Methods for manipulating cancer cell state transitions

Abstract

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

Description

[Technical field]

[0001] The present invention relates to the field of cancer biology. In particular, a method for inducing mesenchymal-epithelial transition (MET) in cancer cells is provided. [Background technology]

[0002] Breast cancer is a heterogeneous disease that is classically stratified by receptor expression status. Therefore, different therapeutic strategies are required to achieve optimal disease control. Estrogen receptor positive (ER+) and ERRB2 / HER2 positive tumors typically respond well to targeted therapies, but 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 pioneering new adjuvant regimens in the clinical management of TNBC, and phase II and III trials of therapeutic agents for TNBC have shown little promise. This highlights the need to explore more effective therapeutic strategies for TNBC. Activation of the epithelial-mesenchymal transition (EMT) program in cancer cells has often been attributed to the acquisition of a resistant phenotype by acquiring cancer stem cell-like properties. Several studies have conceptually suggested that reversal of the EMT program, i.e., mesenchymal-epithelial transition (MET), could result in restored sensitivity of cancer cells to chemotherapy.

[0003] Receptor status is one of the major components of clinical classification, prognosis, and management decisions, and gene expression profiling has been applied to classify breast cancer into several “intrinsic” molecular subtypes: luminal A, luminal B, Her2-rich, basal-like, and claudin-low. Although somewhat controversial, it has been observed that ER+ tumors tend to be luminal-like and express “epithelial” marker genes such as E-cadherin, whereas TNBCs have a basal-like gene signature and express “mesenchymal” markers including claudins and vimentin. Whether the acquisition of “epithelial” or “mesenchymal” features during cell state transitions actually leads to luminal-like or basal-like cancers, respectively, remains unclear. It may be conceivable that MET, beyond a change in cell state, may be capable of transforming basal-like breast cancers into more luminal-like breast cancers characterized by expression of ER or luminal cytokeratins.

[0004] It is well established that the EMT program is regulated by alterations in extracellular and intracellular signaling pathways. For example, exogenous signals such as TGFβ and PDGF lead to the activation of intracellular signaling cascades that ultimately result in 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 less clearly defined. For example, forskolin-induced MET acts through activation of the cAMP pathway leading to epigenetic reprogramming, and TGFβ inhibitors have been reported to mediate MET in germline stem cells. In both cases, the exact mechanisms remain to be elucidated. Moreover, although in principle it is conceivable that biochemical or genetic modifications of potential pathways such as cAMP activation or TGFβ receptor inhibition could help promote MET, their potential clinically relevant applications remain to be seen.

[0005] Although the signaling pathways and transcriptional regulators that control cell states are well understood, other new aspects, especially metabolism, that contribute to cancer cell function, remain less understood. Metabolic alterations have re-emerged as a defining feature of tumors, highlighting the role of metabolic pathways in regulating tumor cell behavior and their therapeutic implications. The majority of research has focused on evaluating the metabolic differences between cancer cells and normal cells, especially the glycolysis pathway. Due to the phenotypic diversity and plasticity between cancer cells, little is known about the association and contribution of their metabolic features to functional adaptation. It is clear that the transition of cancer cells between cell states requires different energy requirements to support such functions. Summary of the Invention [Means for solving the problem]

[0006] Provided herein is a method for inducing mesenchymal-epithelial transition (MET) in cancer cells. In one aspect, a method for inducing mesenchymal-epithelial transition (MET) in cancer cells is provided, comprising contacting the cells with an inducer of mesenchymal-epithelial transition 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. Such cancer cells belong to mesenchymal cancer subtypes.

[0007] In one aspect there is provided an inducer of mesenchymal-epithelial transition (MET) for use in inducing mesenchymal-epithelial transition in cells of a patient.

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

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

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

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

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

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

[0014] In one aspect there is provided the use of an inducer of mesenchymal-epithelial transition (MET) in the manufacture 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 a cell is provided, comprising: a) contacting the cells with a test agent to be screened; and b) detecting expression from the promoter of a gene selected from the group consisting of CD24, CD44, CD166, CDH1, ZEB1, and SERPINE 1; Including, Methods are provided in which an alteration in expression of one or more genes relative to a control indicates that the test agent is capable of inducing mesenchymal-epithelial transition of cells.

[0016] In one aspect, a method is provided for identifying a cancer patient who will be responsive to a combination therapy comprising a mesenchymal-epithelial transition (MET)-inducing compound in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor or modulator of lipid metabolism, comprising detecting a mesenchymal cancer subtype in a sample obtained from the patient, wherein the presence of the mesenchymal cancer subtype indicates that the patient will be responsive to the combination therapy.

[0017] In one aspect, there is provided a method for identifying and treating a cancer patient who is responsive to a combination therapy comprising a mesenchymal-epithelial transition (MET)-inducing compound in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor or modulator of lipid metabolism, comprising detecting a mesenchymal cancer subtype in a sample obtained from the patient, wherein the presence of the mesenchymal cancer subtype indicates that the patient will be responsive to the combination therapy, and treating the patient with the combination therapy.

[0018] In one aspect, a method is provided for inhibiting epithelial-mesenchymal transition (EMT) of a cancer in a subject, 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 a cancer in a subject.

[0019] In one aspect, there is provided an inhibitor or regulator of lipid metabolism for use in inhibiting epithelial-mesenchymal transition (EMT) in a cancer in a subject.

[0020] In one aspect, there is provided the use of an inhibitor or regulator of lipid metabolism in the manufacture of a medicament for inhibiting epithelial-mesenchymal transition (EMT) of a cancer in a subject.

[0021] In one aspect, a method is provided for inhibiting or preventing metastasis of cancer in a subject, the method comprising administering an inhibitor or regulator of lipid metabolism for a time and under conditions sufficient to inhibit or prevent metastasis in the subject.

[0022] In one aspect there is provided an inhibitor or regulator of lipid metabolism for use in inhibiting or preventing metastasis of cancer in a subject.

[0023] In one aspect there is provided the use of an inhibitor or regulator of lipid metabolism in the manufacture of a medicament for inhibiting or preventing metastasis of cancer in a subject.

[0024] Certain specific embodiments are illustrated by the following figures: It should be understood that the following description is merely for purposes of illustrating certain embodiments and is not intended to be limiting with respect to the description. [Brief description of the drawings]

