bexarotene to different concentrations of 5-FU. IC50 values ​​are shown in the table below. Data are expressed as mean + / - SEM, n=3. (L) Schematic diagram of the generation process of SUM159PAC-R cells (top). Dose response graph of SUM159PT versus PACR cells to different PAC concentrations (bottom). IC50 values ​​are shown in the table. Data are presented as mean + / - SEM, n=3.(M) Dose response of SUM159PACR cells treated with DMSO versus bexarotene to different concentrations of 5-FU. IC50 values ​​are shown in the table. Data are presented as mean + / - SEM, n=3.

[0030] Figure 3: Retinoid treatment promotes MET and reduces the tumorigenicity of TNBC cells in animals (A) Tumor measurements of SUM159 pretreated with DMSO, bexarotene or TTNPB before subcutaneous injection into NSG mice (top). Data are expressed as mean diameter + / - SEM, n = 6 for DMSO, n = 8 tumors for bexarotene and TTNPB, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative images of tumors from mice sacrificed at 7 and 8 weeks after implantation (bottom). (B) Tumor size of MCF7-Ras pretreated with DMSO, bexarotene or ATRA before subcutaneous injection into NSG mice (top). Data are expressed as mean diameter + / - SEM, n = 4 for DMSO, n = 6 tumors for bexarotene and ATRA. Representative images of tumors from mice sacrificed at 9 weeks after implantation (bottom). (C) Schematic diagram of mouse xenograft experimental workflow (top). Tumor size from control mice compared to bexarotene-fed mice in NAMEC8-Ras (left), BC2.2 (middle), and BC22.1 (right) (bottom). Data are expressed as mean diameter + / - SEM, control n = 6, bexarotene (NAMEC8-Ras) n = 4, control and bexarotene (BC2.2 and BC22.1) n = 7, *P < 0.05, **P < 0.01. (D) Hematoxylin and eosin staining of NAMEC8-Ras tumor sections formed in control mice compared to bexarotene-fed mice. Images were taken at 200x magnification. Scale bar represents 50 μm. (E) Immunofluorescence staining of EMT and lineage markers in NAMEC8-Ras tumor sections in control mice compared to bexarotene-fed mice. Images were taken at 100x magnification. Scale bar represents 50 μm. (F) Gene expression of retinoic acid-responsive genes in NAMEC8-Ras (top) and BC2.2 (bottom) tumors formed in bexarotene-fed mice relative to tumors in control mice. Data are presented as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01.

[0031] Figure 4: Epigenetic perturbations enhance and increase the persistence of retinoid-induced changes in cell state (A) Dual-glo luciferase activity graph of NAMEC8-SERPINE1prom-Luc cells treated with epigenetic compounds. Data are expressed as mean FF / Ren, n=3. Hits are circled in red. (B) Dual-glo luciferase activity graph of NAMEC8-SERPINE1prom-Luc cells treated with bexarotene, plus a single epigenetic compound. Data are expressed as mean FF / Ren, n=3. Hits are circled in red. (C) Morphological images of NAMEC8 cells treated with DMSO, bexarotene, CI-994, SBHA, bexarotene + C1-994, or bexarotene + SBHA. Images were taken at 50x magnification. Scale bar represents 100 μm. (D) Protein expression of NAMEC8 treated with DMSO, bexarotene, CI-994, SBHA, bexarotene + CI-994, or bexarotene + SBHA. β-actin was used as a loading control. (E) Morphological images of NAMEC8 cells treated with DMSO, bexarotene + CI-994, bexarotene + SBHA (drug "on") and inactivated for 7-10 days (drug "off"). Images were taken at 50x magnification. Scale bar represents 100 μm. (F) Protein expression of NAMEC8 treated with DMSO, bexarotene, CI-994, bexarotene + CI-994, -bexarotene, and -(bexarotene + CI-994). β-actin was used as a loading control. (G) Immunofluorescence staining of KRT5 (top) and KRT8 (bottom) in BC2.2 treated with DMSO, bexarotene, CI-994, bexarotene + CI994, -bexarotene, -(bexarotene + CI994). Images were taken at 100x magnification. Scale bar represents 50 μm. (H) Mammosphere formation assay of NAMEC8 treated with DMSO, bexarotene, CI-994, bexarotene + CI-994, -bexarotene, -(bexarotene + CI-994). Images were taken at 50x magnification. Scale bar represents 100 μm. Graph shows the number of spheres >100 μm. Data are expressed as mean + / - SEM, n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001. (I) Transcriptional regulation of gene expression by retinoid receptors and epigenetic regulators. In the mesenchymal cell state, RAR and RXR often bind to corepressors such as HDACs, thereby inhibiting transcription of epithelial genes. Exposure to bexarotene results in activation of RAR and RXR, causing a change in protein interaction from corepressor to coactivator. HDAC inhibition helps enhance gene transcriptional activity by maintaining histone acetylation at gene promoters.

[0032] Figure 5: Lipid metabolism genes are regulated by retinoid receptors (A) Global heatmaps of ChIP-seq peaks using antibodies to RXRα, RARα, H3K27ac, and p300 in NAMEC8 treated with DMSO, ST (short-term; 7 days) bexarotene, or LT (long-term; 14 days) bexarotene. (B) Graph showing occupancy of RXRα, RARα, H3K27ac, and p300 in gene bodies after ST- and LT-bexarotene treatment in NAMEC8. (C) Gene tracks depict increased RXRα and H3K27ac binding at STRA6, DHRS3, PLIN1, PLIN2, and ACSL5 loci, and decreased RXRα and H3K27ac binding at MGLL loci in ST- and LT-bexarotene-treated NAMEC8. The x-axis shows chromosomal position, and the gene structure is shown below, with each scale unit representing 5 kb. The y-axis shows genome occupancy in rpm per bp. (D) Gene ontology pathway enrichment analysis of genes with enhanced RXRα binding in NAMEC8 after bexarotene treatment. (E) Super-enhancer plot ranked by enhanced H3K27ac signal in NAMEC8 after bexarotene treatment. (F) Heat map of lipid-related genes in NAMEC8, SUM159 cells, MetBr-007DR, BC29.1, and BC22.1 cells after bexarotene treatment. Data represent log2 fold changes. (G) Gene expression of lipid-related genes involved in TAG synthesis after bexarotene treatment relative to DMSO in NAMEC8 (top) and BC2.2 (bottom). Data are presented as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0033] Figure 6: Mesenchymal-epithelial transition limits β-oxidation by directing fatty acids to lipid droplet storage. (a) Relative lipid content measured by LC-MSMS in DMSO versus bexarotene treated NAMEC8 cells. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01. Genes upregulated after bexarotene treatment were validated by RT-PCR (red). (b) Lipid droplet staining in DMSO versus bexarotene treated NAMEC8, SUM159, and BC2.2. Images were taken at 200x magnification. Scale bar represents 50 μm. (c) Mammosphere formation assay in NAMEC8 cells under the following conditions. Treatment with bexarotene, FSG67, or FSG67 + bexarotene (left). Exposure of Ctrl sh or shAGPAT cells in the presence or absence of bexarotene (middle). Treatment in the presence of various combinations of bexarotene and Amidepsine A (right). Images were taken at 50x magnification. Scale bar represents 100 μm. (d) Oxygen consumption rate (OCR) of DMSO- versus bexarotene-treated NAMEC8 cells in the presence or absence of palmitate. Black arrows indicate the rate of fatty acid β-oxidation (FAO). (e) Relative lipid content measured by LC-MSMS in HMLE versus NAMEC8 cells. Data are expressed as mean + / - SEM, n = 3. Downregulated genes in NAMEC8 relative to HMLE verified by RT-PCR are in blue. (f) Lipid droplet staining in HMLE versus NAMEC8. Images were taken at 10x magnification. Scale bar represents 50 μm. Fatty acid β-oxidation (FAO) rates measured by Seahorse in a panel of breast cancer cell lines. Data are expressed as mean + / - SEM, n = 3. (g) Cell viability assay of BT549 and MCF7 cells grown for 6 days in basal medium (DMEM containing 1% FBS, 2.5 mM glucose), basal medium supplemented with 25 mM glucose, 20 μM palmitate or 20 μM lipoic acid. Data are presented as mean + / - SEM, n=3.

[0034] Figure 7: Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. a) Protein expression of NAMEC8 treated with bexarotene, etolimus, bexarotene + etolimus, -bexarotene, -(bexarotene + etolimus). β-Actin was used as a loading control. b) Mammosphere formation assay of NAMEC8 treated with bexarotene, etolimus, bexarotene + etolimus, -bexarotene, -(bexarotene + etolimus). Images were taken at 50x magnification. Scale bar represents 100 μm. The graph shows the number of spheres >100 μm. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, ***P < 0.001, ****P < 0.0001. c) Lipid droplet staining in NAMEC8Ctrlsh, CPT1A-sh1 and sh2 cells treated with DMSO or bexarotene. Images were taken at 100x magnification. The scale bar represents 50 μm. d) Protein expression of NAMEC8Ctrlsh and CPT1A-sh1 cells treated with DMSO or bexarotene. β-Actin was used as a loading control. e) Mammosphere formation assay of NAMEC8Ctrlsh, CPT1A-sh1, and sh2 cells treated with DMSO and bexarotene (left). Images were taken at 50x magnification. The scale bar represents 100 μm. Protein expression of CPT1A knockdown is shown (top right). The chart shows the number of spheres >100 μm (bottom right). Data are expressed as mean + / - SEM, n = 3, **P < 0.01, ***P < 0.001. f) Lipid droplet staining in NAMEC8 blank control, CPT1A-1- and CPT1A-2-overexpressing NAMEC8 cells were treated with DMSO or bexarotene. Images were taken at 200x magnification. The scale bar represents 50 μm. g) Protein expression of NAMEC8 blank control and CPT1A-1 overexpressing cells treated with DMSO or bexarotene. β-actin was used as a loading control. h) Mammosphere formation assay of NAMEC8 blank control, CPT1A-1- and CPT1A-2-overexpressing cells treated with DMSO and bexarotene (left). Images were taken at 50x magnification. Scale bar represents 100 μm. Protein expression of CPT1A overexpression is shown (upper right). The graph shows the number of spheres >100 μm (lower right). Data are expressed as mean + / - SEM, n = 3, ****P < 0.0001.

[0035] Figure 8: Impaired fatty acid oxidation redirects lipids to support the epithelial cell state and prevents EMT. a) Lipid droplet staining in HMLE-TwistER control and 4OHT-induced cells (top), HMLE-Zeb1 control and DOX-induced cells (middle), MCF7-Slug+Sox9 control and DOX-induced cells. Images were taken at 100x magnification. The scale bar represents 50 μm. b) Oxygen consumption rate (OCR) of HMLE-TwistER control and 4-OHT-induced cells in the presence or absence of palmitate (left). The black arrow indicates the ratio of FAO. The ratio of FAO in HMLE-TwistER control and 4OHT-induced cells (right). Data are expressed as mean + / - SEM, n = 3, *P < 0.05. c) Morphology of HMLE-Twist ER treated with etolimus in the presence or absence of 4-OHT. Images were taken at 50x magnification. The scale bar represents 100 μm. d) Protein expression of EMT markers in HMLE-Twist ER treated with edolimus in the presence or absence of 4-OHT. β-actin was used as a loading control. e) Lipid droplet staining in MCF7-Slug+Sox9Ctrlsh and shCPT1A cells + / -DOX induction. Images were taken at 100x magnification. The scale bar represents 50 μm. f) Protein expression in MCF7-Slug+Sox9Ctrlsh and shCPT1A cells + / -DOX induction. β-actin was used as a loading control. g) Schematic diagram of the workflow of mouse xenograft experiments (top). Lower left: Immunofluorescence staining of E-cadherin and vimentin in primary tumors formed in the control group (no DOX) and the DOX group and the edolimus+DOX group (200x magnification). The scale bar represents 50 μm. Whole-body fluorescent lung images (100x magnification) and histology of lung sections of lung metastases (10x magnification) are also shown. White scale bar represents 100 μm, black scale bar represents 50 μm. Bottom right: Graph shows the number of metastases per lung. Data are presented as mean + / - SEM, n = 5 technical replicates, **P < 0.01. h) Model illustrating the different fatty acid metabolism driving epithelial and mesenchymal cell states. In the mesenchymal cell state, β-oxidation is high and lipid storage is minimal. After induction of MET, the epithelial cell state is characterized by suppressed β-oxidation and accumulation of lipid storage.

[0036] Fig. 9 (A) Z-scores of Dual-glo luciferase assays in NAMEC8-SERPINElprom-Luc cells treated with LOPAC (upper left), anticancer drugs (upper right), kinase inhibitors (lower left), and bioactive lipids (lower). Data are presented as mean values, n=3.

[0037] Figure 10. (A) Cell morphology images of SUM159 treated with DMSO, bexarotene (1 μM) or TTNPB (1 μM). Images were taken at 50 times magnification. Scale bar represents 100 μm. (B) Gene expression of EMT and lineage marker genes in SUM159 cells treated with bexarotene relative to DMSO treatment. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (C) Protein expression of EMT and lineage markers in SUM159 cells treated with DMSO compared to bexarotene. β-Actin was used as a loading control. (D) Flow cytometric analysis of CD24 and CD44 expression in SUM159 cells treated with DMSO compared to bexarotene. (E) Mammosphere formation assay of SUM159 cells treated with DMSO, bexarotene or TTNPB. Images were taken at 50 times magnification. The scale bar represents 100 μm. The bar graph shows the number of spheres of >100 μm. The data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01. (F) Gene expression of EMT and lineage marker genes of BC2.2, BC22.1 and BC29.1 cells treated with DMSO by bexarotene. The data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (G) Immunofluorescence staining of EMT and lineage markers in BC22.1 (left) and BC29.1 (right) treated with DMSO or bexarotene. Images were taken at 100 times magnification. The scale bar represents 100 μm. (H) Analysis of mammosphere formation of BC22.1 (top) and BC29.1 (bottom) treated with DMSO compared to bexarotene. Images were taken at 50 times magnification. Scale bar represents 100 μm. Bar graph shows the number of spheres >100 μm. Data are expressed as mean + / - SEM, n = 3, *P < 0.05. (I) Luciferase assay of estrogen receptor element (ERE) reporter gene activity in NAMEC8 cells treated with increasing doses of bexarotene or 1 μM TTNPB. Data are expressed as mean FF / Ren + / - SEM, n = 3, *P < 0.05. (J) Gene expression of estrogen receptor (ER) target genes in NAMEC8 and BC2.2 treated with bexarotene. Data are expressed as mean + / - SEM, n = 3, *P < 0.05. (K) Dose response graph of NAMEC8 treated with DMSO and bexarotene to different concentrations of raloxifene hydrochloride. IC50 values ​​are shown in the table. (L) Gene expression of EMT and lineage marker genes in BC24.1 (left) and BC33.1 (right) in bexarotene treatment relative to DMSO. Data are presented as mean + / - SEM, n = 3. (M) Knockdown of RXRα in NAMEC8 using Ctrl shRNA or RXRα shRNA.β-Actin was used as a loading control. (N) Gene expression of EMT and lineage genes in bexarotene-treated NAMEC8 Ctrl shRNA and RXRαsh1 cells relative to DMSO. Data are expressed as mean + / - SEM, n = 3, *P < 0.05. (O) Protein expression of EMT and lineage markers in NAMEC8 Ctrl shRNA and RXRαsh1 treated with DMSO or bexarotene. β-Actin was used as a loading control. (P) Dose response of DMSO versus bexarotene-treated SUM159 (middle), BC2.2 (right) cells to different concentrations of 5-FU. IC50 values ​​are shown in the table below. Data are expressed as mean + / - SEM, n = 3. (Q) Cell morphology images of NAMEC8 treated with bexarotene in the presence or absence of 2 nM paclitaxel. Images were taken at 50x magnification. (R) Dose response of DMSO and bexarotene-treated NAMEC8 to different concentrations of PAC. IC50 values ​​are shown in the table. Data are expressed as mean + / - SEM, n = 3 (S) Morphology of SUM159 PACR cells treated with DMSO and bexarotene. Images were taken at 50 times magnification. The scale bar represents 100 μm. (T) Gene expression of EMT and lineage marker genes in SUM159 PACR cells treated with bexarotene relative to DMSO. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (U) Protein expression of EMT and lineage markers in SUM159 PACR treated with DMSO and bexarotene. β-Actin was used as a loading control. (V) Mammosphere formation assay of SUM159 PACR cells treated with DMSO or bexarotene. Images were taken at 50 times magnification. The scale bar represents 100 μm. The bar graph shows the number of spheres > 100 μm. Data are presented as mean + / - SEM, n = 3, **P < 0.01.

[0038] Figure 11. (A) Tumor weight of SUM159 pretreated with DMSO, bexarotene or TTNPB at weeks 7 and 8 (top). Tumor volume of SUM159 pretreated with DMSO, bexarotene or TTNPB at weeks 7 and 8 (bottom). Data are expressed as mean + / - SEM, n = 6. (B) Tumor weight (top) and tumor volume (bottom) of MCF7-Ras pretreated with DMSO, bexarotene or ATRA. Data are expressed as mean + / - SEM, n = 6. (C) Cell viability (measured by MTS analysis) of NAMEC8 (left), MCF7 (middle) and MCF10A (right) treated with DMSO, bexarotene or ATRA over 6 days. Data represent mean + / - SEM, n = 3. (D) Images of NAMEC8-Ras, BC2.2 and BC22.1 tumors formed in control mice and bexarotene-fed mice. (E) Tumor weights of NAMEC8-Ras, BC2.2, and BC22.1 tumors formed in control mice and bexarotene-fed mice (top). Tumor volume of NAMEC8-Ras, BC2.2, and BC22.1 tumors formed in control mice and bexarotene-fed mice (bottom). Data are expressed as mean + / - SEM, n = 6. (F) Immunofluorescence staining of EMT and lineage markers in BC2.2 tumor sections of control mice and bexarotene-fed mice. Images were taken at 200x magnification. Scale bar represents 50 μm.