[0025] [Figure 1A] Figure 1 High-throughput chemical library screening identifies retinoids as promoters of mesenchymal-epithelial transition. (A) Generation of reporter cell lines for MET screening using small molecule compound libraries. [Figure 1B] (B) High-throughput chemical library screening identifies retinoids as promoters of mesenchymal-epithelial transition. (C) Dual-Glo luciferase assay of NAMEC8-SERPINE1prom-Luc cells treated with A83-01. Data are presented as mean FF / Ren+ / -SEM, n=3, **P<0.01 by Student's t-test. [Figure 1C]High-throughput chemical library screening identifies retinoids as promoters of mesenchymal-epithelial transition. (C) Dual-Glo luciferase assay of NAMEC8-SERPINE1prom-Luc cells screened with LOPAC, an anticancer drug, kinase inhibitor, and bioactive lipid library. Data are presented as mean FF / Ren, n=3. Hits are circled in red. [Figure 1D] High-throughput chemical library screening identifies retinoids as promoters of mesenchymal-epithelial transition. (D) Scatter plots of primary and secondary screens of 61 compounds. FF / Ren values ​​from both screens were plotted and linear trend lines were calculated. [Figure 1E] High-throughput chemical library screening identifies retinoids as promoters of mesenchymal-epithelial transition. (E) Dual-Glo luciferase assay of 41 compounds performed in a 5-point dose-response. Compounds are grouped into 9 classes according to physiological function. Data are presented as mean FF / Ren, n=3. Grey boxes represent doses that resulted in <50% cell viability from DMSO. [Figure 2A] Figure 1 shows that retinoids induce the transformation of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells 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. [Figure 2B] (B) Retinoids induce the conversion of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells to chemotherapy. (C) Expression of EMT and lineage marker genes in bexarotene-treated NAMEC8 cells compared to DMSO. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001 by Student's t-test. [Figure 2C]Retinoids induce the conversion of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells to chemotherapy. (C) Protein expression of EMT and lineage markers in DMSO-treated vs. bexarotene-treated NAMEC cells. Bexarotene was treated at increasing concentrations of 0.1 μM, 0.5 μM, and 1 μM. β-actin was used as a loading control. [Figure 2D] (D) Immunofluorescence staining of EMT and lineage markers in DMSO-treated vs. bexarotene-treated NAMEC8 cells. Images are taken at 100x magnification. Scale bar represents 50 μm. [Figure 2E] (E) Flow cytometry histogram plots of CD44 staining in DMSO-treated vs. bexarotene-treated NAMEC8 cells. [Figure 2F] Retinoids induce the conversion of basal-like breast cancer cells to luminal-like, enhancing the sensitivity of cancer cells to chemotherapy. (F) Mammosphere formation assay of DMSO-treated vs. bexarotene- or TTNPB-treated NAMEC8 cells. Images are taken at 50x magnification. Scale bar represents 100 μm. Bottom right: Bar graph showing number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, **P<0.01. [Figure 2G] Retinoids induce the conversion of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells to chemotherapy. (G) Protein expression of EMT and lineage markers in BC2.2, BC22.1, BC29.1, MetBr-007 DR, BC24.1, BC33.1: DMSO-treated vs. bexarotene-treated cells. β-actin was used as a loading control. [Figure 2H]Retinoids induce the conversion of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells to chemotherapy. (H) Immunofluorescence staining of EMT and lineage markers in BC2.2:DMSO-treated vs. bexarotene-treated cells. Images are taken at 100x magnification. Scale bar represents 50 μm. [Figure 2I] Retinoids induce the conversion of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells to chemotherapy. (I) Mammosphere formation assay of DMSO-treated vs. bexarotene-treated BC2.2 cells. Images were taken at 50x magnification. Scale bar represents 100 μm. Right: Bar graph showing number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, **P<0.01. [Figure 2J] (J) Cytomorphological images of NAMEC8 cells treated with bexarotene in the presence or absence of 10 μM 5-FU. Images were taken at 50× magnification. [Figure 2K] (K) Dose response graph of NAMEC8 DMSO vs. bexarotene treated cells to various concentrations of 5-FU. IC50 values ​​are shown in the table below. Data are expressed as mean + / - SEM, n=3. [Figure 2L] Figure 1: Retinoids induce the conversion of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells to chemotherapy. (L) Schematic of the process of generating SUM159 PAC-R cells (top). Dose-response graph of SUM159 PT vs. PACR cells to various concentrations of PAC (bottom). IC50 values ​​are shown in the table. Data are expressed as mean + / - SEM, n=3. [Figure 2M]Figure 1 shows that retinoids induce the conversion of basal-like breast cancer cells to luminal-like and enhance the sensitivity of cancer cells to chemotherapy. (M) Dose-response graph of SUM159 PACR for DMSO vs. bexarotene treatment to various concentrations of 5-FU. IC50 values ​​are shown in the table. Data are expressed as mean + / - SEM, n=3. [Figure 3A] Retinoid therapy promotes MET and reduces the tumorigenicity of TNBC cells in animals. (A) Tumor measurements of SUM159 pretreated with DMSO, bexarotene, or TTNPB prior to subcutaneous injection in NSG mice (top). Data are expressed as mean diameter + / - SEM, n=6 for DMSO, n=8 for bexarotene and TTNPB, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Representative images of tumors from mice sacrificed 7 and 8 weeks after implantation (bottom). [Figure 3B] Retinoid therapy promotes MET and reduces the tumorigenicity of TNBC cells in animals. (B) Tumor size of MCF7-Ras pretreated with DMSO, bexarotene, or ATRA prior to subcutaneous injection in NSG mice (top). Data are expressed as mean diameter + / - SEM, n=4 for DMSO, n=6 for bexarotene and ATRA. Representative images of tumors from mice sacrificed 9 weeks after implantation (bottom). [Figure 3C] Retinoid therapy promotes MET and reduces the tumorigenicity of TNBC cells in animals. (C) Schematic of the workflow of mouse xenograft experiments (top). Tumor size of NAMEC8-Ras (left), BC2.2 (middle), and BC22.1 (right) in control versus bexarotene-fed mice (bottom). Data are expressed as mean diameter + / - SEM, n=6 for control, n=4 for bexarotene (NAMEC8-Ras), n=7 for control and bexarotene (BC2.2 and BC22.1), *P<0.05, **P<0.01. [Figure 3D](D) Hematoxylin and eosin staining of NAMEC8-Ras tumors formed in control versus bexarotene-fed mice. Images were taken at 200× magnification. Scale bar represents 50 μm. [Figure 3E] Retinoid therapy promotes MET and reduces the tumorigenic potential of TNBC cells in animals. (E) Immunofluorescence staining of EMT and lineage markers in NAMEC8-Ras tumor sections from control vs. bexarotene-fed mice. Images were taken at 100× magnification. Scale bar represents 50 μm. [Figure 3F] (F) Gene expression of retinoic acid-responsive genes in NAMEC8-Ras (top) and BC2.2 (bottom) tumors formed in bexarotene-fed mice compared to tumors in control mice. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01. [Figure 4A] Figure 1 shows that epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (A) Dual-glo luciferase activity plots of NAMEC8-SERPINE1prom-Luc cells treated with epigenetic compounds. Data are presented as mean FF / Ren, n=3. Hits are circled in red. [Figure 4B] Epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (B) Dual-glo luciferase activity plots of NAMEC8-SERPINE1prom-Luc cells treated with bexarotene + individual epigenetic compounds. Data are presented as mean FF / Ren, n=3. Hits are circled in red. [Figure 4C]Figure 1 shows that epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (C) Cell morphological images of NAMEC8 treated with DMSO, bexarotene, CI-994, SBHA, bexarotene + C1-994, or bexarotene + SBHA. Images were taken at 50x magnification. Scale bar represents 100 μm. [Figure 4D] Epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (D) Protein expression of NAMEC8 treated with DMSO, bexarotene, CI-994, SBHA, bexarotene + C1-994, or bexarotene + SBHA. β-actin was used as a loading control. [Figure 4E] Epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (E) Cell morphological images of NAMEC8 treated with DMSO, bexarotene + CI-994, bexarotene + SBHA (drug "on") and withdrawn for 7-10 days (drug "off"). Images are taken at 50x magnification. Scale bar represents 100 μm. [Figure 4F] Epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (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. [Figure 4G] Epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (G) Immunofluorescence staining of KRT5 (top) and KRT8 (bottom) in BC2.2 treated with DMSO, bexarotene, CI-994, bexarotene+CI994, -bexarotene, -(bexarotene+CI994). Images are taken at 100x magnification. Scale bar represents 50 μm. [Figure 4H]Epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (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 number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 4I] Figure 1 shows that epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes. (I) Transciptional regulation of gene expression by retinoid receptors and epigenetic regulators. In mesenchymal cell states, RAR and RXR tend to associate with co-repressors such as HDACs, thereby inhibiting epithelial gene transcription. Exposure to bexarotene results in activation of RAR and RXR, causing a change in protein interactions from co-repressors to co-activators. HDAC inhibition helps enhance gene transcription activity by maintaining histone acetylation of gene promoters. [Figure 5A] Lipid metabolism genes are regulated by retinoid receptors. (A) Global heatmap of ChIP-seq peaks using RXRα, RARα, H3K27ac, and p300 antibodies in NAMEC8 treated with DMSO, ST (short-term; 7 days) bexarotene, or LT (long-term; 14 days) bexarotene. [Figure 5B] Lipid metabolism genes are regulated by retinoid receptors (B) Plots showing occupancy of RXRα, RARα, H3K27ac, and p300 in gene bodies upon treatment of NAMEC8 with ST- and LT-bexarotene. [Figure 5C]Lipid metabolism genes are regulated by retinoid receptors. (C) Gene tracks showing increased RXRα and H3K27ac binding at STRA6, DHRS3, PLIN1, PLIN2, and ACSL5 loci and decreased RXRα and H3K27ac binding at MGLL locus upon ST- and LT-bexarotene treatment of NAMEC8. The x-axis shows chromosomal location with gene structures illustrated below, each scale unit represents 5 kb. The y-axis shows genome occupancy in rpm per bp. [Figure 5D] Lipid metabolism genes are regulated by retinoid receptors (D) Gene Ontology pathway enrichment analysis of genes that increased binding to RXRα upon bexarotene treatment of NAMEC8. [Figure 5E] Lipid metabolism genes are regulated by retinoid receptors (E) Plot of super-enhancers ranked by increased H3K27ac signal in NAMEC8 treated with bexarotene. [Figure 5F] Lipid metabolism genes are regulated by retinoid receptors. (F) Heatmap of lipid-related genes altered upon bexarotene treatment of NAMEC8, SUM159 cells, MetBr-007DR, BC29.1, and BC22.1 cells. Data represent log2 fold change. [Figure 5G] Lipid metabolism genes are regulated by retinoid receptors. (G) Gene expression of lipid-related genes involved in TAG synthesis upon bexarotene treatment of NAMEC8 (top) and BC2.2 (bottom) compared to DMSO. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 6a]Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. (a) Relative lipid content measured by LC-MSMS in DMSO- vs. bexarotene-treated NAMEC8 cells. Data are presented as mean + / - SEM, n=3, *P<0.05, **P<0.01. Genes upregulated upon bexarotene treatment were validated by RT-PCR (shown in red). [Figure 6b] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. (b) Lipid droplet staining of NAMEC8, SUM159, and BC2.2: DMSO treatment vs. bexarotene treatment. Images were taken at 200× magnification. Scale bar represents 50 μm. [Figure 6c] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. (c) Mammosphere formation assay in NAMEC8 cells following treatment with bexarotene, FSG67, or FSG67+bexarotene (left); exposure of Ctrl sh or shAGPAT cells in the presence or absence of bexarotene (middle); treatment with various combinations of bexarotene and amidepsin A (right). Images were taken at 50× magnification. Scale bar represents 100 μm. [Figure 6d] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. (d) Oxygen consumption rate (OCR) of NAMEC8 DMSO vs. bexarotene in the presence or absence of palmitate. Black arrows indicate the rate of fatty acid β-oxidation (FAO). [Figure 6e] (e) Relative lipid content measured by LC-MSMS in HMLE vs. NAMEC8 cells. Data are presented as mean + / - SEM, n=3. Genes downregulated in NAMEC8 compared to HMLE as validated by RT-PCR are shown in blue. [Figure 6f](f) Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. (b) Lipid droplet staining of HMLE versus NAMEC8. Images were taken at 10× magnification. Scale bar represents 50 μm. [Figure 6g] (g) Rates of fatty acid β-oxidation (FAO) measured by Seahorse in a panel of breast cancer cell lines. Data are presented as mean + / - SEM, n=3. [Figure 6h] (h) Cell viability assay of BT549 and MCF7 grown for 6 days in basal medium (DMEM with 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. [Figure 7a] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. a) Protein expression of NAMEC8 treated with bexarotene, etomoxir, bexarotene+etomoxir, -bexarotene, -(bexarotene+etomoxir). β-actin was used as a loading control. [Figure 7b] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. b) Mammosphere formation assay of NAMEC8 treated with bexarotene, etomoxir, bexarotene+etomoxir, -bexarotene, -(bexarotene+etomoxir). Images were taken at 50x magnification. Scale bar represents 100 μm. Graph shows number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, *P<0.05, ***P<0.001, ****P<0.0001. [Figure 7c]Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. c) Lipid droplet staining of NAMEC8 Ctrlsh, CPT1A-sh1, and sh2 cells treated with DMSO or bexarotene. Images were taken at 100x magnification. Scale bar represents 50 μm. [Figure 7d] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. d) Protein expression of NAMEC8 Ctrlsh and CPT1A-sh1 cells treated with DMSO or bexarotene. β-actin was used as a loading control. [Figure 7e] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. e) Mammosphere formation assay of NAMEC8 Ctrlsh, CPT1A-sh1, and sh2 cells treated with DMSO and bexarotene (left). Images were taken at 50x magnification. Scale bar represents 100 μm. Protein expression shows knockdown of CPT1A (top right). Graph shows number of spheres >100 μm (bottom right). Data are presented as mean + / - SEM, n=3, *P<0.01, ***P<0.001. [Figure 7f] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. f) Lipid droplet staining of NAMEC8 vector control, CPT1A-1-overexpressing, and CPT1A-2-overexpressing NAMEC8 cells treated with DMSO or bexarotene. Images were taken at 200x magnification. Scale bar represents 50 μm. [Figure 7g] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. g) Protein expression in NAMEC8 vector control and CPT1A-1 overexpressing cells treated with DMSO or bexarotene. β-actin was used as a loading control. [Figure 7h]Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. h) Mammosphere formation assay of NAMEC8 vector control, CPT1A-1-overexpressing, and CPT1A-2-overexpressing cells treated with DMSO and bexarotene (left). Images were taken at 50x magnification. Scale bar represents 100 μm. Protein expression shows overexpression of CPT1A (top right). Graph shows number of spheres >100 μm (bottom right). Data are presented as mean + / - SEM, n=3, ****P<0.0001. [Figure 8a] Impairment of fatty acid oxidation re-channels lipids towards supporting an epithelial cell state and blocks EMT. a) Lipid droplet staining of HMLE-Twist ER control vs. 4OHT-induced cells (top row), HMLE-Zeb1 control vs. DOX-induced cells (middle row), MCF7-Slug+Sox9 control vs. DOX-induced cells. Images were taken at 100x magnification. Scale bar represents 50 μm. [Figure 8b] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. b) Oxygen consumption rate (OCR) of HMLE-Twist ER control vs. 4-OHT induced cells in the presence or absence of palmitate (left). Black arrows indicate FAO rate. FAO rate in HMLE-Twist ER control and 4OHT induced cells (right). Data are presented as mean + / - SEM, n=3, *P<0.05. [Figure 8c] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. c) Morphological characteristics of HMLE-Twist ER treated with Etomoxir in the presence or absence of 4-OHT. Images were taken at 50x magnification. Scale bar represents 100 μm. [Figure 8d]Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. d) Protein expression of EMT markers in HMLE-Twist ER treated with Etomoxir in the presence or absence of 4-OHT. β-actin was used as a loading control. [Figure 8e] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. e) Lipid droplet staining of MCF7-Slug+Sox9 Ctrlsh and shCPT1A cells + / - DOX-induced. Images were taken at 100x magnification. Scale bar represents 50 μm. [Figure 8f] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. f) Protein expression in MCF7-Slug+Sox9 Ctrlsh and shCPT1A cells + / - DOX-induced. β-actin was used as a loading control. [Figure 8g] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. g) Schematic of the workflow of mouse xenograft experiments (top row). Bottom left: Immunofluorescence staining of E-cadherin and vimentin in primary tumors formed in control (no DOX) vs. DOX vs. etomoxir+DOX groups (200x magnification). Scale bar represents 50 μm. Whole mount fluorescent lung images (100x magnification) and histological characteristics of lung sections (10x magnification) derived from lung metastases are also shown. White scale bar represents 100 μm, black scale bar represents 50 μm. Bottom right: Graph shows number of metastases per lung. Data are expressed as mean + / - SEM, n=5 technical replicates, **P<0.01. [Figure 8h] FIG. 10: Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. 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. Upon induction of MET, the epithelial cell state is characterized by inhibition of β-oxidation and accumulation of lipid stores. [Figure 9] (A) Dual-glo luciferase assay Z-scores of NAMEC8-SERPINE1prom-Luc cells treated with LOPAC (top left), anticancer drug (top right), kinase inhibitor (bottom left), and bioactive lipid (bottom right). Data are presented as mean, n=3. [Figure 10A] (A) Cell morphology images of SUM159 treated with DMSO, bexarotene (1 μM), or TTNPB (1 μM). Images were taken at a magnification of 50×. Scale bar represents 100 μm. [Figure 10B] (B) Gene expression of EMT and lineage marker genes in bexarotene-treated SUM159 cells compared to DMSO. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 10C] (C) Protein expression of EMT and lineage markers in DMSO- vs. bexarotene-treated SUM159 cells, β-actin was used as a loading control. [Figure 10D] (D) Flow cytometry analysis of CD24 and CD44 expression in DMSO-treated versus bexarotene-treated SUM159 cells. [Figure 10E] (E) Mammosphere formation assay of DMSO-, bexarotene-, or TTNPB-treated SUM159 cells. Images were taken at 50x magnification. Scale bar represents 100 μm. Bar graph shows number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01. [Figure 10F] (F) Gene expression of EMT and lineage marker genes in BC2.2, BC22.1, and BC29.1 bexarotene-treated cells compared to DMSO. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 10G](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× magnification. Scale bar represents 100 μm. [Figure 10H] (H) Mammosphere formation assay of BC22.1 (top) and BC29.1 (bottom) with DMSO vs. bexarotene treatment. Images were taken at 50x magnification. Scale bar represents 100 μm. Bar graph shows number of spheres >100 μm. Data are presented as mean + / - SEM, n=3, *P<0.05. [Figure 10I] (I) Luciferase assay of estrogen receptor element (ERE) reporter activity in NAMEC8 cells treated with increasing doses of bexarotene or 1 μM TTNPB. Data are presented as mean FF / Ren+ / -SEM, n=3, *P<0.05. [Figure 10J] (J) Gene expression of estrogen receptor (ER) target genes in NAMEC8 and BC2.2 treated with bexarotene. Data are presented as mean + / - SEM, n=3, *P<0.05. [Figure 10K] (K) Dose response graph of DMSO-treated vs. bexarotene-treated NAMEC8 to various concentrations of raloxifene HCl. IC50 values ​​are shown in the table. [Figure 10L] (L) Gene expression of EMT and lineage marker genes in bexarotene-treated BC24.1 (left) and BC33.1 (right) compared to DMSO. Data are presented as mean + / - SEM, n=3. [Figure 10M] (M) Knockdown of RXRα in NAMEC8 using Ctrl shRNA or RXRα shRNA, β-actin was used as a loading control. [Figure 10N](N) Gene expression of EMT and lineage genes in NAMEC8 Ctrl shRNA and RXRα sh1 bexarotene treated cells compared to DMSO. Data are presented as mean + / - SEM, n=3, *P<0.05. [Figure 10O] (O) Protein expression of EMT and lineage markers in NAMEC8 Ctrl shRNA and RXRα sh1 treated with DMSO or bexarotene, β-actin was used as a loading control. [Figure 10P] (P) Dose response graphs of SUM159 (middle), BC2.2 (right):DMSO vs. bexarotene treated cells to various concentrations of 5-FU. IC50 values ​​are shown in the table below. Data are expressed as mean + / - SEM, n=3. [Figure 10Q] (Q) Cell morphology images of NAMEC8 treated with bexarotene in the presence or absence of 2 nM paclitaxel. Images were taken at 50× magnification. [Figure 10R] (R) Dose response graph of DMSO-treated vs. bexarotene-treated NAMEC8 against various concentrations of PAC. IC50 values ​​are shown in the table. Data are presented as mean + / - SEM, n=3. [Figure 10S] (S) Morphological characteristics of DMSO-treated vs. bexarotene-treated SUM159 PACR cells. Images were taken at 50× magnification. Scale bar represents 100 μm. [Figure 10T] (T) Gene expression of EMT and lineage marker genes in bexarotene treated SUM159 PACR cells compared to DMSO. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 10U] (U) Protein expression of EMT and lineage markers in DMSO vs. bexarotene treated SUM159 PACR cells, β-actin was used as a loading control. [Figure 10V](V) Mammosphere formation assay of DMSO- or bexarotene-treated SUM159 PACR cells. Images were taken at 50x magnification. Scale bar represents 100 μm. Bar graph shows number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, **P<0.01. [Figure 11A] (A) Tumor mass (top) of SUM159 pretreated with DMSO, bexarotene, or TTNPB at weeks 7 and 8. Tumor volume (bottom) of SUM159 pretreated with DMSO, bexarotene, or TTNPB at weeks 7 and 8. Data are presented as mean + / - SEM, n=6. [Figure 11B] (B) Tumor mass (top) and tumor volume (bottom) of MCF7-Ras pretreated with DMSO, bexarotene, or ATRA. Data are presented as mean + / - SEM, n=6. [Figure 11C] (C) Cell viability (measured by MTS assay) of NAMEC8 (left), MCF7 (middle), and MCF10A (right) treated with DMSO, bexarotene, or ATRA for 6 days. Data represent mean + / - SEM, n=3. [Figure 11D] (D) Images of NAMEC8-Ras, BC2.2, and BC22.1 tumors formed in control versus bexarotene-fed mice. [Figure 11E] (E) Tumor mass of NAMEC8-Ras, BC2.2, and BC22.1 tumors formed in control mice versus bexarotene-fed mice (top row). Tumor volume of NAMEC8-Ras, BC2.2, and BC22.1 tumors formed in control mice versus bexarotene-fed mice (bottom row). Data are presented as mean + / - SEM, n=6. [Figure 11F] (F) Immunofluorescence staining of EMT and lineage markers of BC2.2 tumor sections from control vs. bexarotene-fed mice. Images were taken at 200× magnification. Scale bar represents 50 μm. [Figure 12A](A) Gene expression of EMT and lineage marker genes in NAMEC8 (left) and SUM159 (right) treated with bexarotene, CI-994, or bexarotene + CI-994 compared to DMSO. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 12B] (B) Dual-glo luciferase assay of NAMEC8-SERPINE1prom-Luc cells first treated with DMSO, bexarotene, followed by compound withdrawal for 24, 48, or 72 hours (drug "off"). Data are presented as mean + / - SEM, n=6, **P<0.01, ***P<0.001. [Figure 12C] (C) Dual-glo luciferase assay of NAMEC8-SERPINE1prom-Luc cells first treated with DMSO, bexarotene+CI-994, followed by compound withdrawal for 24, 48, or 72 hours (drug "off"). Data are presented as mean + / - SEM, n=6, ****P<0.0001. [Figure 12D] (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. Graph shows number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 12E] (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 number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 13A] (A) Global heatmap of ChIP-Seq peaks in SUM159 treated with DMSO, ST (short-term; 7 days) bexarotene, or LT (long-term; 14 days) bexarotene using RXRα, RARα, H3K27ac, and p300 antibodies. [Figure 13B] (B) Plots showing occupancy of RXRα, RARα, H3K27ac, and p300 at gene bodies upon treatment of SUM159 cells with ST- and LT-bexarotene. [Figure 13C] (C) Gene tracks showing increased RXRα and H3K27ac binding at the STRA6, DHRS3, PLIN1, PLIN2, and ACSL5 loci, and decreased RXRα and H3K27ac binding at the MGLL locus upon treatment of SUM159 cells with ST- and LT-bexarotene. The x-axis shows chromosomal position with gene structures illustrated below, each scale unit represents 5 kb. The y-axis shows genome occupancy in rpm per bp. [Figure 13D] (D) Gene Ontology pathway enrichment analysis of RNA-Seq derived genes with log2 fold change >1 upon treatment of SUM159 with LT bexarotene. [Figure 13E] (E) Gene Ontology pathway enrichment analysis of RNA-Seq derived genes with log2 fold change >1 upon bexarotene treatment of MetBr-007-DR. [Figure 13F] (F) Plot of super-enhancers ranked by increased H3K27ac signal in SUM159 treated with bexarotene. [Figure 13G] (G) Gene expression of lipid-related genes involved in TAG synthesis upon TTNPB treatment in NAMEC8 compared to DMSO. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01. [Figure 14A]Figure 1: Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. (a) Lipid droplet staining of BC29.1 and BC22.1: DMSO-treated vs. bexarotene-treated. Images were taken at 200x magnification. Scale bar represents 50 μm. [Figure 14b] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. b) Lipid droplet staining of NAMEC8 treated with bexarotene, FSG67, or FSG67+bexarotene (left). Exposure of Ctrl sh or shAGPAT cells in the presence or absence of bexarotene (middle). Treatment with various combinations of bexarotene and amidepsin A (right). Images were taken at 100× magnification. Scale bar represents 50 μm. [Figure 14c] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. c) Protein expression of epithelial and luminal markers in NAMEC8 cells upon treatment with bexarotene, FSG67, or FSG67+bexarotene (left). Exposure of Ctrl sh or shAGPAT cells in the presence or absence of bexarotene (middle). Treatment with various combinations of bexarotene and amidepsin A (right). β-actin was used as a loading control. [Figure 14d] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. d) Graph showing number of spheres >100 μm in mammosphere formation assay with NAMEC8 cells in 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 with various combinations of bexarotene and amidepsin A (right). Images were taken at 50× magnification. Scale bar represents 100 μm. Data are presented as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001. [Figure 14e]Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. e) Oxygen consumption rate (OCR) for SUM159 (left) and BC2.2 (right) in the presence or absence of palmitate: DMSO vs. bexarotene. Black arrows indicate the rate of fatty acid β-oxidation (FAO). [Figure 14f] Figure 1: Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. f) NAMEC8 (left), SUM159 (middle), and BC2.2 (right): FAO rates with DMSO vs. bexarotene treatment. Data are presented as mean + / - SEM, n=3, **P<0.01, ***P<0.001. [Figure 14g] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids into lipid droplet stores. g) Gene expression of lipid-related genes involved in TAG synthesis in NAMEC8 compared to HMLE. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 15a] FIG. 1 shows that induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. a) Graph showing FAO rate in SUM159 treated with bexarotene, etomoxir, and bexarotene + etomoxir. Data are expressed as mean + / - SEM, n=3, **P<0.01, ***P<0.001. [Figure 15b] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. b) Morphological characteristics of NAMEC8 treated with bexarotene, etomoxir, bexarotene+etomoxir, -bexarotene, or -(bexarotene+etomoxir). Images were taken at 100x magnification. Scale bar represents 50 μm. [Figure 15c]Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. c) Gene expression of EMT and retinoid acid responsive genes in NAMEC8 treated with bexarotene, etomoxir, or bexarotene + etomoxir. Data are presented as mean + / - SEM, n=3, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 15d] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. d) Mammosphere formation assay of SUM159 treated with bexarotene, etomoxir, bexarotene+etomoxir, -bexarotene, or -(bexarotene+etomoxir). Images were taken at 50x magnification. Scale bar represents 100 μm. Graph shows number of spheres >100 μm. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01. [Figure 15e] Figure 1 shows that induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. e) Graph showing FAO rates in vector or CPT1A overexpressing SUM159 cells treated with either DMSO or bexarotene. Data are presented as mean + / - SEM, n=3, **P<0.01. [Figure 15f] Figure 1 shows that induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. f) Lipid droplet staining of SUM159 vector, CPT1A-1 overexpressing, and CPT1A-2 overexpressing cells treated with DMSO or bexarotene. Images were taken at 200x magnification. Scale represents 50 μm. [Figure 15g] Figure 1 shows that induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. g) Morphological characteristics of NAMEC8 vector and CPT1A overexpressing cells treated with DMSO or bexarotene. Images were taken at 40x magnification. Scale bar represents 50 μm. [Figure 15h] Induction of mesenchymal-epithelial transition is dependent on the fatty acid modifier carnitine palmitoyltransferase. h) Mammosphere formation assay of SUM159 vector, CPT1A-1 overexpressing, and CPT1A-2 overexpressing cells treated with DMSO and bexarotene (left). Images were taken at 50x magnification. Scale bar represents 100 μm. Protein expression shows overexpression of CPT1A (top right). Graph shows number of spheres >100 μm (bottom right). Data are presented as mean + / - SEM, n=3, ****P<0.0001. [Figure 16a] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. (a) Gene expression of EMT genes in HMLE-Twist ER treated with etomoxir 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. [Figure 16b] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. (b) Gene expression of EMT genes in MCF7-Slug+Sox9 treated with etomoxir in the presence or absence of doxycycline induction compared to control. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 16c] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. (c) Protein expression of EMT markers in MCF7-Slug+Sox9 treated with etomoxir in the presence or absence of doxycycline induction. β-actin was used as a loading control. [Figure 16d]Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. (d) Cytomorphological images of HMLE-Zeb1 treated with etomoxir in the presence or absence of doxycycline induction compared to control. Images were taken at 40x magnification. Scale bar represents 50 μm. [Figure 16e] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. (e) Gene expression of EMT genes in MCF7-Slug+Sox9 Ctrlsh and shCPT1A cells + / - DOX induction compared to Ctrlsh cells. Data are expressed as mean + / - SEM, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 16f] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. (f) Cell morphology images of A549 treated with etomoxir in the presence or absence of TGFβ compared to control. Images were taken at 40× magnification. Scale bar represents 50 μm. [Figure 16g] Impairment of fatty acid oxidation rechannels lipids towards supporting an epithelial cell state and blocks EMT. g) Protein expression of EMT markers in A549 treated with Etomoxir in the presence or absence of TGFβ. β-actin was used as a loading control. [Figure 17A]Figure 1. Epigenetic library screening to identify potent MET inducers. (A) Dual-glo luciferase assay of NAMEC8-SERPINE1 cells screened with epigenetic library to identify MET inducers. Data are expressed as cell viability vs. SERPINE1 promoter activity (average FF / Ren value), n=3. A83-01 (circled in black) was included as a positive control, and compounds with lower SERPINE1 promoter activity than A83-01 were identified as hits (circled in red). [Figure 17B] (B) Epigenetic library screening to identify potent MET inducers. (A) Dual-glo luciferase assay of MET screen hits performed with 6-point dose response. Data are expressed as SERPINE1 promoter activity (mean FF / Ren value) vs. concentration, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant. [Figure 17C] Figure 1. Epigenetic library screening to identify potent MET inducers. (C) Cell morphology images of NAMEC8 treated with DMSO and epigenetic compounds. LLY-283 and GSK591 were used at 20 nM, and UNC0642 and SGC-CBP30 were used at 1 μM. Only SGC-CBP30 treatment resulted in an epithelial-like morphology. Images were taken at 4× magnification. Scale bar represents 100 μm. [Figure 17D] (D) Epigenetic library screening to identify potent MET inducers. (E) qRT-PCR analysis of EMT and basal / luminal genes in NAMEC8 treated with epigenetic hits. mRNA levels are shown relative to DMSO. Data are presented as mean ± SEM, n=3. [Figure 17E]Epigenetic library screening to identify potent MET inducers. (E) Immunoblot analysis of EMT and basal / luminal markers of NAMEC8 upon treatment with epigenetic hits. SGC-CBP30 showed the most dramatic changes compared to other compounds. β-actin was used as a loading control. [Figure 17F] Epigenetic library screening to identify potent MET inducers. (F) Left: Mammosphere formation assay of NAMEC8 upon treatment with epigenetic hits. A significant reduction in sphere formation and sphere size was observed with three of the compounds tested. Images were taken at 4x magnification. Scale bar represents 100 μm. Right: Bar graph representing the number of tumor spheres >50 μm. Data are expressed as mean ± SEM, n=3, *P<0.05, **P<0.01, ns not significant. [Figure 17G] (G) Flow cytometry analysis of CD44 and CD24 staining in NAMEC8, DMSO-treated vs. SGC-CBP30-treated cells. A population of cells with decreased CD44 expression was observed upon SGC-CBP30 treatment, suggesting induction towards a non-stem-like state. [Figure 18A] CREBBP / EP300 bromodomain inhibition induces MET in 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 4× magnification. Scale bar represents 100 μm. [Figure 18B]CREBBP / EP300 bromodomain inhibition induces MET in SUM159 TNBC cell line (B) Immunoblot analysis of EMT and basal / luminal markers in SUM159 treated with epigenetic hits. β-actin was used as a loading control. [Figure 18C] CREBBP / EP300 bromodomain inhibition induces MET in SUM159 TNBC cell line. (C) Top: Mammosphere formation assay of SUM159 upon treatment with epigenetic hits. A significant reduction in sphere 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 representing the number of tumor spheres >50 μm. Data are presented as mean ± SEM, n=3, *p<0.05. [Figure 18D] CREBBP / EP300 bromodomain inhibition induces MET in SUM159 TNBC cell line. (D) Flow cytometry analysis of CD44 and CD24 staining in SUM159, DMSO-treated vs. SGC-CBP30-treated cells. Cells displayed decreased CD44 expression and higher CD24 expression, suggesting induction towards a non-stem-like state. [Figure 19A] CREBBP / EP300 bromodomain inhibition reduces basal-like TNBC tumor burden in vivo. (A) Cytomorphological images of NAMEC8 treated with 1 μM A-485. Epithelial-like morphology was observed upon treatment with A-485. Images were taken at 4× magnification. Scale bar represents 100 μm. [Figure 19B] CREBBP / EP300 bromodomain inhibition reduces tumor burden in basal-like TNBC in vivo (B) Immunoblot analysis of EMT and basal / luminal markers of NAMEC8 upon treatment with A-485. β-actin was used as a loading control. [Figure 19C]CREBBP / EP300 bromodomain inhibition reduces basal-like TNBC tumor burden in vivo. (C) Top: Mammosphere formation assay of NAMEC8 upon treatment with A-485. Reduction in sphere formation and sphere size was observed. Images were taken at 4x magnification. Scale bar represents 100 μm. Bottom: Bar graph representing number of tumor spheres >50 μm. Data are presented as mean ± SEM, n=3, ns not significant. [Figure 19D] CREBBP / EP300 bromodomain inhibition reduces tumor burden of basal-like TNBC in vivo. (G) Flow cytometry analysis of CD44 and CD24 staining in NAMEC8, DMSO-treated vs. A-485-treated cells. Cells exhibited higher CD44 expression treatment, suggesting that A-485 was unable to induce loss of stem-like traits. [Figure 19E] CREBBP / EP300 bromodomain inhibition reduces basal-like TNBC tumor burden in vivo. (E) Immunoblot analysis of histone acetylation markers in NAMEC8 upon treatment with SGC-CBP30 and A-485. H3 and β-actin were used as loading controls. [Figure 19F] CREBBP / EP300 bromodomain inhibition reduces basal-like TNBC tumor burden in vivo. (F) Top: Tumor size of NAMEC8-Ras pretreated with DMSO, SGC-CBP30, and A-485 prior to subcutaneous injection in NSG mice. Data are presented as mean ± SEM, n=3, *p<0.05, ns not significant. Bottom: Images of tumors from mice sacrificed at week 11. SGC-CBP30 treatment significantly reduced tumor formation in mice. [Figure 19G]CREBBP / EP300 bromodomain inhibition reduces tumor burden in basal-like TNBC in vivo. (G) Working model showing CREBBP / EP300-induced cell state transition in TNBC upon bromodomain inhibition. In the absence of bromodomain inhibitors, CREBBP and EP300 could recognize acetylated lysines on histones to mediate mesenchymal gene activation, thus maintaining cells in a mesenchymal state. However, the presence of bromodomain inhibitors would block the readout function of CREBBP and EP300 proteins, thereby reducing the transcriptional regulation of genes required for the maintenance of a mesenchymal cell state. [Figure 20A] Figure 1. Metabolic library screening to identify potent MET inducers. (A) Dual-glo luciferase assay of NAMEC8-SERPINE1 cells screened with metabolic library to identify MET inducers. Data are expressed as cell viability versus SERPINE1 promoter activity (average FF / Ren value) relative to Renilla, n=3. Compounds identified as hits are circled in red. [Figure 20B] (A) Metabolic library screening to identify potent MET inducers (B) Table summarizing the compounds identified as hits, with their respective molecular targets listed in the right column. [Figure 20C] (C) Dual-glo luciferase assay of MET screen hits performed in a 5-point dose response. Data are presented as SERPINE1 promoter activity (mean FF / Ren value) versus concentration, and the C-IN-1 glutaminase inhibitor shows a dose-response effect of decreasing SERPINE1 promoter activity. [Figure 20D](D) Western blot showing GLS1 expression in epithelial and mesenchymal breast cancer cell lines. [Figure 20E] Figure 1. Metabolic library screening to identify potent MET inducers. (E) Cell morphological images of NAMEC8 treated with 500 nM C-IN-1. [Figure 20F] (F) Immunoblot analysis of EMT and basal / luminal markers in NAMEC8 upon treatment with MET hits. [Figure 20G] (G) Western blot showing knockdown of GLS1 in NAMEC8 using three shRNAs. [Figure 20H] Metabolic library screening to identify potent MET inducers. (H) Cellular potency glow assay of NAMEC8 ctrlsh vs. GLS1sh2 and GLS1sh3 treated with paclitaxel. IC50 of GLSsh2 cells is lower than Ctrlsh cells. [Figure 20I] Figure 1. Metabolic library screening to identify potent MET inducers. (I) Western blot of GLS1 knockdown in NAMEC8 Hras cells by sh2 and sh3. [Figure 20J] Metabolic library screening to identify potent MET inducers. (J) Mammosphere assay of NAMEC8 Hras Ctrlsh vs. GLS1sh2 vs. GLS1sh3 cells. Spheres >100um were counted after 7 days. [Figure 20K](K) NAMEC8 Hras Ctrlsh and GLS1sh2 cells were injected subcutaneously into NSG mice and tumor growth was followed for 8 weeks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Inducers of mesenchymal-epithelial transition Provided herein are methods for inducing mesenchymal-epithelial transition (MET) in cancer cells. In one aspect, a method for inducing MET in a cancer cell is provided, comprising contacting the cell with an inducer of mesenchymal-epithelial transition for a time and under conditions sufficient to induce MET in the cell, wherein the cancer cell is a basal-like (or mesenchymal-like) cancer cell.