[0039] Figure 12. (A) Gene expression of EMT and lineage marker genes in NAMEC8 (left) and SUM159 (right) treated with bexarotene, CI-994 or bexarotene + CI-994 relative to DMSO. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (B) Dual-glo luciferase assay of NAMEC8-SERPINE1prom-Luc cells, first treated with DMSO, bexarotene, and then the compound was deactivated for 24 hours, 48 ​​hours, or 72 hours (drug "off"). Data are expressed as mean + / - SEM, n = 6, **P < 0.01, ***P < 0.001. (C) Dual-glo luciferase assay of NAMEC8-SERPINE1prom-Luc cells, first treated with DMSO, bexarotene + CI-994, and then withholding the compounds for 24 h, 48 h, or 72 h (drug “off”). Data are expressed as mean + / - SEM, n = 6, ****P < 0.0001. (D) Mammosphere formation assay of NAMEC8 treated with DMSO, bexarotene, SBHA, bexarotene + SBHA, -bexarotene, -(bexarotene + SBHA). Images were taken at 50x magnification. Scale bar represents 100 μm. The graph shows the number of spheres > 100 μm. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (E) Mammosphere formation assay of SUM159 (top) and BC2.2 (bottom) treated with bexarotene, CI-994, bexarotene + CI-994, -bexarotene or -(bexarotene + CI-994). Images were taken at 50x magnification. Scale bar represents 100 μm. Graph shows the number of spheres >100 μm. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0040] Figure 13. (A) Global heatmap of ChIP-Seq peaks using antibodies to RXRα, RARα, H3K27ac, and p300 in SUM159 treated with DMSO, ST (short-term; 7 days) bexarotene, or LT (long-term; 14 days) bexarotene. (B) Plot showing occupancy of RXRα, RARα, H3K27ac, and p300 in gene bodies in ST- and LT-bexarotene-treated SUM159 cells. (C) Gene tracks depict increased RXRα and H3K27ac binding at the STRA6, DHRS3, PLIN1, PLIN2, and ACSL5 loci, and decreased RXRα and H3K27ac binding at the MGLL locus in ST- and LT-bexarotene-treated SUM159 cells. The x-axis shows chromosomal position, and the gene structure is shown in the figure below, with each scale unit representing 5 kb. The y-axis shows genomic occupancy in rpm per bp. (D) Gene ontology pathway enrichment analysis of genes from RNA-Seq with log2 fold change >1 in SUM159 after LT bexarotene treatment. (E) Gene ontology pathway enrichment analysis of genes from RNA-Seq with log2 fold change >1 in MetBr-007-DR after bexarotene treatment. (F) Super enhancer map ranked by increased H3K27ac signal in SUM159 treated with bexarotene. (G) Gene expression of lipid-related genes involved in TAG synthesis in NAMEC8 after TTNPB treatment compared to DMSO. Data are presented as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01.

[0041] Figure 14: Mesenchymal-epithelial transition limits β-oxidation by directing fatty acids to lipid droplet storage. a) Lipid droplet staining of BC29.1 and BC22.1: DMSO vs. bexarotene treatment. Images were taken at 200x magnification. Scale bar represents 50 μm. b) Lipid droplet staining in NAMEC8 treated under the following conditions: treatment with bexarotene, FSG67, or FSG67+bexarotene (left); Ctrl sh or shAGPAT cells exposed to the presence or absence of bexarotene (middle); treated in the presence of various combinations of bexarotene and AmidepsineA (right). Images were taken at 100x magnification. Scale bar represents 50 μm. c) Protein expression of epithelial and luminal markers in NAMEC8 cells under the following conditions: treatment with bexarotene, FSG67, or FSG67+bexarotene (left); Ctrl sh or shAGPAT cells exposed to the presence or absence of bexarotene (middle); treated in the presence of various combinations of bexarotene and AmidepsineA (right). β-Actin was used as a loading control. d) Graph showing the number of spheres >100 μm in the mammosphere formation assay in NAMEC8 cells under the following conditions: treatment with bexarotene, FSG67, or FSG67+bexarotene (left); Ctrl sh or shAGPAT cells exposed to the presence or absence of bexarotene (middle); treated in the presence of various combinations of bexarotene and AmidepsineA (right). Images were taken at 50x magnification. Scale bar represents 100 μm. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. e) Oxygen consumption rate (OCR) of SUM159 (left) and BC2.2 (right): DMSO vs. bexarotene, in the presence or absence of palmitate. The black arrow indicates the rate of fatty acid β-oxidation (FAO). f) Graph showing FAO ratios in NAMEC8 (left), SUM159 (middle), and BC2.2 (right): DMSO vs. bexarotene treatment. Data are expressed as mean + / - SEM, n = 3, **P < 0.01, ***P < 0.001. g) Gene expression of lipid-related genes involved in TAG synthesis in NAMEC8 relative to HMLE. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0042] Figure 15: Induction of mesenchymal-epithelial transition depends on the fatty acid modifier carnitine palmitoyltransferase. a) The graph shows the ratio of FAO in SUM159 treated with bexarotene, etolimus, and bexarotene + etolimus. Data are expressed as mean + / - SEM, n = 3, **P < 0.01, ***P < 0.001. b) Morphology of NAMEC8 treated with bexarotene, etolimus, bexarotene + etolimus, -bexarotene or -(bexarotene + etolimus). Images were taken at 100x magnification. The scale bar represents 50 μm. c) Gene expression of EMT and retinoid response genes in NAMEC8 treated with bexarotene, etolimus, or bexarotene + etolimus. Data are expressed as mean + / - SEM, n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001. d) Mammosphere formation assay of SUM159 treated with bexarotene, edolimus, bexarotene + edolimus, -bexarotene or -(bexarotene + edolimus). Images were taken at 50x magnification. Scale bar represents 100 μm. The graph shows the number of spheres >100 μm. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01. e) The graph shows the FAO ratio in vehicle or CPT1A overexpressing SUM159 cells treated with DMSO or bexarotene. Data are expressed as mean + / - SEM, n = 3, **P < 0.01. f) Lipid droplet staining of SUM159 vector, CPT1A-1- and CPT1A-2 overexpressing cells treated with DMSO or bexarotene. Images were taken at 200x magnification. Scale represents 50 μm. g) Morphology of NAMEC8 vector and CPT1A overexpressing cells treated with DMSO or bexarotene. Images were taken at 40x magnification. Scale bar represents 50 μm. h) Mammosphere formation assay of SUM159 vector, CPT1A-1 and CPT1A-2 overexpressing cells treated with DMSO and bexarotene (left). Images were taken at 50x magnification. Scale bar represents 100 μm. Protein expression of CPT1A overexpression is shown (top right). The graph shows the number of spheres >100 μm (bottom right). Data are expressed as mean + / - SEM, n = 3, ****P < 0.0001.

[0043] Figure 16: Impairment of fatty acid oxidation redirects lipids to support epithelial cell states and blocks EMT. (a) Gene expression of EMT genes in HMLE-Twist ER treated with etolimus in the presence or absence of 4-OHT compared to control. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (b) Gene expression of EMT genes in MCF7-Slug + Sox9 treated with etolimus in the presence or absence of doxycycline induction relative to control. Data are expressed as mean + / - SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (c) Protein expression of EMT markers in MCF7-Slug + Sox9 treated with etolimus in the presence or absence of doxycycline induction. β-actin was used as a loading control. (d) Cell morphology images of HMLE-Zeb1 treated with edolimus in the presence or absence of doxycycline induction relative to the control. Images were taken at 40x magnification. The scale bar represents 50 μm. (e) Gene expression of EMT genes in MCF7-Slug+Sox9Ctrlsh and shCPT1A cells + / -DOX induction relative to Ctrlsh cells. Data are expressed as mean + / -SEM, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (f) Cell morphology images of A549 treated with edolimus in the presence or absence of TGFβ relative to the control. Images were taken at 40x magnification. The scale bar represents 50 μm. (g) Protein expression of EMT markers in A549 treated with edolimus in the presence or absence of TGFβ. β-actin was used as a loading control.

[0044] Figure 17: Epigenetic library screening to identify effective MET inducers. (A) Dual-glo luciferase assay of NAMEC8-SERPINE1 cells screened with an epigenetic library to identify MET inducers. Data are presented as SERPINE1 promoter activity (average FF / Ren value) against Renilla cell viability, n=3. A83-01 (black circles) served as a positive control, and compounds with lower SERPINE1 promoter activity than A83-01 were identified as hits (red circles). (B) Dual-glo luciferase assay of MET screening results performed with a 6-point dose response. Data are presented as SERPINE1 promoter activity (average FF / Ren value) versus concentration, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. (C) Cell morphology images of NAMEC8 treated with DMSO and epigenetic compounds. LLY-283 and GSK591 were used at 20 nM, UNC0642 and SGC-CBP30 were used at 1 μM. Only SGC-CBP30 treatment resulted in epithelial-like morphology. Images were taken at 4x magnification. Scale bar represents 100 μm. (D) qRT-PCR analysis of EMT and basal / luminal genes of NAMEC8 treated with epigenetic hits. mRNA levels relative to DMSO are shown. Data are expressed as mean ± SEM, n = 3. (E) Immunoblot analysis of EMT and basal / luminal markers in NAMEC8 after epigenetic hit treatment. SGC-CBP30 showed the most dramatic changes compared to other compounds. β-Actin was used as a loading control. (F) Left: Mammosphere formation analysis of NAMEC8 after treatment with epigenetic hits. A significant reduction in mammosphere formation and sphere size was observed in the three tested compounds. Images were taken at 4x magnification. Scale bar represents 100 μm. Right: Bar graph represents the number of tumor spheres >50 μm. Data are expressed as mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ns, not significant. (G) Flow cytometric analysis of CD44 and CD24 staining in NAMEC8, DMSO and SGC-CBP30 treated cells. A cell population with reduced CD44 expression was observed after SGC-CBP30 treatment, indicating the induction of a non-stem cell-like state.

[0045] FIG18 : CREBBP / EP300 bromodomain inhibition induces MET in the SUM159 TNBC cell line. (A) Cell morphology images of SUM159 treated with DMSO and epigenetic compounds. LLY-283 and GSK591 were used at 30 nM, and UNC0642 and SGC-CBP30 were used at 2 μM. Images were taken at 4x magnification. Scale bar represents 100 μm. (B) Immunoblot analysis of EMT and basal / luminal markers in SUM159 after epigenetic hit treatment. β-Actin was used as a loading control. (C) Top: Mammosphere formation analysis of SUM159 after epigenetic hit treatment. A significant reduction in mammosphere formation and sphere size was observed in SGC-CBP30 treated cells. Images were taken at 4x magnification. Scale bar represents 100 μm. Bottom: Bar graph represents the number of tumor spheres >50 μm. Data are presented as mean ± SEM, n=3, *p<0.05. (D) Flow cytometric analysis of CD44 and CD24 staining in SUM159, DMSO and SGC-CBP30 treated cells. Cells expressed reduced CD44 expression and higher CD24 expression, indicating the induction of a non-stem cell-like state.

[0046] FIG19 : CREBBP / EP300 bromodomain inhibition reduces tumor burden of basal-like TNBC in vivo. (A) Cell morphology images of NAMEC8 treated with 1 μM A-485. Epithelial-like morphology was observed after A-485 treatment. Images were taken at 4× magnification. Scale bar represents 100 μm. (B) Immunoblot analysis of EMT and basal / luminal markers in NAMEC8 after treatment with A-485. β-Actin was used as a loading control. (C) Top: Mammosphere formation analysis of NAMEC8 after treatment with A-485. A reduction in sphere formation and sphere size was observed. Images were taken at 4× magnification. Scale bar represents 100 μm. Bottom: Bar graph represents the number of tumor spheres >50 μm. Data are expressed as mean ± SEM, n=3, ns, not significant. (D) Flow cytometric analysis of CD44 and CD24 staining in NAMEC8, DMSO, and A-485 treated cells. Cells expressed higher CD44 expression after treatment, indicating that A-485 failed to induce loss of stem cell-like characteristics. (E) Immunoblot analysis of histone acetylation marks in NAMEC8 after treatment with SGC-CBP30 and A-485. H3 and β-actin were used as loading controls. (F) Top: Tumor size of NAMEC8-Ras pretreated with DMSO, SGC-CBP30, and A-485 before subcutaneous injection into NSG mice. Data are presented as mean ± SEM, n = 3, *p < 0.05, ns, not significant. Bottom: Tumor images from mice sacrificed at week 11. SGC-CBP30 treatment significantly reduced tumor formation in mice. (G) Working model showing CREBBP / EP300-induced TNBC cell state transition after bromodomain inhibition. In the absence of bromodomain inhibitors, CREBBP and EP300 can recognize acetylated lysines on histones to mediate mesenchymal gene activation, thereby maintaining cells in a mesenchymal state. However, the presence of bromodomain inhibitors blocks the read-through function of CREBBP and EP300 proteins, thereby reducing the transcriptional regulation of genes required to maintain the mesenchymal cell state.

[0047] Figure 20: Metabolic library screening for identifying potent MET inducers. (A) Dual-glo luciferase assay of NAMEC8-SERPINE1 cells using metabolic library screening to identify MET inducers. Data are presented as SERPINE1 promoter activity (mean FF / Ren value) against Renilla cell viability, n=3. Compounds identified as hits are circled in red. (B) Compounds identified as hits are summarized in the table, and their respective molecular targets are listed in the right column. (C) Dual-glo luciferase assay of MET screening results performed under a 5-point dose response. Data are presented as SERPINE1 promoter activity (mean FF / Ren value) versus concentration, with C-IN-1 glutaminase inhibitor showing a dose-response effect in reducing SERPINE1 promoter activity. (D) Western blot showing GLS1 expression in epithelial and mesenchymal breast cancer cell lines. (E) Cell morphology images of NAMEC8 treated with 500 nM C-IN-1. (F) Immunoblot analysis of EMT and basal / luminal markers in NAMEC8 after treatment with MET hits. (G) Western blot display of knockdown of GLS1 in NAMEC8 using 3 shRNAs. (H) Luminescence titer assay of NAMEC8 ctrlsh vs. GLS1sh2 and GLS1sh3 cells treated with paclitaxel. IC50 of GLSsh2 cells was lower than that of Ctrlsh cells. (I) Western blot of GLS1 knockdown in NAMEC8Hras cells with sh2 and sh3. (J) Mammosphere assay of NAMEC8HrasCtrlshvsGLS1sh2vsGLS1sh3 cells. Spheres >100um were counted after 7 days. (K) NAMEC8HrasCtrlsh and GLS1sh2 cells were injected subcutaneously into NSG mice and tumor growth was followed over 8 weeks.

[0048] Detailed Description

[0049] Mesenchymal-epithelial transition inducer

[0050] Provided herein are methods for inducing mesenchymal-epithelial transition (MET) in cancer cells. In one aspect, a method for inducing MET in cancer cells is provided, the method comprising contacting the cells with a mesenchymal-epithelial transition inducer for a time and under conditions sufficient to induce MET in the cells, wherein the cancer cell is a basal-like (or mesenchymal-like) cancer cell.

[0051] Cell state transitions can control the functional behavior of cancer cells, controlling their tumorigenicity and response to therapy. Epithelial-mesenchymal transition (EMT) can confer cancer stem cell-like properties and enhance the tumorigenic potential and drug resistance of tumor cells. EMT may be highly activated in TNBC, leading to relapse and chemotherapy resistance. The inventors have discovered that mesenchymal-epithelial transition (MET), the reverse process of EMT, can suppress these phenotypes and sensitize tumors to various standard of care therapies including chemotherapy and immunotherapy. Specifically, the inventors have developed: (i) a technology platform that can be used to identify drugs, chemical probes, and drug targets that can improve cancer response; (ii) novel combination therapies that enhance anti-tumor response; (iii) new drug targets whose activation or inhibition can reverse drug resistance, sensitize tumors to various standard of care therapies including chemotherapy and immunotherapy, and inhibit tumor growth.

[0052] In one embodiment, a method for inhibiting cancer cell growth is provided, the method comprising contacting a cell with a mesenchymal-epithelial transition inducer for a time and under conditions sufficient to inhibit the growth of the cancer cell, wherein the cancer cell is a basal-like (or mesenchymal-like) cancer cell.

[0053] The terms "epithelial" or "epithelial-like" are understood in the art and can be identified by morphological, molecular and / or functional characteristics. For example, compared to mesenchymal cells, epithelial cells or epithelial-like cells generally have a round or cobblestone appearance, express the epithelial marker E-cadherin, divide rapidly and / or have relatively low levels of motility, invasiveness and / or anchorage-dependent growth.

[0054] The terms "mesenchymal" or "mesenchymal-like" are understood in the art and can be identified by morphological, molecular and / or functional characteristics. For example, compared to epithelial cells, mesenchymal or mesenchymal-like cells generally have an elongated or spindle-shaped appearance, express mesenchymal markers vimentin, fibronectin and N-cadherin, divide slowly or not, and / or have relatively high levels of motility, invasiveness and / or anchorage-independent growth.

[0055] The term "epithelial-mesenchymal transition" ("EMT") is known in the art and refers to the process by which epithelial cancer cells assume a mesenchymal phenotype, which is believed to be associated with cancer metastasis. In addition, while not wishing to be bound by any theory, these mesenchymal cells generally exhibit reduced adhesion, increased motility and invasiveness, and are relatively resistant to chemotherapeutic agents and / or radiation (e.g., treatments for rapidly dividing cells).

[0056] The term "mesenchymal-epithelial transition" ("MET") is also known in the art and refers to the reprogramming of cells undergoing EMT to regain one or more epithelial characteristics (e.g., as described above). For example, such cells typically exhibit reduced motility and / or invasiveness and / or rapid division, thereby potentially regaining sensitivity to cytotoxic agents.

[0057] In one embodiment, the method may be an in vitro, in vivo or ex vivo method.

[0058] The MET inducer may be a compound selected from the group consisting of a TGFβ inhibitor, adenosine / adrenergic, GABA or NMDA receptor modulator, neurotransmitter, epigenetic modification compound.

[0059] In one embodiment, the MET inducer is selected from the group consisting of: amotinib (MP-470), Evista (raloxifene hydrochloride), isotretinoin, SB216763, tretinoin (Aberela), 1,3-dipropyl-8-p-sulfophenylxanthine, 5-bromo-2'-deoxyuridine, alprenolol hydrochloride, amiodarone hydrochloride, apomorphine hydrochloride hemihydrate, methylatropine nitrate, cantharidic acid, L-798106, L-aspartic acid, Rhodblock 6. TNP, Bexarotene, PD-161570, SB-525334, 13-cis-retinoic acid, AC-55649, Raloxifene hydrochloride, Retinoic acid, Tazarotene, TTNPB, U0126, Amotinib (MP-470), AG-490, Abitrexate (methotrexate), Roscovitin (Seliciclib, CYC202), PIK-294, U0126-EtOH, PH-797804, Arry-380, Motisanib diphosphate (AMG-706), CH5132799, PIK-93, KN-62, 16,16-dimethyl prostaglandin D2, SB431542, prostaglandin D2 ethanolamide, 6-keto prostaglandin E1, Prostratin, CAY10587, 15-deoxy-12,14-prostaglandin D2, UCM707, D4476, 16-phenoxybutynol prostaglandin A2, KN-93, prostaglandin D2-1-glycerylester, C-8 ceramide, S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (hydrochloride), LLY-283, GSK591, UNC0642, SGC-CBP30, tanshinone I, heme, autophinib, β-lapachone, Vps34-PIK-III, glutaminase C-IN-1, GSK256066, AN-2728, GSK2837808A, acitretin, fenretinide, T0901317, AGI-6780, SW033291 and NCT-503.

[0060] In one embodiment, the MET inducing agent is a retinoid. The retinoid can be, for example, dehydroretinol, didehydroretinol, tretinoin (retinoic acid), isotretinoin, alitretinoin, acetyl ester, acetyl ester, adapalene, bexarotene, 13-cis-retinoic acid, tazarotene, TTNPB, acetyl ester or fenretinide.

[0061] In one embodiment, the retinoid is a retinoic acid receptor alpha (RAR-α) agonist or a retinoic acid X receptor alpha (RXR-α) agonist. The RAR-α agonist may be TTNPB, IRX5183, SY-1425, acitretin or fenretinide. The RXR-α agonist may be bexarotene.

[0062] The MET inducer can be a regulator (e.g., inhibitor or inducer) of a metabolic enzyme or pathway. For example, a regulator of a metabolic enzyme or pathway can be a glutaminase inhibitor, a phosphodiesterase inhibitor, an autophagy regulator, a phospholipase inhibitor, a topoisomerase, a PI3K, a lactate dehydrogenase, 15-PGDH, or a phosphoglycerate dehydrogenase.

[0063] In one embodiment, the MET inducer is a glutaminase inhibitor. The glutaminase inhibitor can be glutaminase C-IN-1, CB839 (Telaglenastat), UPGL00004 or BPTES. The glutaminase inhibitor can be a glutaminase 1 (GLS1) inhibitor. The glutaminase inhibitor can be a glutaminase C inhibitor or a kidney-type glutaminase (KGA) inhibitor.

[0064] In one embodiment, the MET inducer is a phosphodiesterase (PDE) inhibitor. In one embodiment, the PDE inhibitor is a PDE4 inhibitor. In one embodiment, the PDE inhibitor is GSK256066 or AN-2728.

[0065] In one embodiment, the MET inducer is a regulator (eg, inhibitor or inducer) of autophagy. The autophagy regulator can be heme, octofenib, β-lapachone, Vps34-PIK-III, or fenretinide.

[0066] The MET inducer can be a phospholipase inhibitor (e.g., Tanshinone I), topoisomerase (e.g., β-lapachone), PI3K (Vps34-PIK-III, Idelalisib, Copanlisib, Duvelisib, Alpelisib), lactate dehydrogenase (e.g., GSK28378158A), 15-PGDH (e.g., SW033291), or phosphoglycerate dehydrogenase (e.g., NCT-503).

[0067] The MET inducer may be an epigenetic modification compound. The epigenetic modification compound may be an inhibitor of p300 / CREBBP bromodomain, protein arginine methyltransferase 5 (PRMT5) or a histone methyltransferase.

[0068] In one embodiment, the MET inducer is a p300 / CREBBP bromodomain inhibitor. The p300 / CREBBP bromodomain inhibitor may be SGC-CBP30, GNE-272 or GNE-781.

[0069] In one embodiment, the MET inducer is a PRMT5 inhibitor. The inhibitor of PRMT5 can be LLY-283 or GSK591.

[0070] In one embodiment, the MET inducer may be an inhibitor of a histone methyltransferase (eg, G9a and / or GLP). In one embodiment, the histone methyltransferase inhibitor is UNC0642.

[0071] Epithelial-mesenchymal transition inhibitors

[0072] MET inducers can also be inhibitors of EMT. In one embodiment, the MET inhibitor is a compound selected from TGFβ inhibitors, adenosine / adrenergic, GABA or NMDA receptor modulators, neurotransmitters, epigenetic modification compounds. In one embodiment, the EMT inhibitor is retinoids.

[0073] In one embodiment, the EMT inhibitor is selected from the group consisting of: amotinib (MP-470), Evista (raloxifene hydrochloride), isotretinoin, SB 216763, tretinoin (Aberela), 1,3-dipropyl-8-p-sulfophenylxanthine, 5-bromo-2'-deoxyuridine, alprenolol hydrochloride, amiodarone hydrochloride, apomorphine hydrochloride hemihydrate, methylatropine nitrate, cantharidic acid, L-798106, L-aspartic acid, Rhodblock 6. TNP, bexarotene, PD-161570, SB-525334, 13-cis-retinoic acid, AC-55649, raloxifene hydrochloride, retinoic acid, tazarotene, TTNPB, U0126, amotinib (MP-470), AG-490, abitrexate (methotrexate), roscovitine (Seliciclib, CYC202), PIK-294, U0126-EtOH, PH-797804, Arry-380, motisanib diphosphate (AMG-706), CH5132799, PIK-93, KN-62, 16,16-dimethyl prostaglandin D2, SB 431542, prostaglandin D2 ethanolamide, 6-ketoprostaglandin E1, prostaglandin, CAY10587, 15-deoxy-_12,14-prostaglandin D2, UCM707, D 4476, 16-phenoxybutyrrolidone prostaglandin A2, KN-93, prostaglandin D2-1-glyceride, C-8 ceramide and S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (hydrochloride).

[0074] In one embodiment, the EMT inhibitor is an inhibitor or modulator of lipid metabolism (or an inhibitor of fatty acid oxidation).

[0075] The lipid metabolism inhibitor may be an inhibitor of carnitine palmitoyltransferase I (CPT1). In one embodiment, the inhibitor of CPT1 is etolimus, perhexiline, teglicar or hydroxyphenylglycine. In one embodiment, the inhibitor of CPT1 is etolimus.

[0076] The lipid metabolism inhibitor may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0077] The lipid metabolism inhibitor may be an inhibitor of long chain fatty acid-CoA ligase (ACSL). In one embodiment, the inhibitor of ACSL is Triacsin C or thiazolidinedione (TZD).

[0078] The lipid metabolism inhibitor may be an inhibitor of 3-ketoacyl-CoA thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0079] The lipid metabolism inhibitor may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0080] The cancer cells may be basal-like breast cancer or TNBC, but not luminal / ER+ breast cancer. In one embodiment, the cancer cells are selected from triple-negative breast cancer and mesenchymal-like cancers, such as head and neck cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, and colorectal cancer.

[0081] The cancer cell may be a chemotherapy-resistant cancer cell. In other words, the cancer cell may be a cell that responds poorly or substantially unresponsive to anticancer therapy. In one embodiment, the cancer cell is a cell that is resistant to standard of care therapy.

[0082] The cancer cells may also be metastatic cancer cells.

[0083] Drug combinations

[0084] More effective and durable cancer control may be achieved through a combination therapy approach involving retinoids and epigenetic modulators. The inventors have shown that several epigenetic modulators can also induce MET by themselves. Their combination with retinoids can further enhance and maintain the MET transformation phenotype.

[0085] Transformation of mesenchymal cells to an epithelial phenotype via retinoid-induced MET may also sensitize cells to immuno-oncology agents such as immune checkpoint inhibitors. Retinoids may have immunomodulatory effects by enhancing T helper and T regulatory cell populations, increasing type I and II interferon signaling, interferon gamma signaling, increasing cytokine-mediated signaling, and activating the complement cascade (Tables 4A and 4B).

[0086] In one embodiment, the method further comprises: after inducing MET in cancer cells, contacting the cells with a chemotherapeutic agent (e.g., paclitaxel or 5-fluorouracil), an epigenetic modification compound (e.g., a class I HDAC inhibitor (e.g., C1-994 and SBHA)), or an immunomodulator. The method may include contacting the cells with an inducer of mesenchymal epithelial transition simultaneously or sequentially.

[0087] In one embodiment, the method further comprises contacting the cell with a chemotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, 5-fluorouracil, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibrutinib, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.

[0088] In one embodiment, the method further comprises contacting the cell with an epigenetic modification compound. The epigenetic modification compound can be an HDAC inhibitor. The HDAC inhibitor can be a Class I HDAC inhibitor. The HDAC inhibitor can be selected from hydroxamic acid vorinostat (SAHA), belinostat (PXD101), LAQ824 and panobinostat (LBH589); and benzamides: entinostat (MS-275), tekdinostat (CI994) and mocetinostat (MGCD0103).

[0089] In one embodiment, the method further comprises: contacting the cell with an immunomodulatory agent. In one embodiment, the immunomodulatory agent is an immune checkpoint inhibitor.

[0090] As used herein, the term "immune checkpoints" refers to a group of molecules on the cell surface of CD4 and CD8 T cells. These molecules effectively act as "brakes" to downregulate or inhibit anti-tumor immune responses. Immune checkpoint molecules include, but are not limited to, programmed death ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, B7H1, B7H4, OX-40, CD137, CD40, 2B4, IDO1, IDO2, VISTA, CD27, CD28, PD-L2 (B7-DC, CD273), LAG3, CD80, CD86, PDL2, B7H3, HVEM, BTLA, KIR, GAL9, TIM3, A2aR, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), ICOS (inducible T cell co-stimulator), HAVCR2, CD276, VT70CN1 and CD160.

[0091] As used herein, "immune checkpoint inhibitors" refers to any modulator that inhibits the activity of immune checkpoint molecules. Immune checkpoint inhibitors include: small molecule inhibitors, antibodies, antibody derivatives (including Fab fragments and scFv), antibody-drug conjugates, antisense oligonucleotides, siRNA, aptamers, peptides and peptide mimetics. Inhibitory nucleic acids that reduce the expression and / or activity of immune checkpoint molecules can also be used in the methods disclosed herein. One embodiment is a small inhibitory RNA (siRNA) for interfering with or inhibiting the expression of a target gene.

[0092] In one embodiment, the MET inducer is co-administered with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD 70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T-cell co-stimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1 or inhibitors of any combination thereof.

[0093] In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-L1. In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-1. In one embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In one embodiment, the immune checkpoint inhibitor is an inhibitor of LAG3. In one embodiment, the immune checkpoint inhibitor is an inhibitor of TIM3.

[0094] The immunomodulator can also be a cell therapy agent, such as a T cell (eg, a CarT cell), a natural killer (NK) cell, or a dendritic cell.

[0095] In one embodiment, a method for inducing MET in cancer cells is provided, the method comprising: contacting the cells with a mesenchymal-epithelial transition inducer and a chemotherapeutic agent for a time and under conditions sufficient to induce MET in the cells, wherein the cancer cells are basal-like (or mesenchymal-like) cancer cells. The mesenchymal-epithelial transition inducer can be a retinoid, such as bexarotene. The chemotherapeutic agent can be paclitaxel or 5-fluorouracil.

[0096] In one embodiment, a method for inducing MET in cancer cells is provided, the method comprising: contacting the cells with a mesenchymal-epithelial transition inducer and an epigenetic modification compound for a time and under conditions sufficient to induce MET in the cells, wherein the cancer cells are basal-like (or mesenchymal-like) cancer cells. The mesenchymal-epithelial transition inducer can be a retinoid, such as bexarotene. The epigenetic modifier can be a class I HDAC inhibitor, such as C1-994 and SBHA.

[0097] In one embodiment, a method for inducing MET in cancer cells is provided, the method comprising: contacting the cells with a mesenchymal-epithelial transition inducer and an immunomodulator for a time and under conditions sufficient to induce MET in the cells, wherein the cancer cells are basal or basal-like (or mesenchymal-like) cancer cells. The mesenchymal-epithelial transition inducer can be a retinoid, such as bexarotene. The immunomodulator can be an immune checkpoint inhibitor, such as an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA-4 antibody.

[0098] Lipid metabolism pathways

[0099] The inventors have shown that epithelial and mesenchymal cell states are defined by different metabolic pathways. A key finding is related to differences in lipid metabolic pathways that support epithelial and mesenchymal cell states. These findings reveal important and novel therapeutic targets, specifically metabolic genes that can be activated or inhibited to: (i) target or kill more aggressive mesenchymal subtype tumors; (ii) lead to MET conversion; (iii) block EMT and disease progression (i.e., tumor growth, invasion, metastasis, and resistance).

[0100] Without being bound by theory, the inventors found that mesenchymal tumor cells rely more on the fatty acid oxidation (FAO) metabolic pathway by utilizing fatty acids as a source of energy substrates, while epithelial tumor cells tend to store fatty acids as lipid droplets. In order to target mesenchymal tumor cells, key genes involved in FAO (such as ACSL, CPT1, 3-KAT, mitochondrial thiolase and MGLL) can be targeted. Inhibition of these genes can selectively kill mesenchymal tumor cells. Inhibitors of ACSL include TriacsinC (preclinical) and thiazolidinediones (TZD) (used in diabetes clinics). Inhibitors of CPT1 include etomomus (clinical trials that failed due to liver toxicity), perhexiline (clinical use for angina pectoris), hydroxyphenylglycine (preclinical). Inhibitors of 3-KAT include trimetazidine, ranolazine (clinical use for angina pectoris). Inhibitors of mitochondrial thiolase include 4-bromocrotonic acid (preclinical). Inhibitors of MGLL include JZL184 (preclinical).

[0101] Furthermore, the combination of these FAO pathway inhibitors with standard of care therapies may more effectively target mesenchymal tumor cells, thereby inhibiting tumor growth, reducing drug resistance, blocking metastasis, and improving anticancer responses.

[0102] The inventors further discovered that disrupting fatty acid oxidation can further block cancer metastasis. The use of the inhibitor of FAO targeting the gene CPT1, etolimus, can prevent breast cancer metastasis. Using CPT1 inhibitors or inhibitors targeting FAO to inhibit FAO can be used as an effective anti-cancer metastasis agent for cancers with a strong tendency to metastasize and cancers that rely on FAO, such as ovarian cancer, breast cancer, oral cancer, etc.

[0103] The method as defined herein may also include: after inducing MET in cancer cells, contacting the cells with an inhibitor or regulator of lipid metabolism. The lipid metabolism inhibitor may be an inhibitor of the fatty acid oxidation (FAO) pathway. In one embodiment, the lipid metabolism inhibitor is an inhibitor of an enzyme selected from the group consisting of long-chain fatty acid-CoA ligase (ACSL), 3-ketoacyl-CoA thiolase (3-KAT), mitochondrial thiolase and monoacylglycerol lipase (MGLL), or an activator of an enzyme selected from the group consisting of glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), prostatic acid phosphatase (PPAP), diacylglycerol acyltransferase (DGAT).

[0104] The lipid metabolism inhibitor may be an inhibitor of carnitine palmitoyltransferase I (CPT1). In one embodiment, the inhibitor of CPT1 is etolimus, perhexiline or hydroxyphenylglycine.

[0105] The lipid metabolism inhibitor may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0106] The lipid metabolism inhibitor may be an inhibitor of long chain fatty acid-CoA ligase (ACSL). In one embodiment, the inhibitor of ACSL is Triacsin C or thiazolidinedione (TZD).

[0107] The lipid metabolism inhibitor may be an inhibitor of 3-ketoacyl-CoA thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0108] The lipid metabolism inhibitor may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0109] In one embodiment, a method for inducing MET in cancer cells is provided, the method comprising: contacting the cells with a mesenchymal-epithelial transition inducer and a lipid metabolism inhibitor for a time and under conditions sufficient to induce MET in the cells, wherein the cancer cells are basal-like (or mesenchymal-like) cancer cells. The MET inducer can be a retinoid, such as bexarotene. The threo lipid metabolism inhibitor can be an inhibitor of CPT1, such as etolimus.

[0110] In one embodiment, a method for inducing MET in cancer cells is provided, the method comprising: contacting the cells with a mesenchymal-epithelial transition inducer and an inhibitor of CPT1 for a time and under conditions sufficient to induce MET in the cells, wherein the cancer cells are basal-like (or mesenchymal-like) cancer cells.

[0111] In one embodiment, a method of inducing MET in a cancer cell is provided, the method comprising contacting the cell with a combination of bexarotene and etolimus for a time and under conditions sufficient to induce MET in the cancer cell.

[0112] The methods defined herein may comprise treating cancer in a patient or sensitizing a patient to an anti-cancer therapy.

[0113] Unless otherwise indicated, the term "treatment" as used herein refers to partially or completely reversing, alleviating, inhibiting or preventing the growth of a tumor, tumor metastasis or other cancerous or tumor cells in a cancer patient. Unless otherwise indicated, the term "treatment" as used herein refers to the act of treating.

[0114] The terms "subject," "patient," and "individual" are used interchangeably. As used herein, they refer to an animal. By way of example only, a subject may be, but is not limited to, a mammal, including, but not limited to, a human. These terms do not require supervision (whether continuous or intermittent) by a medical professional.