[0027] Cell state transitions can control the functional behavior of cancer cells and determine their tumorigenicity and responsiveness to therapy. Epithelial-mesenchymal transition (EMT) can confer cancer stem cell-like properties and enhance tumorigenicity and drug resistance of tumor cells. EMT can be highly activated in TNBC and may contribute to recurrence and chemotherapy resistance. The inventors have discovered that the reverse process of EMT, mesenchymal-epithelial transition (MET), can suppress this phenotype and sensitize tumors to various standard treatment modalities, including chemotherapy and immunotherapy. In particular, the inventors have developed (i) technology platforms useful for identifying drugs, chemical probes, and drug targets that can improve cancer response; (ii) novel combination therapies that enhance anti-tumor response; and (iii) new drug targets whose activation or inhibition can reverse resistance, sensitize tumors to various standard treatment modalities, including chemotherapy and immunotherapy, and inhibit tumor growth.

[0028] In one embodiment, a method is provided for inhibiting the growth of cancer cells, comprising contacting the cells with an inducer of mesenchymal-epithelial transition for a time and under conditions sufficient to inhibit the growth of the cancer cells, wherein the cancer cells are basal-like (or mesenchymal-like) cancer cells.

[0029] The terms "epithelial" or "epithelial-like" are understood in the art and can be distinguished by morphological, molecular, and / or functional characteristics. For example, epithelial or epithelial-like cells generally have a rounded or cobblestone appearance, express the epithelial marker E-cadherin, divide rapidly, and / or exhibit relatively low levels of motility, invasiveness, and / or anchorage-independent growth compared to mesenchymal cells.

[0030] The terms "mesenchymal" or "mesenchymal-like" are understood in the art and can be distinguished by morphological, molecular, and / or functional characteristics. For example, mesenchymal or mesenchymal-like cells generally have an elongated or spindle-shaped appearance, express the mesenchymal markers vimentin, fibronectin, and N-cadherin, are slowly dividing or non-dividing, and / or exhibit relatively high levels of motility, invasiveness, and / or anchorage-independent growth compared to epithelial cells.

[0031] The term "epithelial-mesenchymal transition" ("EMT") is known in the art and refers to the process by which epithelial cancer cells adopt a mesenchymal phenotype that is believed to be associated with metastasis. Additionally, without wishing to be bound by any theory, such mesenchymal cells generally display reduced adhesion, increased motility and invasiveness, and are relatively resistant to chemotherapeutic agents and / or radiation (e.g., treatments that target rapidly dividing cells).

[0032] The term "mesenchymal to epithelial transition" ("MET") is also known in the art and refers to the reprogramming of cells that have undergone 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 divide rapidly, which may restore sensitivity to cytotoxic agents.

[0033] In one embodiment, the method may be an in vitro, in vivo, or ex vivo method.

[0034] The inducer of MET may be a compound selected from the group consisting of TGFβ inhibitors, adenosine / adrenergic, GABA or NMDA receptor modulators, neurotransmitters, epigenetic modulating compounds.

[0035] In one embodiment, the inducer of MET is selected from the group consisting of: amvatinib (MP-470); Evista (raloxifene HCl); isotretinoin; SB216763; tretinoin (Aberela); 1,3-dipropyl-8-p-sulfophenylxanthine; 5-bromo-2'-deoxyuridine; alprenolol hydrochloride; amiodarone hydrochloride; apomorphine hydrochloride hemihydrate; atropine methyl nitrate; cantharidic acid; L-798106; L-aspartic acid; Rhodblock 6 6);TNP;Bexarotene;PD-161570;SB-525334;13-cis-Retinoic acid;AC-55649;Raloxifene hydrochloride;Retinoic acid;Tazarotene;TTNPB;U0126;Amvatinib (MP-470);AG-490;Avitrexate (methotrexate);Roscovitine (seliciclib, CYC202);PIK-294;U0126-EtOH;PH-797804;Arry-380;Motesanib diphosphate (AMG-706);CH5132799;PIK-93;KN-62;16,16-dimethyl prostaglandin D2;SB431542;Prostaglandin D2 ethanolamide;6-keto prostaglandin E1;Prostratin;CAY1 0587;15-deoxy-_12,14-prostaglandin D2;UCM707;D4476;16-phenoxytetranorprostaglandin A2;KN-93;prostaglandin D2-1-glyceryl ester;C-8 ceramide;S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (hydrochloride);LLY-283,GSK591,UNC0642;SGC-CBP30;tanshinone I;hemin;autofinib;β-lapachone;Vps34-PIK-III;glutaminase C-IN-1;GSK256066;AN-2728;GSK2837808A;etretinate;fenretinide;T0901317;AGI-6780;SW033291;and NCT-503.

[0036] In one embodiment, the inducer of MET is a retinoid, which may be, for example, dehydroretinol, didehydroretinol tretinoin (retinoic acid), isotretinoin, alitretinoin, etretinate, acitretin, adapalene, bexarotene, 13-cis-retinoic acid, tazarotene, TTNPB, etretinate, or fenretinide.

[0037] In one embodiment, the retinoid is a retinoic acid receptor alpha (RAR-α) agonist or a retinoid X receptor alpha (RXR-α) agonist. The RAR-α agonist may be TTNPB, IRX5183, SY-1425, etretinate, or fenretinide. The RXR-α agonist may be bexarotene.

[0038] An inducer of MET can be a metabolic enzyme or pathway modifier (e.g., an inhibitor or inducer). For example, the metabolic enzyme or pathway modifier can be a glutaminase inhibitor, a phosphodiesterase inhibitor, a modifier of autophagy, an inhibitor of phospholipase, a topoisomerase, a PI3K, lactate dehydrogenase, 15-PGDH, or a phosphoglycerate dehydrogenase.

[0039] In one embodiment, the inducer of MET is a glutaminase inhibitor. The glutaminase inhibitor may be Glutaminase C-IN-1, CB839 (Telaglenastat), UPGL00004, or BPTES. The glutaminase inhibitor may be a glutaminase 1 (GLS1) inhibitor. The glutaminase inhibitor may be a glutaminase C inhibitor or a kidney-type glutaminase (KGA) inhibitor.

[0040] In one embodiment, the inducer of MET 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.

[0041] In one embodiment, the inducer of MET is a modulator (e.g., an inhibitor or inducer) of autophagy. The modulator of autophagy may be hemin, autofinib; β-lapachone; Vps34-PIK-III, or fenretinide.

[0042] The inducer of MET may be a phospholipase (e.g., Tanshionone I), a topoisomerase (e.g., β-lapachone), a PI3K (Vps34-PIK-III, idelalisib, copanlisib, duvelisib, alpelisib), a lactate dehydrogenase (e.g., GSK2837808A), a 15-PGDH (e.g., SW033291), or a phosphoglycerate dehydrogenase (e.g., NCT-503). The inducer of MET may be an epigenetic modifying compound. The epigenetic modifying compound may be an inhibitor of p300 / CREBBP bromodomain, protein arginine methyltransferase 5 (PRMT5), or histone methyltransferase.

[0043] In one embodiment, the inducer of MET is a p300 / CREBBP bromodomain inhibitor. The p300 / CREBBP bromodomain inhibitor may be SGC-CBP30, GNE-272, or GNE-781.

[0044] In one embodiment, the inducer of MET is an inhibitor of PRMT5. The inhibitor of PRMT5 may be LLY-283 or GSK591.

[0045] In one embodiment, the inducer of MET may be an inhibitor of histone methyltransferase (e.g., G9a and / or GLP). In one embodiment, the inhibitor of histone methyltransferase is UNC0642.

[0046] Inhibitors of epithelial-mesenchymal transition An inducer of MET can also be an inhibitor of EMT. In one embodiment, the inhibitor of MET is a compound selected from the group consisting of TGFβ inhibitors, adenosine / adrenergic, GABA or NMDA receptor modulators, neurotransmitters, epigenetic modulating compounds. In one embodiment, the inhibitor of EMT is a retinoid.

[0047] In one embodiment, the inhibitor of EMT is selected from the group consisting of: amuvatinib (MP-470); Evista (raloxifene HCl); isotretinoin; SB216763; tretinoin (Aberela); 1,3-dipropyl-8-p-sulfophenylxanthine; 5-bromo-2'-deoxyuridine; alprenolol hydrochloride; amiodarone hydrochloride; apomorphine hydrochloride hemihydrate; atropine methyl nitrate; cantharidic acid; L-798106; L-aspartic acid; roadblock 6; TNP; bexarotene; PD-161570; SB-525334; 13-cis-retinoic acid; AC-55649; raloxifene hydrochloride; retinoic acid; tazarotene; TTNPB; U0126; amuvatinib (MP-470); AG-490; Avitre methotrexate;roscovitine (seliciclib, CYC202);PIK-294;U0126-EtOH;PH-797804;Arry-380;motesanib diphosphate (AMG-706);CH5132799;PIK-93;KN-62;16,16-dimethyl prostaglandin D2;SB431542;prostaglandin D2ethanolamide;6-keto toprostaglandin E1; prostratin; CAY10587; 15-deoxy-_12,14-prostaglandin D2; UCM707; D4476; 16-phenoxytetranorprostaglandin A2; KN-93; prostaglandin D2-1-glyceryl ester; C-8 ceramide; and S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (hydrochloride).

[0048] In one embodiment, the inhibitor of EMT is an inhibitor or regulator of lipid metabolism (or an inhibitor of fatty acid oxidation).

[0049] The inhibitor of lipid metabolism may be an inhibitor of carnitine palmitoyltransferase I (CPT1). In one embodiment, the inhibitor of CPT1 is etomoxir, perhexiline, teglicar, or oxfenicin. In one embodiment, the inhibitor of CPT1 is etomoxir.

[0050] The inhibitor of lipid metabolism may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0051] The inhibitor of lipid metabolism 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).

[0052] The inhibitor of lipid metabolism may be an inhibitor of 3-ketoacyl-coenzyme A thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0053] The inhibitor of lipid metabolism may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0054] The cancer cells may be basal-like breast cancer or TNBC, but are not luminal / ER+ breast cancer. In one embodiment, the cancer cells are selected from the group consisting of 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.

[0055] The cancer cells may be chemotherapy-resistant cancer cells. In other words, the cancer cells may be cancer cells that are poorly responsive or essentially unresponsive to anti-cancer therapy. In one embodiment, the cancer cells are cancer cells that are resistant to standard treatment therapies.

[0056] The cancer cells may also be metastatic cancer cells.

[0057] Drug Combinations Combination therapy including retinoids and epigenetic regulators can achieve stronger and more durable cancer control. We have shown that some epigenetic modifiers themselves can induce MET. When combined with retinoids, epigenetic modifiers can further enhance and maintain the MET-transformed phenotype.

[0058] Conversion of mesenchymal cells to an epithelial phenotype by retinoid-induced MET can also sensitize cells to immuno-oncological drugs such as immune checkpoint inhibitors. Retinoids can exert immunomodulatory effects by enhancing T helper and T regulatory cell populations, increasing type I and type II interferon signaling, interferon gamma signaling, increasing cytokine-mediated signaling, and activating the complement cascade (Table 4A and Table 4B).

[0059] In one embodiment, the method further comprises, after inducing MET in the cancer cells, contacting the cells with a chemotherapeutic agent (such as paclitaxel or 5-fluorouracil), an epigenetic modifying compound (such as a class I HDAC inhibitor (e.g., C1-994 and SBHA)), or an immunomodulatory agent. The method may also comprise contacting the cells simultaneously or sequentially with an inducer of mesenchymal-epithelial transition.

[0060] In one embodiment, the method further comprises contacting the cell with a chemotherapeutic agent, which can be selected from chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, 5-fluorouracil, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.

[0061] In one embodiment, the method further comprises contacting the cell with an epigenetic modifying compound. The epigenetic modifying compound may be an HDAC inhibitor. The HDAC inhibitor may be a class I HDAC inhibitor. The HDAC inhibitor may also be selected from the group consisting of hydroxamic acids vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589); and benzamides: entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103).

[0062] 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.

[0063] As used herein, the term "immune checkpoint" refers to a group of molecules on the cell surface of CD4 and CD8 T cells that effectively act as a "break" 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 collagenous structure), PS (phosphatidylserine), ICOS (inducible T cell costimulator), HAVCR2, CD276, VTCN1, CD70, and CD160.

[0064] "Immune checkpoint inhibitors" as used herein refers to any modulator that inhibits the activity of an immune checkpoint molecule. Immune checkpoint inhibitors include small molecule inhibitors, antibodies, antibody derivatives (including Fab fragments and scFv), antibody-drug conjugates, antisense oligonucleotides, siRNAs, aptamers, peptides, and peptidomimetics. 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) to disrupt or inhibit the expression of a target gene.

[0065] In one embodiment, the inducer of MET is co-administered with an immune checkpoint inhibitor, which may be any of the following: 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, C Inhibitors of D70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagenous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof.

[0066] 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.

[0067] The immunomodulatory agent may also be a cell therapy such as a T cell (eg, CarT cell), a natural killer (NK) cell, or a dendritic cell.

[0068] In one embodiment, a method for inducing MET in a cancer cell is provided, comprising contacting the cell with an inducer of mesenchymal-epithelial transition and a chemotherapeutic agent for a time and under conditions sufficient to induce MET in the cell, wherein the cancer cell is a basal-like (or mesenchymal-like) cancer cell. The inducer of mesenchymal-epithelial transition may be a retinoid, such as bexarotene. The chemotherapeutic agent may be paclitaxel or 5-fluorouracil.