[0115] By "sensitizing" a cancer or cancer cell (e.g., cancer stem cells and / or cancer cells with a mesenchymal phenotype) to an anti-cancer therapy or "increasing the sensitivity of a cancer or cancer cell to an anti-cancer therapy," it is meant that a lower dose of the anti-cancer therapeutic drug is effective in treating the cancer and / or the cancer or cancer cell responds more strongly to the same dose of the anti-cancer drug than would have occurred in the absence of the methods of the present invention.

[0116] In one aspect, a mesenchymal-epithelial transition (MET) inducing agent for inducing mesenchymal-epithelial transition (MET) in cells of a patient is provided.

[0117] In one aspect, there is provided a use of a mesenchymal-epithelial transition (MET) inducer in the preparation of a medicament for inducing mesenchymal-epithelial transition in cells of a patient.

[0118] In one embodiment, a method for inhibiting epithelial-mesenchymal transition (EMT) in cancer cells is provided, the method comprising: contacting the cells with an epithelial-mesenchymal transition (EMT) inhibitor at a time and under conditions sufficient to inhibit epithelial-mesenchymal transition, wherein the cancer cells are basal-like (or mesenchymal-like) cancer cells. This may include blocking the disease progression (i.e., tumor growth, invasion, metastasis, and resistance) of cancer cells.

[0119] In one embodiment, an epithelial-mesenchymal transition (EMT) inhibitor for inhibiting epithelial-mesenchymal transition (EMT) in cancer cells is provided.

[0120] In one embodiment, there is provided a use of an epithelial-mesenchymal transition (EMT) inhibitor in the preparation of a medicament for inhibiting epithelial-mesenchymal transition in cancer cells.

[0121] In inhibiting EMT, the inhibitor may be an inhibitor of CPT1, such as etolimus, perhexiline or hydroxyphenylglycine.

[0122] In inhibiting EMT, the inhibitor may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0123] In inhibiting EMT, the inhibitor may be an inhibitor of long chain fatty acid-CoA ligase (ACSL). In one embodiment, the inhibitor of ACSL is Triacsin C or thiazolidinedione (TZD).

[0124] In inhibiting EMT, the inhibitor may be an inhibitor of 3-ketoacyl-CoA thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0125] In inhibiting EMT, the inhibitor may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0126] In one aspect, a method for treating cancer in a patient in need thereof is provided, the method comprising administering to the patient a mesenchymal-epithelial transition (MET) inducer in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator, or a lipid metabolism inhibitor or regulator at a time and under conditions sufficient to treat the patient's cancer.

[0127] The mesenchymal-epithelial transition inducer can be administered simultaneously or sequentially with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator, or a lipid metabolism inhibitor or regulator.

[0128] In one aspect, a mesenchymal-epithelial transition (MET) inducer for treating cancer in a patient in need thereof is provided, wherein the mesenchymal-epithelial transition (MET) inducer is administered to the patient in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator or a lipid metabolism inhibitor.

[0129] In one aspect, provided is a use of a mesenchymal-epithelial transition (MET) inducer in the preparation of a medicament for treating cancer in a subject, wherein the medicament is administered to the patient in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator or a lipid metabolism inhibitor.

[0130] In one aspect, a method of sensitizing a cancer patient to an anti-cancer therapy is provided, the method comprising administering a retinoid to the patient at a time and under conditions that sensitize the patient to the anti-cancer therapy.

[0131] In one aspect, a mesenchymal-epithelial transition (MET) inducer for sensitizing a cancer patient to an anti-cancer therapy is provided.

[0132] In one aspect, a mesenchymal-epithelial transition (MET) inducer for use in the preparation of a medicament for sensitizing a patient to an anti-cancer therapy is provided.

[0133] In one embodiment, a method for inducing mesenchymal-epithelial transition (MET) in a subject is provided, the method comprising administering an inhibitor or regulator of lipid metabolism under the time and conditions of inducing mesenchymal-epithelial transition. Inhibitors or regulators of lipid metabolism can also be used for (I) targeting or killing more aggressive mesenchymal subtype tumors or (ii) blocking EMT and disease progression (i.e., tumor growth, invasion, metastasis, and resistance).

[0134] The inhibitor or regulator of lipid metabolism can be an inhibitor of the fatty acid oxidation pathway. In one embodiment, the inhibitor or regulator of lipid metabolism is an inhibitor of an enzyme selected from the group consisting of long-chain fatty acid-CoA ligase (ACSL), 3-ketoacyl-CoA thiolase (3-KAT), mitochondrial thiolase and monoacylglycerol lipase (MGLL), or an activator of an enzyme selected from the group consisting of glycerol-3-phosphate acetyltransferase (GPAT), 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), prostatic acid phosphatase (PPAP), diacylglycerol acyltransferase (DGAT).

[0135] The lipid metabolism inhibitor may be an inhibitor of carnitine palmitoyltransferase I (CPT1). In one embodiment, the inhibitor of CPT1 is etolimus, perhexiline or hydroxyphenylglycine. In one embodiment, the inhibitor of CPT1 is etolimus.

[0136] The lipid metabolism inhibitor may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0137] The lipid metabolism inhibitor may be an inhibitor of long chain fatty acid-CoA ligase (ACSL). In one embodiment, the inhibitor of ACSL is Triacsin C or a thiazolidinedione (TZD).

[0138] The lipid metabolism inhibitor may be an inhibitor of 3-ketoacyl-CoA thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0139] The lipid metabolism inhibitor may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0140] Lipid metabolism inhibitors can be used as a single agent or in combination with another anticancer agent such as standard of care chemotherapeutic agents or radiation therapy to treat cancer.

[0141] In one embodiment, a mesenchymal-epithelial transition (MET) lipid metabolism inhibitor or regulator for inducing cancer in a subject is provided.

[0142] In one embodiment, there is provided a use of a lipid metabolism inhibitor or regulator in the preparation of a medicament for inducing mesenchymal-epithelial transition (MET) in cancer in a subject.

[0143] In one embodiment, a method of blocking or preventing cancer metastasis in a subject is provided, the method comprising administering a lipid metabolism inhibitor or regulator for a time and under conditions sufficient to block or prevent metastasis in the subject.

[0144] In one embodiment, a lipid metabolism inhibitor or regulator for blocking or preventing metastasis of cancer in a subject is provided.

[0145] In one embodiment, there is provided a use of a lipid metabolism inhibitor or regulator in the preparation of a medicament for blocking or preventing cancer metastasis in a subject.

[0146] In one embodiment, a method of inhibiting epithelial-mesenchymal transition (EMT) of cancer in a subject is provided, the method comprising: administering an inhibitor or regulator of lipid metabolism for a time and under conditions sufficient to inhibit epithelial-mesenchymal transition (EMT) of cancer in the subject.

[0147] In one embodiment, a lipid metabolism inhibitor or regulator for use in inhibiting epithelial-mesenchymal transition (EMT) in cancer in a subject is provided.

[0148] In one embodiment, there is provided a use of a lipid metabolism inhibitor or regulator in the preparation of a medicament for inhibiting epithelial-mesenchymal transition (EMT) of cancer in a subject.

[0149] In one embodiment, a method for characterizing cancer aggressiveness is provided, the method comprising: detecting a mesenchymal-like lipid signature in a sample obtained from a patient. Also provided is a method for diagnosing cancer, comprising detecting a mesenchymal-like lipid signature in a sample obtained from a patient.

[0150] In one aspect, a method for identifying an agent that induces mesenchymal-epithelial transition in a cell is provided, the method comprising: a) contacting a cell with a test agent to be screened; and b) detecting the expression of a promoter of a gene selected from the group consisting of CD24, CD44, CD166, CDH1, ZEB1 and SEPINE1; wherein a change in the expression of one or more genes compared to a control indicates that the test agent is capable of inducing mesenchymal-epithelial transition in the cell.

[0151] The change in expression of one or more genes can be an increase or decrease in expression of one or more genes compared to a control. The control can be a cell that has not been contacted with the test agent to be screened.

[0152] The cells are reporter cell lines that have been engineered to express a measurable marker from the promoter of the gene whose expression is to be detected.

[0153] Also provided herein are reporter gene expression constructs capable of expressing a measurable marker from the promoter of a gene selected from the group consisting of CD24, CD44, CD166, CDH1, ZEB1, and SERPINE1.

[0154] This article presents a platform that involves the use of genetically modified cancer cell lines in high-throughput small molecule / drug library screening to detect potent MET inducers. The cell lines allow detection of changes in cell state while excluding generally cytotoxic drugs. This is different from other common screens that detect loss of cell viability phenotypes, as most anticancer drug screens are designed to measure cell killing resulting from drug treatment.

[0155] Provided herein are engineered reporter cell lines comprising a reporter expression construct as defined herein. Also provided herein are kits (or platforms) for identifying agents that induce mesenchymal-epithelial transition in cells.

[0156] Control of cancer cell state, i.e., epithelial or mesenchymal, can be used to improve cancer cell responsiveness to therapy. Treatment with retinoids and other compounds that modulate the MET pathway induces the MET pathway in basal-like breast cancer or TNBC, but not luminal / ER+ breast cancer. This provides an important stratification strategy for selecting patient populations that are more likely to respond to retinoid therapy. These patients have basal-like breast cancer or TNBC with an activated EMT program. This MET approach can be extended to mesenchymal-like tumors in other solid cancers, including gastric cancer, colorectal cancer, head and neck cancer, lung cancer, pancreatic cancer, and liver cancer, all of which may contain tumors with mesenchymal features.

[0157] For breast cancer, patients can be stratified by: (i) IHC or protein expression of tumor biopsy / resection, during which the expression of ER, PR, HER2 will be assessed to distinguish between luminal, HER2, and basal subtypes of breast cancer; (ii) using molecular diagnostics, such as PAM50, which includes a gene expression signature indicating a mesenchymal phenotype; (iii) using IHC or protein expression of tumor biopsy / resection, during which the expression of mesenchymal markers such as vimentin, fibronectin, slug, zeb1, cytokeratin 5, cytokeratin 14, and epithelial markers such as E-cadherin, EpCam, cytokeratin 8, cytokeratin 18, mucin 1, will be assessed.

[0158] In one aspect, a method is provided for identifying a cancer patient who responds to a combination therapy, the combination therapy comprising a compound that induces mesenchymal-epithelial transition (MET) in combination with a chemotherapeutic agent, an epigenetic modification compound, an immunomodulator, or a lipid metabolism inhibitor, the method comprising: detecting a mesenchymal subtype of the cancer in a sample obtained from the patient, wherein the presence of the mesenchymal subtype of the cancer indicates that the patient responds to the combination therapy.

[0159] The mesenchymal subtype of cancer may be referred to as basal-like (or mesenchymal-like) cancer.

[0160] In one embodiment, the method comprises treating the patient with a combination therapy.

[0161] In one aspect, a method is provided for identifying and treating a cancer patient who is responsive to a combination therapy comprising a compound that induces mesenchymal-epithelial transition (MET) in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator, or a lipid metabolism inhibitor, the method comprising: a) detecting a mesenchymal subtype of the cancer in a sample obtained from the patient, wherein the presence of the mesenchymal subtype of the cancer indicates that the patient is responsive to the combination therapy; and b) treating the patient with the combination therapy.

[0162] Provided herein are methods for stratifying a patient population and identifying a subgroup of patients that may respond to a combination therapy. The method may include detecting a subgroup of patients having a mesenchymal cancer subtype in a sample obtained from each patient.

[0163] Provided herein are methods of monitoring a cancer patient's response to a combination therapy comprising a compound that induces mesenchymal-epithelial transition (MET) in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator, or a lipid metabolism inhibitor.

[0164] Some patients may receive retinoids as neoadjuvant therapy or in combination with various standard of care therapies. Standard of care therapies may differ for various cancers. MET is characterized by changes in EMT markers, a shift from basal to luminal cytokeratins, suppression of cancer stem cell properties, and enhanced sensitivity to SOC therapies, leading to improved clinical responses. Patients’ tumor response to retinoid combination therapy can be monitored by: (i) IHC or protein expression of tumor biopsy / resection, during which the expression of ER, PR, HER2 will be assessed to distinguish between luminal, HER2, and basal subtypes of breast cancer; (ii) using molecular diagnostics, such as PAM50, which includes a gene expression signature that shows a mesenchymal phenotype; (iii) using IHC or protein expression of tumor biopsy / resection, during which the expression of mesenchymal markers such as vimentin, fibronectin, slug, zeb1, cytokeratin 5, cytokeratin 14, and epithelial markers such as E-cadherin, EpCam, cytokeratin 8, cytokeratin 18, mucin 1, will be assessed. As an endpoint clinical outcome, clinical measures of response, such as tumor size, will be used. Similar approaches will be used for other cancers, but using cancer-specific molecular markers.

[0165] In this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of stated elements or integers or method steps or groups of elements or integers or method steps but not the exclusion of any other elements or integers or method steps or groups of other elements or integers or method steps.

[0166] As used in this specification, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one method, as well as two or more methods; reference to "an agent" includes one agent, as well as two or more agents; reference to "the disclosure" includes single and multiple aspects of the disclosure teachings; and so forth. Aspects taught and enabled herein are encompassed by the term "invention." Any variations and derivatives contemplated herein are encompassed by "forms" of the invention.

[0167] Example

[0168] Materials and methods

[0169] Cell lines and cell culture

[0170] A human mammary epithelial cell line (HMLE) was generated as disclosed and immortalized using a retroviral vector to express the catalytic subunit of human telomerase, hTERT, and the SV-40 large T antigen. NAMEC8 was generated as disclosed. Briefly, HMLE cells were grown to 50% confluence and then differentially trypsinized with 0.05% trypsin for 1 minute. Isolated cells were carefully collected and re-seeded into 24-well plates at approximately 200 cells per well. Upon expansion, selected wells were screened for cell populations that were mesenchymal in morphology and could proliferate stably. Clonal and non-clonal NAMEC cell lines were generated. NAMEC8 is a non-clonal line derived from at least 200 mesenchymal cells. HMLE cells and NAMECs were maintained in MEGM (Lonza).

[0171] HMLE-Twist-ER cells were generated as disclosed. For EMT induction, 100 nM 4-hydroxytamoxifen ethanol solution was added to the culture medium every 48 hours to induce Twist expression. Control cells were treated with ethanol. MCF7-Slug+Sox9 cells were generated as described above. HMLE-Zeb1 cells were generated by infection with FUW-Zeb1 and rTTA. Doxycycline was added to the culture medium every 48 hours to induce Zeb1 expression. To generate tumorigenic cells from NAMEC8, cells were infected with pWZL-Blast-Ras. MCF7, MCF7-Ras, SUM159 remained unchanged as described above. All cell cultures were maintained at 37°C and 5% CO2.

[0172] Primary human cells

[0173] Breast tissue biopsies from cancer patients were obtained from the National Cancer Centre Singapore (NCCS) with patient consent and according to an IRB-approved protocol (2015 / 2976). Tissues were digested with 1 mg / ml collagenase IV (Gibco) and 1 mg / ml dispase II (Thermo Fisher Scientific) in F12 medium for 2 hours while shaking in a 37°C incubator. The cell suspension was then passed through a cell strainer and centrifuged at 1500 rpm for 10 minutes. The cells were then washed twice and resuspended in culture medium and then seeded onto irradiated 3T3 feeder cells as previously described. When confluent, subculture was performed by first lifting the feeder layer using 0.15% trypsin for 2 minutes. After removing the feeder cells, 0.25% trypsin was added to isolate tumor progenitor cells.

[0174] Establishment and treatment of patient-derived breast cancer xenografts

[0175] Primary human breast cancer samples were obtained from the National Cancer Centre Singapore (NCCS) with patient consent and under an IRB-approved protocol (2015 / 2976). All animal experiments were approved by the Agency for Science, Technology and Research-BioResource Centre IACUC (protocol number 171286), Singapore. Patient-derived breast tumor fragments (approximately 3 × 1 × 1 mm) were inserted bilaterally into the inguinal mammary fat pad of 6-8 week old NSG female mice to initially establish tumors and, once established, subsequently expanded in NSG mice. In this study, established BC2.2 tumor fragments were implanted into a cohort of 6-8 week old female NSG mice. For BC22.1, 1 × 10 6 Cells were injected subcutaneously into both flanks of NSG mice.

[0176] Generation of reporter cell lines for compound screening

[0177] pGreenFire1-mCMV was digested with ClaI and SpeI and gel purified. The promoter sequences of genes Zeb1, Serpine1, CDH1, CD24, CD44, CD166 were amplified from human genomic DNA using AccuPrime Taq DNA polymerase (ThermoFisher Scientific) and pre-designed primers containing ClaI and SpeI restriction enzyme sites. The PCR products were then digested with restriction enzymes and gel purified. The vector was ligated to the PCR insert using Quick Ligase (Fermentas) and transformed into Stbl2 competent cells. Colonies were picked and inoculated into tuberculosis culture medium containing ampicillin. The plasmid was then isolated using a plasmid miniprep kit (Qiagen) and sent for sequencing (1stBASE Laboratories). After verifying the plasmid sequence, HEK293 cells were transfected to generate lentivirus. FuGENE6 (Promega) was used for transfection. Viral supernatants were harvested at 24 and 48 hours. NAMEC8 cells were then infected twice with the virus using polybrene (8 μg / μl) and checked for good transduction efficiency using the Dual-Glo Luciferase System (Promega).

[0178] Compound library screening

[0179] The reserve plate of the compound library is prepared in DMSO with 1mM concentration by the GIS CHiP facility. NAMEC8-SERPINE1prom-Luc cells are seeded into 384-well plates at a density of 700 cells / well in a total volume of 50 μl using an automatic dispensing system (BioTek). Then 0.5 μl of compound is added to the wells using a robot needle transfer with a Bravo workstation (Agilent Technologies) on the second day. Cells are then grown for 3 days, and promoter-luciferase activity is measured using the Dual-Glo luciferase system (Promega).

[0180] Dual-Glo Luciferase Assay

[0181] Measure according to the manufacturer's instructions. In order to measure the luciferase activity in 384-well plates, first remove the culture medium from the plate. Firefly luciferase activity is determined by adding 30 μl Dual-Glo luciferase substrate (Promega) in the well and hatching for 10 minutes before using TECAN M1000 Pro plate reader to measure luminescence. Then 15 μl Dual-GloStop & Glo substrate is added to the well, hatched for 10 minutes and measured on the plate reader to determine the Renilla luciferase activity. Then calculate the promoter luciferase activity by adopting firefly luciferase instead of Renilla luciferase.

[0182] ERE-luciferase activity assay

[0183] ERE-luciferase constructs were kindly provided by Alan Prem Kumar, CSI, Singapore. To measure ERE-luciferase activity, cells were seeded 24 hours before transfection. ERE-luciferase (1 μg) and NSV-Renilla (0.1 μg) were packaged and transfected into NAMEC8 using Lipofectamine 2000. 24 hours after transfection, cells were re-seeded into 96-well plates for drug treatment. The activities of firefly luciferase and Renilla luciferase were measured 24 hours after treatment using the Dual-Glo Luciferase System (Promega).

[0184] Lentiviral shRNA and overexpression constructs

[0185] Lentiviral shRNAs were from the RNAiConsortium (TRC) collection of the Broad Institute. The clone IDs of the shRNAs used were: TRCN0000072181 (GFP control), TRCN0000330783 (RXRα shRNA1), TRCN0000021616 (RXRα shRNA2), TRCN0000021618 (RXRα shRNA3), TRCN050201020302030203020202020021616. Each shRNA was cloned into the lentiviral plasmid pLKO.1 (Addgene).

[0186] For overexpression of CPT1A, the ORF of CPT1A was digested from pCMV6-CPT1A (Origene Cat# RC200485) and cloned into pMN-GFP vector (from Qiang Lab, GIS). Positive clones were confirmed by sequencing and used for retroviral production. For overexpression of Zeb1, the ORF of Zeb1 was cloned into FUW-LPT2 vector. Positive clones were confirmed by sequencing and used for lentiviral production.

[0187] To produce lentivirus, the plasmid was packaged into HEK293T cells with packaging plasmid using FuGENE6 (Promega). To produce retrovirus, the plasmid was packaged into PlatA cells with packaging plasmid using FuGENE6 (Promega). Virus was harvested at 48 hours and transduced into target cells. Infected cells were selected with 2 μg / ml puromycin for 7 days.

[0188] RNA isolation, reverse transcription and real-time PCR analysis

[0189] Cells were rinsed twice in ice-cold PBS. Total RNA was extracted using Trizol (Invitrogen) and column purified using RNeasy kit (Qiagen). cDNA was synthesized with 500ng total RNA at 37°C for 2 hours using SuperScript III first-strand synthesis system (Thermo Fisher Scientific), and then diluted 5 times. mRNA levels were measured using ABI Prism 7900HT sequence detection system 2.2 (Applied Biosystems) using gene-specific primers listed below. Results were normalized to GAPDH and analyzed using SDS2.2 software.

[0190] Proliferation assay

[0191] To measure cell growth rate, 1000 cells were plated in triplicate on 96-well plates. Cell Titre Glo reagent (Promega) or Cell viability was measured using the One Solution Cell Proliferation Assay Kit (MTS) (Promega).

[0192] Protein extraction and immunoblotting

[0193] Cells were harvested and scraped from culture dishes in ice-cold PBS. They were then centrifuged at 12,000 rpm for 5 minutes at 4°C. The pellets were then snap-frozen in liquid nitrogen or lysed in ice-cold RIPA lysis buffer (Thermo Scientific) and placed on ice for 30 minutes. The lysate was then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was then aspirated and placed in a new tube. Protein concentration was determined using Coomaisse Blue. 50-80 μg of total protein was mixed with NuPAGETM LDS sample buffer and heated at 70°C for 10 minutes. The samples were loaded into NuPAGETM 4-12% Bis-Tris gels (precast polyacrylamide gels; InvitrogenTM). Gel electrophoresis was performed at 200V for 1.5 hours and then blotted onto a nitrocellulose membrane (Trans-Blot TurboTM transfer kit; Bio-Rad). The membrane was blocked with 5% skim milk Tris-buffered saline (TBS; 1st BASE) containing 0.1% Tween-20 (Santa Cruz) and then incubated with the primary antibody overnight at 4°C. Thereafter, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature. Finally, the immunoreactive proteins were visualized using enhanced chemiluminescence (ECL) reagents (Thermo Scientific, 34075) and ChemidocTM Imager (Bio-Rad, 17001401).

[0194] Fluorescence activated cell sorting (FACS)

[0195] Cells were trypsinized until they detached from the cell culture plate. Neutralization medium (DMEM containing 20% ​​FBS) was then added to inactivate trypsin. The cells were then centrifuged at 1200 rpm for 3 minutes and the cell pellet was resuspended in PBS / 2% FBS. The cells were stained with DAPI (1:2000) and filtered through a 40 μm filter before sorting. FACS was completed on a BD AriaFusion flow cytometer.

[0196] Staining for CD24 and CD44

[0197] Cells were collected by FACS and resuspended in 100 μl of a solution containing CD24-PE and CD44-APC (BD-Biosciences) antibodies diluted in PBS with 2% FBS (1:40 dilution). The cells were stained for 45 minutes, vortexing every 10 minutes to ensure uniform staining. After antibody incubation, the cells were washed once and resuspended in 300 μl of PBS with 2% FBS. The cells were then filtered through a 40 μm filter and analyzed on a BDLSR Fortessa X-20 cell analyzer.

[0198] Mammosphere cells

[0199] Detection

[0200] For mammosphere culture, cells were seeded at 700 cells / well in 96-well ultra-low adhesion plates in Mammocult medium (Stem Cell Technologies) supplemented with 4 μg / ml heparin, 0.5 μg / ml hydrocortisone and 1% methylcellulose. Spheres (>100 μm) were counted after 7-10 days.

[0201] Immunofluorescence (IF) staining

[0202] Cell cultures were fixed in 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 and then blocked with 1% bovine serum albumin in PBST. Cells were incubated in specific primary antibodies followed by appropriate secondary antibodies conjugated to Alexa Fluor-488 or -594 (Molecular Probes). Cell nuclei were then stained with DAPI and then observed and imaged using an Axio Observer D1 epifluorescence microscope with a built-in AxioCamMR3 camera (Zeiss) using optimal filters and a magnification of 100-200x.

[0203] Generation of SUM159PACR cells

[0204] SUM159 cells were exposed to increasing amounts of PAC starting at 10 nM. The medium containing fresh drug was replaced every two days, and the cells were split when they reached 80-90% confluence. The concentration of PAC was increased by 10 nM at each split until the cells could survive at 100 nM. The cells were then subjected to a cell viability assay to measure the IC50. The IC50 values ​​were determined using GraphPad Prism software. Cells were considered resistant to PAC when the IC50 value increased at least 10-fold compared to the parental cell line.

[0205] IC 50 In vitro measurements

[0206] Cells were seeded at a density of 2000 cells / well in 96-well plates. Drugs (5-FU / PAC) were added the next day in a 10-point dose response. Cells were grown for 3 days before measuring cell viability using Cell Titer Glo reagent (Promega). Data were normalized to blank controls. Contour plots were generated and IC values ​​were determined using GraphPad Prism software. 50 value.

[0207] In vivo experiments using NSG mice

[0208] All animal experiments were approved by the IACUC of the Bioresource Center of the Agency for Science, Technology and Research, Singapore (protocol number 140940). 6 The cells were suspended in 100 μl of culture medium containing 50% Matrigel and injected subcutaneously into both flanks of female NSG mice. Tumors in NSG mice were examined by palpation once a week and harvested when they reached approximately 15 mm in diameter. For treatment of NSG mice, 1 × 10 5 NAMEC8-Ras cells were suspended in 100 μl of medium containing 50% matrigel and injected subcutaneously into both sides of NSG mice. Tumor formation in NSG mice was examined by palpation, and when the tumor was about 3 mm long, the mice were randomly divided into two groups: a blank group (5% DMSO in corn oil) and a treatment group (bexarotene, 100 mg / kg / day oral administration). Tumor size was measured weekly using a vernier caliper, and mice were sacrificed when the tumor reached about 15 mm or after about 4 weeks of treatment. For patient-derived cells, 2×10 6 cells.

[0209] Orthotopic tumor transplantation of MCF7-Slug+Sox9 cells labeled with tdTomato fluorescent protein was performed as described previously. 6 The cells were resuspended in 30 μl of medium containing 50% matrigel and injected into the mammary fat pad of NSG mice. The mice were randomly divided into 3 groups (control group, DOX group, and etolimus + DOX group). Mice were administered blank control (0.9% w / v NaCl) or etolimus (20 mg / kg) by intraperitoneal injection. Injections were given every day for 3 weeks. Doxycycline was then given through drinking water for 2 weeks. Mice were killed 10 weeks after cessation of doxycycline treatment and lung metastases were analyzed under a fluorescent microscope.

[0210] Hematoxylin and eosin staining of tumor sections

[0211] Tumor sections were fixed overnight in 4% paraformaldehyde, immersed in 70% ethanol, and sent to the IMCB Advanced Molecular Pathology Laboratory for paraffinization and hematoxylin and eosin staining.

[0212] Immunofluorescence (IF) staining of tumor sections

[0213] Tumor sections were fixed overnight in 4% paraformaldehyde, immersed in 70% ethanol, and sent to the IMCB Advanced Molecular Pathology Laboratory for paraffinization. For IF staining, slides were dewaxed and rehydrated in PBS. The tissue was surrounded with a hydrophobic barrier using a barrier pen and blocked with blocking buffer (1% bovine serum albumin; BSA; 1% horse serum and 0.2% TritonX-100 in PBS) for 1 hour. It was then washed with PBS and incubated in the specific primary antibody overnight at 4°C. The slides were washed with PBS and incubated with the appropriate secondary antibody conjugated with Alexa Fluor-488 or -594 (Molecular Probes) for 1 hour at room temperature. The slides were then covered with DAPI (Vector Laboratories) and coverslips were used. The tissue was mounted on a slide with mounting medium. The coverslip was permanently sealed with nail polish to extend storage time. The slides were observed and imaged using an Axio Observer D1 epifluorescence microscope with a built-in AxioCamMR3 camera (Zeiss) using optimal filters and 100-200x magnification. Fluorescence image analysis and fluorescence overlay images were obtained using AxioVision Rel4.8 imaging software.

[0214] Chromatin immunoprecipitation-sequencing (ChIP-Seq)

[0215] ChIP assays were performed as described previously. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature and then inactivated by the addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 200-500 bp were immunoprecipitated using anti-RARα, anti-RXRα, anti-H3K27ace, or anti-p300 antibodies. ChIP-enriched DNA was then decross-linked and subjected to library preparation using the Illumina TruSeq ChIP kit and then analyzed using Illumina sequencing.

[0216] Transcriptome sequencing (RNA-Seq)

[0217] Cells were treated with bexarotene or DMSO and lysed with Trizol. Total RNA was extracted using the Qiagen RNeasy kit (#74106). RNA integrity and concentration were measured on a bioanalyzer, and libraries were prepared using the Illumina True-Seq RNA kit and then analyzed using Illumina sequencing.

[0218] Genomic data analysis

[0219] For ChIP-Seq data processing, reads were aligned to the human genome (hg19) using the BWA aligner. Aligned reads were deduplicated using Picard Markduplicates.jar. Peak calling was performed using MACS2peak-caller with default parameters. Differential binding was performed using DiffBind. H3K27ac super-enhancers were identified using the ROSE algorithm as described. ChIP-Seq data are deposited in GEO (accession number: GSE119824).

[0220] For RNA-Seq data processing, reads were aligned to the human reference genome GRCh37.p13 using TopHat2, and read counts were performed using featureCounts. Lowly expressed genes (i.e., genes with fewer than 10 counts in less than 60% of the samples) were excluded from further analysis. The library size was then scaled by the trimmed M-value mean (TMM) method. Differentially expressed genes were identified using the R package limma, and gene ontology pathway analysis was performed using the GREAT software. RNA-Seq data are deposited in the SRA database (accession number: SRP152713).

[0221] Lipid extraction

[0222] Lipids were extracted from cell pellets using butanol:methanol (1:1) as the extraction solvent. The extraction solvent included an internal standard mixture consisting of the following components: 17:0LPC (lysophosphocholine), C14LPE (lysophosphoethanolamine), C17ceramide, C17phosphocholine, C17phosphoserine, C17:0PG, C8-ceramide, DMPE (1,2-dimyristoyl-glycero-3-phosphoethanolamine), DMPG (1,2-dimyristoyl-glycero-3-phospho-(1′rac-glycerol), DMPS (dimyristoyl-glycero-phosphoserine), 12:0DG (1,2-dilauroyl-sn-glycerol), C8GluCer (D-glucosyl-β1-1′-N-octanoyl-D-erythro-sphingosine), 24:0SM (N-lignanyl-D-erythro-sphingosine phosphocholine), 24:1SM (N-ceranyl-D-erythro-sphingosine phosphocholine), d5-TAG 48∶0. All standards were from Avanti Polar Lipids (Alabaster, AL, USA) except d5-TAG 48∶0 which was from CDN Isotope. All standards were added to a final concentration of 100 ng / ml. 100 μl of extraction solvent (including standards) was added to 1×10 7 cells. The mixture was vortexed for 15 seconds. The sample was then sonicated in an ultrasonic bath for 60 minutes at room temperature. The mixture was then centrifuged at 14000 g for 5 minutes. The supernatant was transferred to a new tube and kept at -80 °C until analysis.

[0223] Lipid analysis

[0224] Samples were analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system (Agilent 1290 UHPLC connected to an Agilent 6490 QQQ). Separation was performed using a reverse phase column (Agilent ZORBAX RRHD Eclipse Plus C18, 2.1×50 mm, 1.8 μm). Gradient elution was performed using mobile phase A (40% acetonitrile / 60% water, containing 10 mM ammonium formate) and mobile phase B (90% isopropanol / 10% acetonitrile, containing 10 mM ammonium formate). Quality control (QC) samples were generated by pooling aliquots of lipid extracts from each sample. During the analysis, one QC sample was injected for every 10 samples to monitor the performance of the experimental procedure. Lipids were quantified in positive multiple reaction monitoring (MRM) mode. MS parameters: gas temperature 300°C, gas flow rate 5 L / min, sheath gas flow rate 11 L / min, sheath gas temperature 250°C. Data were extracted using MassHunter quantitative analysis software (Agilent). Lipids were normalized using internal standards and cell number. Graphs and statistical analysis were completed using GraphPad Prism and the Mann-Whitney U test.

[0225] Lipid droplet staining

[0226] Intracellular lipid droplets were visualized with the fluorescent Bodipy dye. Cells in culture were fixed in 4% paraformaldehyde for 15 min at room temperature, rinsed twice with PBS, permeabilized with 0.2% TritonX-100 in PBS for 10 min, and incubated with Bodipy diluted in PBS (final concentration 1 μg / ml) overnight at 4°C. Cells were washed with PBS and stained with DAPI. Fluorescence images were acquired using an Axio Observer D1 epifluorescence microscope with a built-in AxioCamMR3 camera (Zeiss) using optimal filters and 100-200× magnification.

[0227] Fatty acid β-oxidation assay

[0228] FAO rates were measured using the mitochondrial stress test on an XF96 analyzer (Seahorse Bioscience) according to the manufacturer's instructions. Cells were seeded in complete medium in 96-well XF96 cell culture plates at a density of 9000 cells / well. The cells were then incubated with XF modified DMEM assay medium (Seahorse Bioscience) and 1 mM glutamine, 0.5 mM glucose, 1% FBS, and 0.5 mM carnitine were added the next day for 18 hours. On the day of the assay, cells were incubated in FAO assay medium (KHB: 111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 2 mM MgSO4, 1.2 mM NaH2PO4, supplemented with 2.5 mM glucose, 0.5 mM carnitine, 5 mM HEPES, adjusted to pH 7.4) 45 minutes before analysis in a non-CO2 incubator. Seahorse cells were tested for mitochondrial stress with compounds (2 μM oligomycin, 2 μM FCCP, 2 μM rotenone). To inhibit FAO, etolimus (40 μM) was added 15 minutes before the assay. XF palmitate-BSAFAO substrate or BSA was added to the cells just before starting the assay. The plate was then placed in the XF96 analyzer and the XF CellMito stress test was run using the recommended protocol. The ratio of FAO was calculated by the difference in maximum respiration when the palmitate-BSA FAO substrate was supplemented relative to the BSA control.

[0229] Statistical analysis

[0230] GraphPad Prism (GraphPad Software) v7.0 was used for statistical analysis. Unless otherwise stated, all data are expressed as mean ± standard error of the mean. Student's t-test (two-tailed) was used to compare two groups and calculate p-values. P < 0.05 was considered statistically significant. The significance level is indicated in the relevant figure legends.

[0231] Example 1

[0232] To broadly understand the molecular basis and therapeutic utility of the MET process, we performed a high-throughput small molecule library screen to identify classes of agents with MET-promoting properties in an unbiased manner. This led us to identify retinoids as the most potent MET promoters, whose potency and durability could be further enhanced by epigenetic enhancement with HDAC inhibitors. Surprisingly, treatment with retinoids resulted in a basal-to-luminal-like transition, with a shift in cytokeratin expression, and re-expression of ER in several patient-derived tumor cell models, in addition to classical MET marker genes and phenotypic changes. Administration of retinoids to tumor-bearing animals induced MET-like tumor architectural changes and arrested tumor growth. Furthermore, we revealed for the first time that cell-state transitions in breast cancer are defined by the reprogramming of lipid metabolism, specifically the involvement of retinoids and their regulation of the switch between β-oxidation and lipid storage. As revealed by genome-wide analyses, retinoids bind and target key lipid metabolism genes via retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This results in the redirection of fatty acid utilization for β-oxidation in the mesenchymal cell state to lipid droplet storage in the epithelial cell state. Disruption of key metabolic enzymes mediating this transition leads to inhibition of the MET process. Conversely, perturbation of β-oxidation using the CPT1 inhibitor, etolimus, redirects fatty acid transitions and promotes a more epithelial phenotype, and is also able to prevent EMT-driven breast cancer metastasis in animal models.