[0069] In one embodiment, a method for inducing MET in a cancer cell is provided, comprising contacting the cell with an inducer of mesenchymal-epithelial transition and an epigenetic modifying compound for a time and under conditions sufficient to induce MET in the cell, wherein the cancer cell is a basal-like (or mesenchymal-like) cancer cell. The inducer of mesenchymal-epithelial transition may be a retinoid, such as bexarotene. The epigenetic modifying agent may be a class I HDAC inhibitor, such as Cl-994 and SBHA.

[0070] In one embodiment, a method for inducing MET in a cancer cell is provided, comprising contacting the cell with an inducer of mesenchymal-epithelial transition and an immunomodulatory agent for a time and under conditions sufficient to induce MET in the cell, wherein the cancer cell is a basal or basal-like (or mesenchymal-like) cancer cell. The inducer of mesenchymal-epithelial transition may be a retinoid, such as bexarotene. The immunomodulatory agent may be an immune checkpoint inhibitor, such as an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA-4 antibody.

[0071] Lipid Metabolic Pathway The inventors have shown that epithelial and mesenchymal cell states are defined by distinct metabolic pathways. The main findings relate to the distinct lipid metabolic pathways that support either epithelial or mesenchymal cell states. These findings reveal important new therapeutic targets, in particular metabolic genes that can be either activated or inhibited to (i) target or kill more aggressive mesenchymal subtype tumors, (ii) induce MET transformation, and (iii) block EMT and disease progression (i.e., tumor growth, invasion, metastasis, and resistance).

[0072] Without being bound by theory, the inventors have found that mesenchymal tumor cells are more dependent on the fatty acid oxidation (FAO) metabolic pathway, since they use fatty acids as a source of energy substrate, whereas epithelial tumor cells tend to store fatty acids as lipid droplets. To target mesenchymal tumor cells, the 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 triacsin C (preclinical) and thiazolidinediones (TZDs) (already in clinical use for diabetes). Inhibitors of CPT1 include etomoxir (clinical trials failed due to hepatotoxicity), perhexiline (clinical use for angina pectoris), and oxfenicine (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).

[0073] Furthermore, the combination of such FAO pathway inhibitors with standard of care therapies may result in more potent targeting of mesenchymal tumor cells, thereby inhibiting tumor growth, reducing resistance, preventing metastasis, and improving anti-cancer responses.

[0074] The present inventors have further found that disrupting fatty acid oxidation can further inhibit cancer metastasis. The use of the FAO inhibitor etomoxir, which targets the CPT1 gene, can inhibit breast cancer metastasis. Inhibition of FAO, either by CPT1 inhibitors or inhibitors that target FAO, can be effective as an anti-metastatic agent against cancers with a high propensity for metastasis, as well as cancers that are highly FAO-dependent, such as ovarian, breast, and oral cancers.

[0075] The method as defined herein further comprises contacting the cells with an inhibitor or regulator of lipid metabolism after inducing MET in the cancer cells. The inhibitor of lipid metabolism may be an inhibitor of the fatty acid oxidation (FAO) pathway. In one embodiment, the inhibitor of lipid metabolism is an inhibitor of an enzyme selected from the group consisting of long-chain fatty acid CoA ligase (ACSL), 3-ketoacyl coenzyme A 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), and diglyceride acyltransferase (DGAT).

[0076] The inhibitor of lipid metabolism may be an inhibitor of carnitine palmitoyltransferase I (CPT1). In one embodiment, the inhibitor of CPT1 is etomoxir, perhexiline, or oxfenicine.

[0077] The inhibitor of lipid metabolism may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0078] The inhibitor of lipid metabolism 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).

[0079] The inhibitor of lipid metabolism may be an inhibitor of 3-ketoacyl-coenzyme A thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0080] The inhibitor of lipid metabolism may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0081] In one embodiment, a method for inducing MET in a cancer cell is provided, comprising contacting the cell with an inducer of mesenchymal-epithelial transition and an inhibitor of lipid metabolism for a time and under conditions sufficient to induce MET in the cell, wherein the cancer cell is a basal-like (or mesenchymal-like) cancer cell. The inducer of MET may be a retinoid, such as bexarotene. The inhibitor of lipid metabolism may be an inhibitor of CPT1, such as etomoxir.

[0082] In one embodiment, a method for inducing MET in a cancer cell is provided, comprising contacting the cell with an inducer of mesenchymal-epithelial transition and an inhibitor of CPT1 for a time and under conditions sufficient to induce MET in the cell, wherein the cancer cell is a basal-like (or mesenchymal-like) cancer cell.

[0083] In one embodiment, a method for inducing MET in a cancer cell is provided, comprising contacting the cell with a combination of bexarotene and etomoxir for a time and under conditions sufficient to induce MET in the cancer cell.

[0084] A method as defined herein may include treating cancer in a patient or sensitizing a patient to an anti-cancer therapy.

[0085] The term "treat," as used herein, unless otherwise indicated, means to reverse, ameliorate, inhibit, or prevent, either partially or completely, tumor growth, tumor metastasis, or other carcinogenic or neoplastic cell progression in a patient with cancer. The term "treatment," as used herein, unless otherwise indicated, refers to the act of treating.

[0086] The terms "subject," "patient," and "individual" are used interchangeably. As used herein, such terms 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. Such terms do not require the supervision (whether continuous or intermittent) of a medical professional.

[0087] "Sensitizing" a cancer or cancer cells (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 cells to an anti-cancer therapy means that a lower dosage of the anti-cancer therapy drug is effective in treating the cancer or killing the cancer cells and / or the cancer or cancer cells are more responsive to the same dosage of the anti-cancer drug compared to their response in the absence of the methods of the invention.

[0088] In one aspect there is provided an inducer of mesenchymal-epithelial transition (MET) for use in inducing mesenchymal-epithelial transition in cells of a patient.

[0089] In one aspect there is provided the use of an inducer of mesenchymal-epithelial transition (MET) in the manufacture of a medicament for inducing mesenchymal-epithelial transition in cells of a patient.

[0090] In one embodiment, a method is provided for inhibiting epithelial-mesenchymal transition (EMT) of a cancer cell, comprising contacting the cell with an inhibitor of epithelial-mesenchymal transition (EMT) for a time and under conditions sufficient to inhibit EMT of the cancer cell, where the cancer cell is a basal-like (or mesenchymal-like) cancer cell, which may include blocking disease progression (i.e., tumor growth, invasion, metastasis, and resistance) of the cancer cell.

[0091] In one embodiment, there is provided an inhibitor of epithelial-mesenchymal transition (EMT) for use in inhibiting EMT of cancer cells.

[0092] In one embodiment there is provided the use of an inhibitor of epithelial-mesenchymal transition (EMT) in the manufacture of a medicament for inhibiting epithelial-mesenchymal transition of cancer cells.

[0093] When inhibiting EMT, the inhibitor may be an inhibitor of CPT1, for example, etomoxir, perhexiline, or oxfenicine.

[0094] When inhibiting EMT, the inhibitor may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0095] When 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 a thiazolidinedione (TZD).

[0096] When inhibiting EMT, the inhibitor may be an inhibitor of 3-ketoacyl-coenzyme A thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0097] When inhibiting EMT, the inhibitor may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0098] In one aspect, a method is provided for treating cancer in a patient in need thereof, comprising administering to the patient an inducer of mesenchymal-epithelial transition (MET) in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor or modulator of lipid metabolism for a time and under conditions to treat cancer in the patient.

[0099] The inducer of mesenchymal-epithelial transition may be administered simultaneously or sequentially with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor or regulator of lipid metabolism.

[0100] In one aspect, there is provided an inducer of mesenchymal-epithelial transition (MET) for use in treating cancer in a patient in need thereof, wherein the inducer of mesenchymal-epithelial transition (MET) is to be administered to the patient in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor or modulator of lipid metabolism.

[0101] In one aspect, there is provided the use of an inducer of mesenchymal-epithelial transition (MET) in the manufacture of a medicament for treating cancer in a subject, wherein the medicament is to be administered to a patient in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor or regulator of lipid metabolism.

[0102] In one aspect, a method is provided for sensitizing a cancer patient to an anti-cancer therapy, the method comprising administering to the patient a retinoid for a time and under conditions to sensitize the patient to the anti-cancer therapy.

[0103] In one aspect, an inducer of mesenchymal-epithelial transition (MET) is provided for use in sensitizing a cancer patient to an anti-cancer therapy.

[0104] In one aspect there is provided the use of an inducer of mesenchymal-epithelial transition (MET) in the manufacture of a medicament for sensitizing a patient to an anti-cancer therapy.

[0105] In one embodiment, a method is provided for inducing mesenchymal-epithelial transition (MET) in a subject, comprising administering an inhibitor or regulator of lipid metabolism for a time and under conditions to induce mesenchymal-epithelial transition, and the inhibitor or regulator of lipid metabolism can be used to (i) target or kill more aggressive mesenchymal subtype tumors, or (ii) block EMT and disease progression (i.e., tumor growth, invasion, metastasis, and resistance).

[0106] The inhibitor or regulator of lipid metabolism may be an inhibitor of 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 coenzyme A 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), and diglyceride acyltransferase (DGAT).

[0107] The inhibitor of lipid metabolism may be an inhibitor of carnitine palmitoyltransferase I (CPT1). In one embodiment, the inhibitor of CPT1 is etomoxir, perhexiline, or oxfenicine. In one embodiment, the inhibitor of CPT1 is etomoxir.

[0108] The inhibitor of lipid metabolism may be an inhibitor of mitochondrial thiolase. In one embodiment, the inhibitor of mitochondrial thiolase is 4-bromocrotonic acid.

[0109] The inhibitor of lipid metabolism 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).

[0110] The inhibitor of lipid metabolism may be an inhibitor of 3-ketoacyl-coenzyme A thiolase (3-KAT). In one embodiment, the inhibitor of 3-KAT is trimetazidine or ranolazine.