[0233] High-throughput small molecule library screening identifies retinoids as promoters of mesenchymal-epithelial transition

[0234] Acquisition of a mesenchymal cell state in breast cancer is often associated with a more aggressive phenotype that is also chemoresistant and cancer stem-like. We reasoned that conversion of a mesenchymal to a more epithelial cell state through the mesenchymal-epithelial transition (MET) process might reduce tumorigenicity and chemoresistance in cancer cells as a form of combination therapy for breast cancer. To identify small molecule compounds with the ability to promote MET, we utilized the mesenchymal breast cell line NAMEC8, which responds to perturbations of the cell state, coupled to a promoter firefly luciferase reporter as a readout of cell state changes (Figure 1A). We tested several gene promoters, including CD24, CD44, CD166, CDH1, ZEB1, and SERPINE1, which have previously been shown to be associated with either epithelial or mesenchymal cell phenotypes. From other studies, TGFβ can promote EMT in several cancer cell lines, and inhibition of the TGFβ pathway can block EMT or modestly induce a MET-like phenotype. Therefore, we used the TGFβR inhibitor A83-01 as a positive control to evaluate the response of these gene promoters to changes in cell state (Figure 1B). Among them, SERPIN Elprom-Luc showed the most dynamic response when treated with A83-01, as it was downregulated by at least 50%.

[0235] We screened four compound libraries (LOPACs, anticancer, kinase inhibitors, bioactive lipids) containing >2700 compounds to identify molecular classes with MET-promoting properties (Figure 1C). Of note, in the mesenchymal NAMEC8SERPINE1prom-Luc reporter cells, we further included a Renilla-luciferase reporter under the control of a constitutive CMV promoter as a readout of cell number. The goal was to identify compounds that altered cell state without adversely affecting cell viability (Figure 1A). From this primary screen, the “hit rate” was 2.19% (61 compounds). Z-scores were also calculated and plotted (Figure 8A). These were validated in secondary screening and dose-response measurements, indicating a compliance rate of 83.6% (51 compounds), thus highlighting the robustness of the reporter-based screen (Figures 1D, E and Table 1). At least 8 classes of compounds were represented, including TGFβ inhibitors, which we previously used as positive controls. Interestingly, compounds such as adenosine / adrenergic, GABA and NMDA receptor agonists and antagonists, as well as several neurotransmitters not previously implicated in cell state transitions, were identified as potential modulators. Among these categories, retinoids appeared to induce the most significant reduction in the SERPINElprom-Luc reporter gene in a dose-dependent manner (Figure 1E).

[0236] Table 1 List of MET inducers identified in the preliminary screening

[0237]

[0238]

[0239] Retinoids induce transformation of basal-like breast cancer cells into luminal-like cells and enhance their sensitivity to chemotherapy

[0240] We chose to include retinoids as MET inducers for several reasons. First, they represent the most potent class of MET-inducing compounds in our screen. Second, they are clinically feasible agents that are used as differential agents for acute promyelocytic leukemia, although their utility and efficacy for other cancers remain unclear. Finally, the manner and mechanism by which retinoids induce cell state changes or promote differentiation are not well elucidated. From our screen, we selected the strongest RARα (TTNPB) and RXRα (bexarotene) agonists for validation studies as well as biochemical and pathway characterization. Consistent with the downregulation of SERPINE1prom-Luc reporter activity, which marks a mesenchymal cell state, treatment of NAMEC8 and another mesenchymal TNBC cell line, SUM159, with bexarotene or TTNPB resulted in a significant change in morphology from scattered, spindle-shaped, and fibroblast-like to an epithelial-like morphology characterized by compact clusters of epithelial islands (Figures 2A and 9A). This morphological change was associated with downregulation of mesenchymal signature markers (ZEB1, SERPINE1, FN1, SLUG) and a concomitant increase in the epithelial marker CDH1 (Figs. 2B, C, D and 9B, C). For the first time, we noted a shift in cytokeratin expression from KRT5 (typically associated with basal-like, triple-negative breast cancer) to KRT8, which is expressed in the luminal, ER+ subtype of breast cancer. Mucin 1 (MUC1), a signature marker of luminal breast cancer that is uncommon in TNBC, was also upregulated. These data provide the first indication that, in addition to MET, these cancer cells, thought to represent basal-like, triple-negative breast cancer, may have acquired luminal-like features. Because a mesenchymal phenotype has been shown to be associated with a cancer stem cell-like state, we examined the expression of CD44 and the mammosphere-forming ability of retinoid-treated cells. Both NAMEC8 and SUM159 cells showed a significant reduction in surface CD44 expression by flow cytometry and a significant reduction in mammosphere-forming ability (Figs. 2E, F and 9D, E).

[0241] To further evaluate the effects of retinoids beyond classical cancer cell lines, which tend to be homogeneous, divergent from their parental tumors, or have altered plasticity, we performed bexarotene treatment of triple-negative and ER+ breast tumors from biopsies or resections (Table 2). Consistent with our previous findings, bexarotene treatment of patient-derived triple-negative breast cancer cell lines (BC2.2, BC22.1, BC29.1, MetBR007-DR) increased E-cadherin expression while decreasing SLUG expression to varying degrees. More notably, however, we continued to observe a shift in cytokeratin expression from KRT5 to KRT8, as well as an increase in MUC1 expression (Figs. 2G, H and 9F, G). The mammosphere-forming capacity of patient-derived cancer cells was also reduced following retinoid treatment (Figs. 2I and 9H). Surprisingly, in three of the tumor cell lines, we noted upregulation of estrogen receptor (ER) expression, a hallmark of ER+ luminal breast cancers. The expression of the estrogen response element (ERE)-luciferase reporter gene was also increased after treatment with bexarotene or TTNPB ( Fig. 9 I). In support of this, ER target genes such as ABCA3, NRIP1, TFF1, and CREB1 were significantly upregulated by bexarotene treatment in two cancer cell lines (NAMEC8 and BC2.2) ( Fig. 9 J). Since other basal-like cancer cells had now acquired ER expression, we wondered whether they also acquired sensitivity to anti-ER therapy, which is given to breast cancer patients with ER+ tumors. Interestingly, bexarotene-treated cancer cells appeared to become sensitive to low doses of the selective ER modulator raloxifene hydrochloride ( Fig. 9 K). Nevertheless, we noted a modest two-fold difference in drug sensitivity, thus suggesting that the switch from basal-like to a true luminal phenotype may be incomplete. Interestingly, when bexarotene was applied to patient-derived tumor cell lines (BC24.1 and BC33.1) generated from ER+, luminal-like breast tumors, epithelial / mesenchymal or luminal / basal markers were expressed (Figures 2G and 9L), indicating that the retinoid exerts its effects in mesenchymal-like, but not in epithelial-like breast cancer cells.

[0242] Next, we wanted to determine whether retinoids were indeed acting and signaling through retinoic acid receptors (RXRα or RARα). Using shRNA targeting RXRα, we achieved stable knockdown in NAMEC8 ( Fig. 9 M). The increase in epithelial-luminal markers and loss of mesenchymal basal markers induced by bexarotene were now abolished in RXRα knockdown cells ( Fig. 9 N, O). These results confirm that retinoid-induced changes in cell state are dependent on the RXRα receptor and its downstream activities.

[0243] Acquisition of a mesenchymal cell phenotype via EMT has been shown to confer resistance to a broad range of chemotherapies. We reasoned that the opposite phenomenon, MET, might sensitize other mesenchymal cancer cells to chemotherapeutic agents, thereby overcoming resistance. Not surprisingly, exposure to MET-inducing bexarotene was able to confer at least a 4- to 6-fold increase in sensitivity to the standard of care for TNBC, 5-FU or paclitaxel, in both classical mesenchymal-like cancer cell lines (NAMEC8 and SUM159) and two patient-derived TNBC cell lines (BC2.2 and BC22.1) (Figures 2J, K and 9P, Q, R).

[0244] In the context of breast cancer management, the use of retinoids in combination with chemotherapy in treatment-naive patients may not be feasible. For retinoids to be useful as therapeutic agents in TNBC, they need to be evaluated in cancer cells that are resistant to chemotherapy. To test this, we generated paclitaxel-resistant (PACR) SUM159 cells, which displayed >20-fold increased paclitaxel resistance compared to parental (PT) cells ( Figure 2L ). SUM159 PACR cells switched from a mesenchymal-like to an epithelial-like phenotype, exhibited a clustered morphology, and showed elevated expression of epithelial and luminal markers after treatment with bexarotene ( Fig. 9 S, T, U). Bexarotene-treated PACR cells also had reduced mammosphere formation ability ( Fig. 9 V), indicating a reduction in cancer stem cell-like properties. More importantly, they acquired sensitivity to an alternative chemotherapeutic drug, 5-fluorouracil (5-FU), suggesting that drug-resistant cancer cells may respond to this treatment strategy (Figure 2M).

[0245] Table 2. Molecular information of patient-derived breast cancer cell lines

[0246]

[0247]

[0248] Retinoids promote MET in vivo and reduce the tumorigenicity of animal TNBC cells

[0249] We reasoned that the switch from a mesenchymal to an epithelial cell state might result in a loss of tumorigenicity in basal-like, mesenchymal-like breast cancer cells. Ex vivo treatment of SUM159 cells with either bexarotene or TTNPB indeed severely impaired their tumorigenic potential for up to 7 weeks after transplantation into immunocompromised NSG mice, although tumor growth accelerated after this time (Figures 3A and 10A), indicating that short-term (14 days) ex vivo treatment is insufficient to mediate permanent or durable changes in cell state. It also suggests that continued forced treatment with bexarotene might be necessary to arrest tumor growth. Interestingly, when the luminal epithelial-like breast cancer cell line MCF7-Ras was similarly exposed to either bexarotene or all-trans retinoic acid (ATRA), no effect on tumor cell growth was observed (Figures 3B and 10B). Notably, the reduction in tumorigenicity was not attributable to the effects of the retinoids in arresting cell proliferation, as they maintained a similar growth capacity to control cells (Figure 10C). This clearly highlights the utility of retinoid-induced changes in cell state in basal breast cancers, but not in luminal breast cancers.

[0250] To evaluate the efficacy of retinoids in tumor-bearing animals, we first transplanted NAMEC8-Ras cells and two other patient-derived TNBC cell lines (BC2.2 and BC22.1) into NSG mice and allowed tumors to reach a diameter of ∼3 mm by week 4. Bexarotene was administered daily by oral gavage to one of the groups during the study, while a vehicle control was administered to the other group. The tumor growth rate and burden were significantly reduced in tumor-bearing mice fed bexarotene compared with vehicle-treated mice (Figs. 3C and 10D, E). To understand the reason for this reduced growth, we collected the residual tumors for analysis. Immunohistochemistry of the tumors showed that control tumors were grade 3 carcinomas with ductal features, while tumors from bexarotene-treated mice were non-adherent and separate. The latter also showed the presence of spindle cells with focal necrosis, and the fibrovascular core indicated the possible presence of papillary or pseudopapillary components (Fig. 3D), thus indicating a change in the cell state. To confirm this, we performed immunofluorescence staining for EMT markers in tumor sections. Tumors from mice treated with bexarotene at this time showed expression of E-cadherin and loss of fibronectin, thus indicating the emergence of a MET phenotype in vivo as a result of the first therapeutic intervention. In addition, in these tumors, there was a shift in cytokeratin expression from basal-associated KRT5 to luminal-associated KRT8 (Figures 3E and 10F). Since retinoids activate target genes such as STRA6, CRAPBP2, DHRS3, ANGPTL4, IP6K3, and ACSL5 through DNA-binding RXR and RAR receptors, we analyzed their expression in tumors from treated animals. All target genes were upregulated by at least twofold (Figure 3F), thus confirming that the switch in cell state was mediated through retinoid target gene activity.

[0251] Example 2

[0252] Epigenetic perturbations enhance and increase the persistence of retinoid-induced changes in cell state

[0253] Although retinoids can promote MET in mesenchymal breast cancer cells, the phenotype appears to be reversible, at least partially, in the absence of sustained exposure to retinoids. This suggests an inherent plasticity of cancer cells, perhaps as a result of dynamic epigenetic remodeling. We hypothesized that one might be able to enhance the epithelial cell state by modifying epigenetics in conjunction with retinoid-induced cell state transitions. First, we tested whether epigenetic modifying compounds could induce MET on their own. In a manner similar to our previously described screening for MET compounds, we screened 35 epigenetic modifying compounds, six of which were able to downregulate SERPINE1prom-Luc activity in mesenchymal NAMEC8 reporter cells. While these reduced activity by 15–53%, the vast majority did not reach the same magnitude caused by bexarotene treatment (Figure 4A). We next assessed whether the combination of epigenetic compounds with bexarotene might have a greater effect on MET. Interestingly, several epigenetic compounds were able to produce an additive benefit with bexarotene, inducing a more dramatic downregulation of SERPINE1prom-Luc activity (Figure 4B). We focus on C1-994 and SBHA, both class I HDAC inhibitors (HDACi)

[0254] The combination of HDACi and bexarotene resulted in more robust changes in epithelial cell morphology and related EMT markers (Figures 4C, D and 11A). For example, the reduction in ZEB1 and SLUG was more intense, while the gain in KRT8 and MUC1 was more pronounced. Because retinoids alone do not result in a stable MET phenotype, we evaluated whether the combination might prolong the maintenance of the epithelial cell state after discontinuation of both drugs. In fact, the reduction in SERPINE1prom-Luc activity in NAMEC8 cells treated with retinoids alone was restored three days after discontinuation, while it continued to decrease in cells treated with a combination of retinoids and HDACi (Figures 11B, C). More importantly, when the compounds were discontinued after 7 days of treatment, cancer cells that had undergone MET were able to maintain the epithelial phenotype for at least another 7 days (Figures 4E, F, G). In addition, the ability to form mammospheres was also continuously suppressed (Figures 4H and 11D, E). In contrast, cells treated with bexarotene alone gradually reverted to a more mesenchymal cell phenotype and partially reestablished the ability to form mammospheres. In the absence of retinoid ligands, RARs and RXRs bind DNA while associating with transcriptional co-repressors including NCoR and HDACs, leading to gene silencing. When exposed to retinoids, they bind to the receptors and lead to the recruitment of co-activators, such as histone acetyltransferase p300. At the same time, inhibition of HDACs may allow the maintenance of epigenetics that promote gene transcription, resulting in more efficient and durable MET (Figure 4I).

[0255] Lipid metabolism genes are regulated by retinoid receptors

[0256] RAR and RXR are DNA-binding transcription factors that function by regulating target gene expression in the presence or absence of retinoids. Pathways reported to be associated with retinoid signaling include TGFβ, NFkB, and Nrf2 27-29 To understand how RAR and RXR program cell state transitions, we performed ChIP-seq to identify sets of their target genes in an unbiased manner. We exposed mesenchymal NAMEC8 and SUM159 cancer cells to short-term (ST: 7 days) and long-term (LT: 14 days) retinoid treatment and then performed genome-wide analysis of target gene binding sites of RXR, RAR, RAR, and p300. Exposure to the RXR ligand bexarotene resulted in a subset of genes being bound by both RXR and RAR, as both can form heterodimers. These genes also showed an increase in p300 binding and H3K27ac (histone 3, lysine 27 acetylation) modification, which marks gene enhancers (Figures 5A, B and 12A, B). For example, well-known retinoid target genes such as STRA6 and DHRS3, which are initially unbound by RXR in the mesenchymal cell state, but later become bound by MET (Figures 5C and 12C).

[0257] We next sought to identify functional pathways regulated by RXR by mapping the top RXR-bound genes using gene ontology analysis. This revealed that lipid metabolism was a major pathway perturbed during MET—a phenomenon that had not been previously investigated (Figure 5D). To further test whether RXR-bound genes correlated with transcriptional changes caused by RXR activation, we performed RNA-seq on cancer cell lines and patient-derived cancer cells treated with bexarotene to detect gene expression changes. Consistent with the above analysis, genes upregulated by at least two-fold showed significant enrichment in pathways related to lipid metabolism (Figures 12D, E). Super-enhancers are signatures of key genes that help define cell states and cell lineages. Analysis of H3K27ac marks revealed key genes that contained super-enhancers during MET. These included retinoid target genes, such as DHRS3, RXR, and GDF15, as well as lipid metabolism genes, such as B4GALT5 and ACSL1 (Figures 5E and 12F). For example, lipid metabolism genes such as PLIN1, PLIN2, ACSL5, and MGLL showed significant RXR recruitment and increased H3K27ac modification after bexarotene treatment (Figures 5C and 12C); this further emphasizes their inferred role in MET.

[0258] To elucidate which specific aspects of lipid metabolism might be altered during the cell state changes, we examined in more detail the list of RXR target genes that showed consistent upregulation or downregulation in multiple cancer cell lines and patient-derived cancer cells treated with bexarotene (Figure 5F). Interestingly, many lipid-related genes form pathways for the production of triglycerides (TAGs) from fatty acids. Genes involved in TAG synthesis (ACSL4, ACSL5, AGPAT4, AGPAT9, GPAM, DGAT2, PLIN1) were upregulated, while the lipase MGLL involved in TAG degradation was strongly downregulated (Figures 5G and 12G). Therefore, these results strongly suggest a reprogramming of lipid metabolism during MET.

[0259] Mesenchymal-epithelial transition limits β-oxidation by directing fatty acids to lipid storage

[0260] A major fate of fatty acids is their conversion and storage as TAGs. Key lipid metabolism enzymes, long-chain fatty acid-CoA ligase (ACSL), glycerol-3-phosphate acetyltransferase (GPAT), 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), prostatic acid phosphatase (PPAP), and diacylglycerol lipid acyltransferase (DGAT), were all upregulated after retinoid treatment and may help drive the conversion of fatty acids into intermediates such as diacylglycerol (DAG) and TAG (Figure 4a). To demonstrate this, we used lipid mass spectrometry-based lipidomics to determine the fate of fatty acids and their downstream intermediates upon cell state changes. Indeed, lipid species such as phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), which are downstream metabolites of fatty acids, were significantly increased after bexarotene treatment. Accumulation of DAG and TAG species was also observed (Figure 6a). TAGs can be stored in lipid droplets when accumulated. Interestingly, significantly abundant lipid droplets were observed in mesenchymal cancer cells that had undergone MET, whereas control-treated cells did not contain as many lipid droplets (Fig. 6b and Fig. 14a), suggesting that the fate of fatty acids may differ in the mesenchymal and epithelial cell states.

[0261] We sought to functionally test whether the intermediate lipid enzymes involved in the TAG synthesis pathway are indeed required for the regulation of MET. Pharmacological inhibition of GPAT (using FSG67) and DGAT (using AmidepsineA) as well as shRNA knockdown of AGPAT9 all resulted in a significant reduction in the accumulation of lipid droplets during retinoid treatment compared to either the blank treatment or shRNA knockdown controls (Fig. 14b). This was due to blockade of MET, as the increase in epithelial luminal gene markers such as E-cadherin, KRT8, and MUC1 was suppressed when GPAT, DGAT, or AGPAT9 were functionally disrupted, even in the presence of retinoid treatment (Fig. 14c). Associated with MET blockade was a corresponding rescue of the mammosphere-forming capacity of these mesenchymal NAMEC8 cells, clearly highlighting the importance of the TAG synthesis pathway for the epithelial cell state (Fig. 6c and Fig. 14d).

[0262] Since more epithelial cell states appear to be associated with greater lipid storage, we reasoned that in the mesenchymal cell state, fatty acids may be used as an energy source for alternative biochemical pathways, such as fatty acid β-oxidation (FAO). β-Oxidation is a catabolic process by which fatty acids are broken down in the mitochondria to produce acetyl-CoA, which fuels the TCA cycle. Here, we used oxygen consumption rate (OCR) as a readout of TCA cycle activity. Using palmitate as a source of fatty acids, control-treated cells in the mesenchymal state clearly favored palmitate for energy production. In contrast, bexarotene-treated cells that had switched to an epithelial state showed a lower OCR when supplemented with palmitate (Fig. 6d and Fig. 14e,f), thus suggesting that fatty acid utilization and fate differ between cancer cells in different cell states. Mesenchymal cancer cells tend to rely on β-oxidation, while cancer cells in the epithelial state appear to shunt fatty acids to lipid droplet storage.

[0263] Having observed this shift in fatty acid utilization from β-oxidation to lipid storage in retinoid-induced MET, we wondered whether cancer cells in their native cell state also exhibited this metabolic preference. Comparing two isogenic cell lines stably residing in either the epithelial (HMLE) or mesenchymal (NAMEC8) cell state, the former indeed displayed a greater abundance of lipid species favoring TAG production and, ultimately, lipid droplet formation (Fig. 6e, f and Fig. 14g). A higher reliance on β-oxidation was reflected by higher FAO rates in mesenchymal cancer cell lines (BT549, MDA-MB-468, and SUM159) compared to epithelial cancer cell lines (MCF7 and T47D) (Fig. 6g). Importantly, the mesenchymal cancer cell line BT549 had a growth advantage when supplemented with palmitic acid or lipoic acid under nutrient-starved conditions, whereas the epithelial cancer cell line MCF7 did not exhibit this metabolic preference (Fig. 6h).

[0264] Induction of mesenchymal-epithelial transition depends on the fatty acid modifier carnitine palmitoyltransferase

[0265] If cancer cells do rely on β-oxidation to maintain a mesenchymal phenotype, we hypothesized that inhibiting β-oxidation might help promote or stabilize an epithelial phenotype by limiting metabolic pathways that cancer cells rely on. Carnitine palmitoyltransferase I (CPT1) is a key mitochondrial enzyme responsible for catalyzing the transfer of acyl groups from long-chain fatty acyl-CoA to form acylcarnitines. Essentially, this allows for the transport of fatty acids into the mitochondria to fuel the TCA cycle and utilize fatty acids as an energy source. We first showed that exposing mesenchymal cancer cells to the CPT1 inhibitor etolimus, alone or in combination with bexarotene, completely inhibited FAO activity (Fig. 15a). Although we demonstrated that bexarotene alone could induce MET, etolimus alone could not do so. However, the combination of bexarotene and etolimus exerted a more potent MET phenotype that was more persistent and sustained even after at least 7 days of drug removal (Fig. 7a and Fig. 15b, c). Cells treated with the combination therapy also maintained the inhibitory effect on mammosphere formation after drug cessation (Fig. 7b and Fig. 15d).

[0266] To show that the enhancement of MET by addition of etolimus was accurate, we knocked down CPT1A, the major isoform of CPT1, in NAMEC8. Knockdown of CPT1A resulted in increased accumulation of lipid droplets after bexarotene treatment (Figure 7c), which corresponded to a stronger MET phenotype, as shown by a clear decrease in mesenchymal / basal proteins and an increase in luminal proteins (Figure 7d). The greater inhibition of mammosphere formation after bexarotene treatment in CPT1A knockdown cells compared to control cells also showed an enhanced MET phenotype (Figure 7e).

[0267] If mesenchymal cancer cells indeed metabolize fatty acids by β-oxidation, we reasoned that the increased activity of CPT1 through its overexpression would overcome the effects of bexarotene and the induction of an epithelial phenotype. We overexpressed CPT1A in NAMEC8 cells and, surprisingly, this was sufficient to overcome the bexarotene-dependent inhibition of FAO (Fig. 15e). In both cell lines overexpressing CPT1A, cells clearly displayed the absence of lipid droplets even after bexarotene treatment (Fig. 7f and Fig. 15f), further indicating the presence of active FAO in CPT1A-overexpressing cells. This corresponds to the maintenance of a mesenchymal phenotype in CPT1A-overexpressing cells after bexarotene treatment, as evidenced by the growth of bexarotene-treated cells in a dispersed manner (Fig. 15g), the maintenance of mesenchymal markers, and the suppression of the induction of epithelial / luminal markers (Fig. 7g). In mammosphere formation assays performed in CPT1A-overexpressing cell lines, mammosphere formation was rescued even in the presence of bexarotene treatment (Fig. 7h and Fig. 15h); this suggests that the enhancement of fatty acid import into mitochondria for β-oxidation is sufficient to overcome the effects of the cell state changes induced by bexarotene. Thus, while upstream triggers for cell state transitions are necessary, reprogramming of metabolic requirements is essential for strengthening the cell state and appears to be sufficient to overcome the effects of MET inducers.

[0268] Impaired fatty acid oxidation redirects lipids toward a supportive epithelial state and blocks EMT

[0269] Since MET programs the fate of fatty acids toward lipid storage, we reasoned that the reverse process, namely EMT, would lead to the opposite outcome. Using three different cell lines containing inducible expression of the EMT transcription factors Twist, Zeb1, and Slug, respectively (HMLE-Twist-ER, HMLE-Zeb1, MCF7-Slug), we observed abundant lipid droplets when cells were in an epithelial state, which were lost as the cells transitioned to a mesenchymal state (Figure 8a). Using HMLE-Twist-ER cells, we also demonstrated that activation of the EMT program could reprogram the metabolic requirements of cancer cells from lipid storage to β-oxidation, as measured by OCR (Figure 8b and Figure 16a).

[0270] Since changes in metabolic reprogramming during EMT translate into changes in FAO, we hypothesized that inhibition of CPT1 may have an effect on EMT progression. Exposing HMLE-Twist ER cells to 4-OHT induced EMT, and treatment with edolimus was able to significantly prevent this transition (Figure 8c, d). This was replicated in another inducible EMT cell line model, MCF7-Slug+Sox9, where edolimus also blocked Slug+Sox9-induced EMT in MCF7 breast cancer cells (Figure 16b, c, d) 32. The importance of CPT1 to the EMT program was also confirmed by knockdown of CPT1A, which resulted in the inability to induce EMT progression and thus maintain an epithelial phenotype (Figure 8e, f and Figure 16e). This emphasizes the importance of fatty acid utilization in controlling and programming cell states. The role of edolimus in blocking TGFβ-induced EMT is also shown in the lung cancer cell line A549 (Figure 16f and g).

[0271] Finally, we sought to functionally test whether direct perturbations of metabolic pathways, in this case fatty acid oxidation, could alter the EMT-induced metastatic process in an in vivo animal model. We utilized the cell line model MCF7-Slug+Sox9, in which doxycycline-induced concomitant expression of the EMT transcription factors Slug and Sox9 in pre-established MCF7 tumor xenografts can strongly induce EMT in vivo and promote metastasis of otherwise non-metastatic tumor cells from the fat pads of NSG mice to the lungs (Figure 8g) 32. Here, MCF7-Slug+Sox9 cells were transplanted into the fat pads of NSG mice and drug treatment was initiated one week after implantation. One group of mice was administered etolimus (20 mg / kg per day) before doxycycline administration. Another group of mice was administered doxycycline and sham-treated with a blank control at the same time. After 10 weeks, immunofluorescence analysis showed that the primary tumors of control-treated mice were still epithelial in nature and expressed E-cadherin (Figure 8g). In contrast, tumors from doxycycline-treated mice showed hallmarks of EMT, including expression of the mesenchymal marker vimentin and concomitant loss of E-cadherin. However, tumors from doxycycline-treated mice that also received etolimus showed retention of epithelial features, demonstrating blockade of EMT in vivo. More importantly, extensive metastatic nodules were detected in the lungs of doxycycline-treated mice, while the experimental group receiving etolimus had a similar number of metastatic lesions as control-treated mice (Figure 8g). This provides the first proof of concept that fatty acid oxidation is required for activation of the EMT program to generate metastases, and that pharmacological inhibition of this key metabolic pathway may help control breast cancer metastasis.

[0272] Epigenetic regulation controls cell state transitions

[0273] Since EMT is a reversible process, epigenetic mechanisms such as histone modifications and DNA methylation have been found to be important in regulating the expression of EMT-responsive genes. For example, epigenetic silencing of E-cadherin during activation of the EMT program has been well elucidated. In addition, studies have also reported that various epigenetic regulators, such as histone deacetylase inhibitors (HDACi), are able to exert differentiation effects on cancer cells. Therefore, to elucidate the molecular link between EMT and epigenetic regulation, we performed MET and inhibition of EMT screening using the Structural Genomics Consortium (SGC) Epigenetic Chemical Probe Library (Cayman), a comprehensive epigenetic library composed of a variety of epigenetic compounds.

[0274] For the MET screening, good hits corresponded to hits that could induce lower SERPINE1 promoter activity compared to A83-01. The primary screening revealed several hits that were able to induce MET without adversely affecting cell viability (Figure 17A). These hits included inhibitors of protein arginine methyltransferase 5 (PRMT5), G9a / GLP histone methyltransferase, and CREBBP / EP300 bromodomain. They were then examined at six different concentrations to evaluate their dose-dependent changes in SERPINE1 promoter activity with increasing drug concentration, and the MET induction potential of each compound was compared (Figure 17B). All four compounds showed a significant dose-dependent decrease in SERPINE1 promoter activity with increasing concentration. Afterwards, they were subsequently subjected to in vitro validation experiments to test their efficacy in promoting MET in mesenchymal TNBC cells.

[0275] Systematic validation of epigenetic hits identifies CREBBP / EP300 bromodomain inhibitors as potent MET inducers

[0276] From the MET screening, the four most effective hits in reducing SERPINE1 promoter activity (Figure 17C, circled in red) were selected for in vitro validation studies. Namely, these compounds included: PRMT5 inhibitors - LLY-283 and GSK591, inhibitors of histone methyltransferases G9a and GLP - UNC0642, and CREBBP and EP300 bromodomain inhibitors - SGC-CBP30. These compounds were first treated in mesenchymal NAMEC8 cells to morphologically verify whether they could induce changes in cell state. As shown in Figure 3C, treatment with SGC-CBP30 alone resulted in a significant change in cell morphology, from scattered spindles and fibroblast-like cells to compact epithelial island clusters, which are characteristic of epithelial-like morphology. This suggests that SGC-CBP30 may be a stronger agent in inducing MET in mesenchymal cells compared to the other hits. On the other hand, the other three compounds were not observed to induce epithelial-like morphology in NAMEC8 (Figure 17C).

[0277] Cells treated with hits were then evaluated for changes in transcriptional and protein expression of EMT and basal / luminal markers (Figures 17D and E). Similar to the morphological observations, SGC-CBP30 proved to be most effective in inducing an epithelial phenotype. SGC-CBP30 significantly increased the expression of the epithelial marker E-cadherin and downregulated several mesenchymal markers, fibronectin, ZEB1, and SLUG. In contrast, no significant changes were observed in cells treated with LLY-283, GSK591, and UNC0642. In addition, since the mesenchymal phenotype is often associated with basal-like cells and the interest of the current study is in TNBC, basal and luminal markers were also evaluated during drug treatment. It was observed that SGC-CBP30 treatment increased mucin 1 (MUC1), a glycoprotein commonly expressed in luminal breast cancer (Figures 17D and E). Therefore, this suggests that SGC-CBP30 can not only induce MET, but also mediate the acquisition of luminal-like features in basal-like mesenchymal cells.

[0278] When the cells were resuspended in mammosphere culture medium, it was observed that pretreatment with LLY-283 (p < 0.01), GSK591 (p < 0.01), and SGC-CBP30 (p < 0.05) significantly reduced the size and number of spheres formed. In contrast, cells pretreated with DMSO were able to efficiently form spheres (Figure 17F). Therefore, this indicates that the in vitro stemness of mesenchymal NAMEC8 cells was reduced after treatment with these compounds. In addition, the typical characteristic of mammary stem cell-like cells is the presence of CD44high / CD24- / low cell surface markers. Therefore, after treatment with SGC-CBP30, the expression of these cell surface markers was analyzed by flow cytometry, as this compound showed a stronger MET-inducing potential in various validation assays. Figure 3G shows that a cell population with lower CD44 expression was observed by SGC-CBP30 treatment, indicating that the expression of this classic stem cell marker was suppressed. This indicates the loss of stem cell properties when NAMEC8 cells are treated with SGC-CBP30. The reduction in stem cell-like properties after treatment strongly suggests a transition to a mesenchymal cell state, as stem cell-like features are associated with a mesenchymal phenotype.

[0279] To ensure that the effects of these hits in promoting cell state transitions could be recapitulated in cancer cells, these compounds were further evaluated in the TNBC cell line SUM159. Similar to what was demonstrated in NAMEC8, SGC-CBP30 was observed to be a stronger MET inducer compared to the other hits, increasing epithelial features and reducing stem cell properties in SUM159 (Figure 18A-D). When SUM159 cells were analyzed by flow cytometry for the expression of CD44 and CD24 cell surface markers after SGC-CBP30 treatment, the cells exhibited a slight decrease in CD44 expression and a population of cells gained CD24 expression (Figure 3I). This indicates that upon inhibition of the CREBBP / EP300 bromodomain in SUM159, a transition from a stem cell-like state (CD44high / CD24- / low) to a non-stem cell-like state (CD44low / CD24high) was observed. Therefore, these data suggest that SGC-CBP30 can not only reduce mesenchymal and basal-like features, but also induce the loss of TNBC stem cells, thereby potentially reducing its tumorigenicity and invasiveness.

[0280] Inhibition of the histone acetyltransferase domain of CREBBP / EP300 proteins fails to induce efficient MET transformation

[0281] Since CREBBP and EP300 contain both bromodomains and HAT domains, we next explored whether inhibition of the HAT domains of these proteins would induce a similar phenotype as inhibition of the bromodomains. When NAMEC8 was treated with A-485, a potent and selective HAT inhibitor of CREBBP / EP300, tight clusters of epithelial islands were observed, indicating epithelial-like cell morphology (Figure 19A). A-485 treatment also demonstrated upregulation of E-cadherin expression and a slight reduction in mesenchymal markers (Figure 19B). These data suggest that, similar to SGC-CBP30, inhibition of the HAT domains of these proteins appears to promote MET in mesenchymal cells. However, when the in vitro stem cell properties of A-485-treated cells were assessed by mammosphere assay, the sphere counts were not significantly different from those of DMSO-treated cells (Figure 19C), indicating that A-485 has limited potential in reducing the stem cell properties of mesenchymal cells. Interestingly, when the expression of CD44 and CD24 cell surface markers of NAMEC8 cells was analyzed after A-485 treatment, a significant increase in CD44 expression was observed (Figure 19D). Therefore, the data indicate that inhibition of the HAT domain of CREBBP / EP300 proteins can only induce an epithelial-like phenotype in NAMEC8 cells, but cannot reduce stem cell properties in vitro.

[0282] Since CREBBP and EP300 are acetyltransferases, we next assessed the three most common acetylated histone marks, lysine 14, 18, and 27 of histone H3 (H3K14ac, H3K18ac, and H3K27ac), after treatment with SGC-CBP30 and A-485. The data showed that all acetylation marks were reduced after SGC-CBP30 treatment, but the reduction was less than that after A-485 treatment (Figure 19E). Since SGC-CBP30 was shown to be more effective than A-485 in MET conversion, this suggests that cell state transition may not only be attributed to changes in acetylation status, but the involvement of bromodomains is also essential. However, more work is needed to clarify whether bromodomains and HATs do regulate different target genes and proteins, and to explore the effects of bromodomain inhibition on MET conversion. By clarifying the mechanism of how the bromodomains of CREBBP / EP300 specifically induce cell state transitions in mesenchymal cells, it may serve as a new treatment for TNBC.

[0283] CREBBP / EP300 bromodomain inhibition reduces tumor burden in basal-like TNBC in vivo

[0284] After establishing that CREBBP / EP300-mediated epigenetic changes can induce MET and reduce the stem-like properties of basal-like, mesenchymal TNBC cells, we next investigated the tumorigenic potential of TNBC in response to CREBBP / EP300 bromodomain and HAT inhibition to determine if it could reduce tumor burden in mice. When NAMEC8-Ras cells were pretreated with SGC-CBP30 and A-485 before subcutaneous injection into immunodeficient NODscidγ (NSG) mice, SGC-CBP30 treatment alone resulted in significantly smaller tumor cells after 11 weeks compared to DMSO treatment. Tumor formation was still observed after A-485 pretreatment (Figure 19F). This complements the in vitro findings that SGC-CBP30 is more effective in MET transformation of mesenchymal cells compared to A-485. Therefore, this suggests that altering gene regulation of TNBC cells through CREBBP / EP300 bromodomain inhibition reduces the tumorigenicity of the cells. However, the in vivo studies need to be repeated to ensure that this observation is consistent and reproducible. Furthermore, histological analysis of tumor tissues is needed to determine if there are any morphological differences between tumors formed by DMSO and drug-treated cells. Nevertheless, our initial in vivo data suggest that inhibition of the bromodomains of CREBBP / EP300 proteins can reduce the tumorigenic potential of highly aggressive TNBC.

[0285] Together, these data suggest that CREBBP and EP300 proteins may play a role in maintaining the mesenchymal cell state. Inhibition of the bromodomains of these two coactivator proteins may result in reduced transcriptional regulation and epigenetic modifications of genes necessary to maintain cells in a mesenchymal state. Therefore, we hypothesized that the bromodomains of CREBBP and EP300 may regulate mesenchymal genes associated with super enhancers. These genes would be hyperacetylated, leading to greater transcriptional activation, as mediated by the CREBBP / EP300 bromodomains (Figure 19G). Inhibition of the bromodomains would hinder the binding of coactivators and transcription factors to the promoters of these mesenchymal genes, leading to reduced acetylation and subsequent gene transcription. Therefore, we hypothesized that the induction of MET may be due to reduced transcription of genes that are important for maintaining the mesenchymal cell state (Figure 19G).

[0286] Phenotypic screening of MET inducers targeting metabolic targets

[0287] Phenotypic Screening of MET Inducers Against Metabolic TargetsNAMEC8-SERPINE1 cells were used to screen a 303-compound library (MedChem Express) against metabolic targets to identify MET inducers. Fifteen compounds were identified as strong MET inducers, and we selected six compounds for in vitro orthogonal validation: glutaminase C-IN-1, GSK256066, GSK2837808A, AGI-6780, SW033291, and NCT-503 (Figures 20A and B). Dual-glo luciferase assays were performed in NAMEC8-SERPINE1 cells treated with the six selected compounds at increasing concentrations from 10 nM to 2 uM. A dose-dependent decrease in SERPINE1 promoter activity was observed with compounds glutaminase C-iN-1, GSK256066, and AGI-6780 (Figure 20C).

[0288] To investigate any possible association between glutaminase and the mesenchymal phenotype, Western blot analysis of glutaminase was performed in a panel of epithelial and mesenchymal breast cancer cell lines. Mesenchymal breast cancer cell lines were found to express higher levels of GLS1 compared to epithelial breast cancer cell lines, suggesting that GLS1 may be a determinant of the mesenchymal cell state and a potential biomarker for triple-negative breast cancer (Figure 20D).

[0289] For in vitro validation of MET, NAMEC8 cells were treated with C-IN-1. Morphological changes from the dispersed distribution usually observed in mesenchymal cells to compact epithelial island clusters were induced after C-IN-1 treatment (Figure 20E). Corresponding changes in molecular markers were observed by Western blotting after C-IN-1 treatment, with a decrease in mesenchymal proteins Zeb1, fibronectin, Slug, and Snail, an increase in epithelial E-cadherin, a decrease in basal cytokeratin 5, and an increase in luminal markers cytokeratin 8, mucin 1, and ER (Figure 20F). This provides molecular evidence for the induction of MET after glutaminase inhibition.

[0290] GLS1 was knocked down in NAMEC8 using 3 shRNAs (Figure 20G) and then treated with paclitaxel to study changes in chemosensitivity after MET induction. A decrease in the IC50 for paclitaxel was observed in GLS1sh2 cells compared to Ctrlsh cells (Figure 20H), indicating increased chemosensitivity after conversion to an epithelial phenotype. Mammosphere assays were also performed as an in vitro assay of stem cell properties. A decrease in the number of spheres formed was observed in GLS1sh2 and sh3 cells compared to Ctrlsh cells, indicating reduced stem cell properties (Figure 20I). Gene knockout of GLS1 was also performed in NAMEC8-HRas cells and injected subcutaneously into the flanks of NSG mice. Reduced tumor growth was observed in GLS1sh2 cells compared to Ctrlsh cells (Figures 20J and K). This indicates a reduced tumorigenic potential after induction of MET.

[0291] Example 3

[0292] discuss

[0293] Alterations in cell state represent an emerging approach to control cancer cell responses to therapy. Retinoids have been used as first-line adjuvants with curative intent in acute promyelocytic leukemia and cutaneous T-cell lymphoma. However, in solid tumors, we demonstrate that retinoids act as inducers of cell state transitions, which allow otherwise resistant breast cancer cells to respond better to standard therapy and thus may be used clinically in combination therapies. Furthermore, retinoid-induced MET also converts cells from a more aggressive mesenchymal state to a less metastatic epithelial state. In small cohort studies of patients with metastatic breast cancer, retinoid treatment did not produce responses, likely due to a lack of tumor molecular subtype stratification and patient selection. Based on the new information generated by our study, we show that basal-like TNBC, which is more mesenchymal in nature, is more likely to respond. This highlights the need for biomarkers that can stratify potential responders from non-responders.

[0294] Immune checkpoint blockade therapy has shown significant benefits for certain cancers. The appeal of harnessing the immune system lies in its commonality—no matter how diverse the molecular changes in a cancer, the immune system has a natural advantage in its intrinsic ability to recognize and attack cancer cells. While immune checkpoint (PD1, PD-L1) inhibitors are effective in treating certain human cancers, such as melanoma and lung cancer, they have not achieved similar success in ER+ / PR+ breast cancer; they are currently in active clinical studies for metastatic TNBC. Keynote012 reported an 18% response rate with pembrolizumab monotherapy in a phase 1 study. Keynote086, a phase 2 pembrolizumab monotherapy study showed a 5% response rate. Some studies have shown a potential association between cancer cell state and the expression of immune markers such as PD-L1 in cell-based models. The transition from a mesenchymal to an epithelial cell state may trigger intrinsic molecular changes that now cause tumor cells to become more vulnerable to attack by immune cells, thereby improving the success of immunotherapy for breast cancer.

[0295] Mechanistically, several pathways may be able to achieve the conversion to the MET phenotype. In our study, retinoids were the most potent class of such inducers identified in an unbiased manner. We demonstrated for the first time that they act by rewiring lipid metabolism during cell-state transitions (Figure 7I). Whether lipid metabolism is indeed a driver or a consequence of cell-state changes remains to be determined. Inhibition of β-oxidation with etolimus was not sufficient to induce MET, suggesting that other pathways are also necessary or that simultaneous inhibition of several enzymes of the FAO pathway is required. However, CPT1A overexpression alone was sufficient to block bexarotene-induced MET, and the effects of bexarotene were prolonged by etolimus treatment, suggesting that metabolic reprogramming is a necessary feature that enables cells to transition between cell states as well as to maintain the transition. In addition to TNBC, MET conversion may be broadly applicable to cancers that have mesenchymal features and trigger their response to other therapies.

[0296] Why do differences in cancer cell states impose unique metabolic requirements? In embryonic stem cells, glutamine and α-ketoglutarate (αKG) support pluripotency by increasing the ratio of αKG to succinate, promoting DNA and histone demethylation. In cancer, cell states control proliferation, invasiveness, and tolerance to stress, which require stringent requirements for specific metabolites. For example, asparagine availability generally promotes EMT-driven metastasis or tumor growth, whereas cisplatin-resistant cancer cells appear to rely on glutamine for nucleotide biosynthesis. In our study, directing fatty acids to β-oxidation for energy production may allow cancer cells in the mesenchymal state to tolerate fluctuations in nutrient levels during invasion and metastasis, whereas the more proliferative epithelial state may preferentially employ aerobic glycolysis even under glucose-rich conditions, which is typical of bulk tumor cells. Thus, addiction to specific metabolic pathways during different steps of cancer progression could be used to target cell states and open new avenues for antimetabolite therapeutic strategies.

[0297] Table 3: Key resources

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306] Table 4: Gene ontology pathway enrichment analysis of upregulated genes after bexarotene treatment in NAMEC8. Immune response pathways and genes are highlighted below.

[0307] A GO biological process

[0308] Regulation of cell proliferation (CO: 0042127)

[0309] Cytokine-mediated signaling pathways (CO: 0019221)

[0310] Regulation of angiogenesis (CO: 0045765)

[0311] Positive regulation of cell death (GO:0010942)

[0312] Glycosaminoglycan biosynthesis process (GO: 006024)

[0313] Positive regulation of angiogenesis (GO: 0045766)

[0314] Cellular response to reduced oxygen levels (GO:0036294)

[0315] Positive regulation of cell proliferation (GO: 0008284)

[0316] Cellular response to type I interferon (CO: 0071357)

[0317]

[0318] B) WikiPathway analysis of upregulated genes after bexarotene treatment in NAMEC8. Immune response pathways and genes are highlighted below.

[0319] BWikiPathwavs

[0320] Nuclear receptor meta-pathway WP2882

[0321] II Interferon Signaling (IFNG) WP619

[0322] Pathway WP4018 for clear cell renal cell carcinoma

[0323] Benzopyrene Metabolism WP696

[0324] VEGFA-VEGFR2 signaling pathway WP3888

[0325] Serotonin transporter activity WP1455

[0326] Complement activation WP545

[0327] Photodynamic therapy-induced HIF-1 survival signal WP3614

[0328] Hematopoietic stem cell differentiation WP2849

[0329] Prostaglandin Synthesis and Regulation WP98

[0330] Sequence Listing <110> A*STAR <120> Methods to control cell state transitions in cancer <130> S61013719 <160> 68 <170> PatentIn version 3.5 <210> 1 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Forward primer for CDH1 <400> 1 atggctcaag aaacatttgt cat 23 <210> 2 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for CDH1 <400> 2 gagcttgcgg cccgaat 17 <210> 3 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> CD24 forward primer <400> 3 gagaacagga tgccccttag aat 23 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for CD24 <400> 4 aaatctgcgt gggtaggagc a 21 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CD44 forward primer <400> 5 atcttgccac agccactgat 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for CD44 <400> 6 tcggaagttg gctgcagttt 20 <210> 7 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Forward primer for SERPINE 1 <400> 7 cgggcagctc gaagaagtg 19 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for SERPINE 1 <400> 8 tagaggaata gcgggggatc at 22 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer for CD166 <400> 9 agggggagga gagagagatt cg 22 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for CD166 <400> 10 gcggagatca agaggcagaa 20 <210> 11 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Forward primer for ZEB1 <400> 11 cggcggatcg attcagctta tttattcca 29 <210> 12 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for ZEB1 <400> 12 cggtaaacta gtgatcctct cgcttgtgt 29 <210> 13 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer for CDH1 <400> 13 ttgcaccggt cgacaaagga c 21 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for CDH1 <400> 14 tggattccag aaacggaggc c 21 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> VIM forward primer <400> 15 acccgcacca acgagaaggt 20 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for VIM <400> 16 attctgctgc tccaggaagc g 21 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SLUG forward primer <400> 17 taccgctgct ccattccacg 20 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for SLUG <400> 18 catgggggtc tgaaagcttg g 21 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> GAPDH forward primer <400> 19 tggcaaattc catggcaccg 20 <210> 20 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for GAPDH <400> 20 cgccccactt gattttggag g 21 <210> twenty one <211> 19 <212> DNA <213> Artificial sequence <220> <223> Forward primer for SERPINE1 <400> twenty one gcaccacaga cgcgatctt 19 <210> twenty two <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for SERPINE1 <400> twenty two acctctgaaa agtccacttg c 21 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for ZEB1 <400> twenty three tgcactgagt gtggaaaagc 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for ZEB1 <400> twenty four tggtgatgct gaaagagacg 20 <210> 25 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Forward primer for MUC1 <400> 25 tgccgccgaa agaactacg 19 <210> 26 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for MUC1 <400> 26 tggggtactc gctcatagga t 21 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for KRT5 <400> 27 caaggttgat gcactgatgg 20 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for KRT5 <400> 28 tgtcagagac atgcgtctgc 20 <210> 29 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for KRT8 <400> 29 cagaagtcct acaaggtgtc ca 22 <210> 30 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for KRT8 <400> 30 ctctggttga ccgtaactgc g 21 <210> 31 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer for FN1 <400> 31 gagaatggac ctgcaagccc a 21 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for FN2 <400> 32 agtgcaagtg atgcgtccgc 20 <210> 33 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> GREB1 forward primer <400> 33 atgggaaatt cttacgctgg ac 22 <210> 34 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for GREB1 <400> 34 cactcggcta ccaccttct 19 <210> 35 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer for ABCA3 <400> 35 ctcctctgga agaactacac cc 22 <210> 36 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for ABCA3 <400> 36 gggcacattt tccgactgaa tc 22 <210> 37 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer for NRIP1 <400> 37 ggatcaggta ctgccgttga c 21 <210> 38 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Reverse primer for NRIP1 <400> 38 ctggaccatt actttgacag gtg 23 <210> 39 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer for TFF1 <400> 39 ccctcccagt gtgcaaataa g 21 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for TFF1 <400> 40 gaacggtgtc gtcgaaacag 20 <210> 41 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> LIPE forward primer <400> 41 tcagtgtcta ggtcagactg g 21 <210> 42 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for LIPE <400> 42 aggcttctgt tgggtattgg a 21 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for PNPLA2 <400> 43 ggcttcctcg gcgtctacta 20 <210> 44 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PNPLA2 <400> 44 tttaccaggt tgaaggaggg g 21 <210> 45 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer for ACSL4 <400> 45 catccctgga gcagatactc t 21 <210> 46 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for ACSL4 <400> 46 tcacttagga tttccctggt cc 22 <210> 47 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer for ACSL5 <400> 47 ctcaacccgt cttacctctt ct 22 <210> 48 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for ACSL5 <400> 48 gcagcaactt gttaggtcat tg 22 <210> 49 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer for AGPAT4 <400> 49 ctcagggcta atcatcaaca cc 22 <210> 50 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for AGPAT4 <400> 50 gcttgagatg caataggaca gt 22 <210> 51 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Forward primer for AGPAT9 <400> 51 cgctggttct cggcttcat 19 <210> 52 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for AGPAT9 <400> 52 tggcccactc taaagttttc ac 22 <210> 53 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> GPAM forward primer <400> 53 gatgtaagca cacaagtgag ga 22 <210> 54 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Reverse primer for GPAM <400> 54 tccgactcat taggctttct ttc 23 <210> 55 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer for PPAP2A <400> 55 ggcaggttgt ccttctattc ag 22 <210> 56 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PPAP2A <400> 56 cagtgtgggg cgtaagagt 19 <210> 57 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Forward primer for DGAT2 <400> 57 attgctggct catcgctgt 19 <210> 58 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Reverse primer for DGAT2 <400> 58 gggaaagtag tctcgaaagt agc 23 <210> 59 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer for PLIN1 <400> 59 tgtgcaatgc ctatgagaag g 21 <210> 60 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PLIN1 <400> 60 agggcgggga tcttttcct 19 <210> 61 <211> 20 <212> DNA <213> Artificial sequence <220> <223> MGLL forward primer <400> 61 atgccagagg aaagttcccc 20 <210> 62 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for MGLL <400> 62 cgtctgcatt gaccaggtg 19 <210> 63 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for ESR1 <400> 63 cccactcaac agcgtgtctc 20 <210> 64 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Reverse primer for ESR1 <400> 64 cgtcgattat ctgaatttgg cct 23 <210> 65 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer for PTGS1 <400> 65 cgccagtgaa tccctgttgt t 21 <210> 66 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PTGS1 <400> 66 aaggtggcat tgacaaactc c 21 <210> 67 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer for HADHB <400> 67 ctgtccagac caaaacgaag aa 22 <210> 68 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for HADHB <400> 68 cgatgcaaca aacccgtaag c 21

Claims

1. An in vitro or ex vivo method for inducing mesenchymal-epithelial transition, referred to as MET, in cancer cells, the method comprising: The cells are contacted with a mesenchymal-epithelial transition inducer for a time and under conditions sufficient to induce MET in the cells, wherein the cancer cells are triple-negative breast cancer cells, and wherein the MET inducer is SGC-CBP30.

2. The method of claim 1, wherein: The cancer cells are those that are resistant to standard of care therapy.

3. The method of claim 1, wherein: The method further comprises: after inducing MET in the cancer cells, contacting the cells with a chemotherapeutic agent, an epigenetic modifying compound, or an immunomodulatory agent.

4. The method of claim 3, wherein: The chemotherapeutic agent is paclitaxel or 5-fluorouracil.

5. The method of claim 3, wherein: The epigenetic modifying compound is a class I HDAC inhibitor selected from CI-994 or SBHA.

6. The method of claim 1, wherein: The method further comprises contacting the basal-like or mesenchymal-like cancer with a lipid metabolism modulator.

7. The method of claim 6, wherein: The lipid metabolism regulator is etolimus.

8. The method of claim 6, wherein: The lipid metabolism regulators are inhibitors of enzymes involved in the fatty acid oxidation pathway.

9. The method of claim 6, wherein: The lipid metabolism regulator is selected from glycerol-3-phosphate acetyltransferase, 1-acylglycerol-3-phosphate O-acyltransferase, and diacylglycerol acyltransferase.

10. Use of a mesenchymal-epithelial transition, or MET, inducer in the preparation of a medicament for treating triple-negative breast cancer in a subject, wherein: The MET inducer is SGC-CBP30; And wherein the drug is administered to the patient in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulator or a lipid metabolism inhibitor.

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