[0111] The inhibitor of lipid metabolism may be an inhibitor of monoacylglycerol lipase (MGLL). In one embodiment, the inhibitor of MGLL is JZL184.

[0112] The inhibitors of lipid metabolism can be administered as single agents or in combination with another anti-cancer agent, such as a standard of care chemotherapy agent or radiation therapy to treat cancer.

[0113] In one embodiment there is provided an inhibitor or regulator of lipid metabolism for use in inducing mesenchymal-epithelial transition (MET) in a cancer in a subject.

[0114] In one embodiment there is provided the use of an inhibitor or regulator of lipid metabolism in the manufacture of a medicament for inducing mesenchymal-epithelial transition (MET) of cancer in a subject.

[0115] In one embodiment, a method is provided for inhibiting or preventing metastasis of cancer in a subject, the method comprising administering an inhibitor or regulator of lipid metabolism for a time and under conditions sufficient to inhibit or prevent metastasis in the subject.

[0116] In one embodiment there is provided an inhibitor or regulator of lipid metabolism for use in inhibiting or preventing metastasis of cancer in a subject.

[0117] In one embodiment there is provided the use of an inhibitor or regulator of lipid metabolism in the manufacture of a medicament for inhibiting or preventing metastasis in a subject.

[0118] In one embodiment, a method is provided for inhibiting epithelial-mesenchymal transition (EMT) of a cancer in a subject, comprising administering an inhibitor or regulator of lipid metabolism for a time and under conditions sufficient to inhibit epithelial-mesenchymal transition (EMT) of the cancer in the subject.

[0119] In one embodiment there is provided an inhibitor or regulator of lipid metabolism for use in inhibiting epithelial-mesenchymal transition (EMT) in a cancer in a subject.

[0120] In one embodiment there is provided the use of an inhibitor or regulator of lipid metabolism in the manufacture of a medicament for inhibiting epithelial-mesenchymal transition (EMT) of cancer in a subject.

[0121] In one embodiment, a method is provided for characterizing the aggressiveness of cancer, 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.

[0122] In one aspect, a method for identifying an agent that induces mesenchymal-epithelial transition of a cell is provided, comprising: a) contacting the cells with a test agent to be screened; and b) detecting expression from the promoter of a gene selected from the group consisting of CD24, CD44, CD166, CDH1, ZEB1, and SERPINE 1; Including, Methods are provided in which an alteration in expression of one or more genes relative to a control indicates that the test agent is capable of inducing mesenchymal-epithelial transition of cells.

[0123] The change in expression of one or more genes compared to a control may be an increase or decrease in expression of one or more genes. The control may be cells that have not been contacted with the test agent being screened.

[0124] The cells may be a reporter cell line that has been genetically engineered to express a measurable marker from the promoter of the gene whose expression is to be detected.

[0125] Also provided herein is a reporter expression construct capable of expressing a measurable marker from the promoter of a gene selected from the group consisting of CD24, CD44, CD166, CDH1, ZEB1, and SERPINE 1.

[0126] Herein, a platform is provided that encompasses the use of genetically modified cancer cell lines in high-throughput small molecule / drug library screening to detect potent inducers of MET. The cell lines can detect changes in cell state while excluding common cytotoxic drugs. This is different from other common screens that detect loss of cell viability phenotypes, since most anti-cancer drug screens are designed to measure cell death resulting from drug treatment.

[0127] Provided herein is a genetically engineered reporter cell line comprising a reporter expression construct as defined herein.Also provided herein is a kit (or platform) for identifying agents that induce mesenchymal-epithelial transition of cells.

[0128] Control of cancer cell state, epithelial or mesenchymal, can be used to improve cancer cell responsiveness to therapy. Treatment of basal-like breast cancer or TNBC, but not luminal / ER+ breast cancer, with retinoids and other pathway-modulating compounds induced MET. This provides an important stratification strategy to select patient populations likely to respond to retinoid treatment. These patients have basal-like breast cancer or TNBC harboring an activated EMT program. This MET approach can be extended to mesenchymal-like tumors of other solid cancers, including gastric, colorectal, head and neck, lung, pancreas, and liver. All of these cancers may be composed of tumors with a mesenchymal signature.

[0129] In the case of breast cancer, patient stratification can be performed by (i) IHC or protein expression of tumor biopsies / resections, during which expression of ER, PR, HER2 is assessed, thus differentiating between luminal, HER2, and basal subtypes of breast cancer; (ii) using molecular diagnostics such as PAM50, which contains a gene expression signature indicative of a mesenchymal phenotype; and (iii) using IHC or protein expression of tumor biopsies / resections, during which 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 is assessed.

[0130] In one aspect, a method is provided for identifying a cancer patient who will be responsive to a combination therapy comprising a mesenchymal-epithelial transition (MET)-inducing compound in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor of lipid metabolism, comprising detecting a mesenchymal cancer subtype in a sample obtained from the patient, wherein the presence of the mesenchymal cancer subtype indicates that the patient will be responsive to the combination therapy.

[0131] Mesenchymal cancer subtypes can refer to basal-like (or mesenchymal-like) cancers.

[0132] In one embodiment, the method comprises treating the patient with a combination therapy.

[0133] In one aspect, a method for identifying and treating a cancer patient who is responsive to a combination therapy comprising a mesenchymal-epithelial transition (MET) inducing compound in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor of lipid metabolism, comprising: a) detecting mesenchymal-like cancer in a sample obtained from a patient, the presence of a mesenchymal cancer subtype indicating that the patient will be responsive to the combination therapy; and b) treating the patient with a combination therapy. A method is provided that includes:

[0134] Provided herein is a method for stratifying a patient population and identifying a subgroup of patients more likely to respond to a combination therapy, the method optionally comprising detecting a subgroup of patients having a mesenchymal cancer subtype in a sample obtained from each patient.

[0135] Provided herein are methods for monitoring a cancer patient's response to a combination therapy comprising a mesenchymal-epithelial transition (MET)-inducing compound in combination with a chemotherapeutic agent, an epigenetic modifying compound, an immunomodulatory agent, or an inhibitor of lipid metabolism.

[0136] Selected groups of patients can receive retinoids either as neoadjuvants or in combination with various standard of care regimens, which may differ for different cancers. MET is characterized by alterations in EMT markers, basal to luminal cytokeratin switching, suppression of cancer stem cell properties, enhanced sensitivity to SOC therapy, and thus improved clinical responses. The response of patient tumors to combination therapy with retinoids can be monitored by (i) IHC or protein expression of tumor biopsies / resections during which ER, PR, and HER2 expression will be assessed, thus differentiating between luminal, HER2, and basal subtypes of breast cancer; (ii) using molecular diagnostics such as PAM50, which contains a gene expression signature indicative of a mesenchymal phenotype; and (iii) using IHC or protein expression of tumor biopsies / resections during which 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, and mucin 1 will be assessed. Clinical measurements of response, such as tumor size, will be used as the endpoint clinical outcome. Similar approaches will be used for other cancers, but with cancer-specific molecular markers.

[0137] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not to the exclusion of any other element or integer or method step or group of elements or integers or method steps.

[0138] As used in the subject specification, the singular forms "a," "an," and "the" include plural embodiments unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes a single method as well as two or more methods, reference to an "agent" includes a single agent as well as two or more agents, reference to "the disclosure" includes single and multiple embodiments taught by the disclosure, and so forth. The embodiments taught and enabled herein are encompassed by the term "invention." Any variants and derivatives contemplated herein are encompassed by the "forms" of the invention. EXAMPLES

[0139] Materials and Methods Cell lines and cell culture A human mammary epithelial cell line (HMLE) was generated as described and immortalized using a retroviral vector to express the catalytic subunit of the human telomerase enzyme, hTERT, and the SV-40 large T antigen. NAMEC8 was generated as described. Briefly, HMLE cells were grown to 50% confluency followed by differential trypsinization with 0.05% trypsin for 1 min. Detached cells were carefully collected and replated in 24-well plates at approximately 200 cells per well. Once expanded, wells were screened for populations that were morphologically mesenchymal and capable of stable growth. 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 NAMEC were maintained in MEGM (Lonza).

[0140] HMLE-Twist-ER cells were generated as described. For EMT induction, 100 nM 4-hydroxytamoxifen in ethanol 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 previously. 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 were maintained as described previously. All cell cultures were incubated at 37°C with 5% CO 2 It was maintained at .

[0141] Primary human cells Breast tissue biopsies of cancer patients were obtained from the National Cancer Centre Singapore (NCCS) under an IRB approved protocol (2015 / 2976) with patient consent. Tissues were digested with 1mg / ml collagenase IV (Gibco) and 1mg / ml dispase II (Thermo Fisher Scientific) in F12 medium for 2 hours with shaking in a 37°C incubator. The cell suspension was then passed through a cell strainer and centrifuged at 1500 rpm for 10 minutes. Cells were then washed twice and resuspended in medium before being seeded on irradiated 3T3 feeders as previously described. Once confluent, they were passaged by first lifting the feeders using 0.15% trypsin for 2 minutes. After removal of the feeder cells, 0.25% trypsin was added to detach the tumor precursor cells.

[0142] Establishment and therapy of patient-derived breast cancer xenografts Primary human breast cancer samples were obtained from the National Cancer Centre, Singapore (NCCS) under an IRB approved protocol (2015 / 2976) with patient consent. All animal studies were approved by the Agency for Science, Technology and Research, Singapore Biological Resources Centre IACUC (protocol no. 171286). Patient-derived breast tumor fragments (approximately 3 × 1 × 1 mm) were inserted bilaterally into the inguinal mammary fat pads of 6-8 week old NSG female mice for initial establishment of tumors, which were then expanded and propagated in NSG mice once established. In this study, established tumor fragments of BC2.2 were implanted into cohorts of 6-8 week old female NSG mice. For BC22.1, 1 × 10 6 The cells were injected subcutaneously into both flanks of NSG mice.

[0143] Generation of reporter cell lines for screening compounds pGreenFire1-mCMV was cleaved using ClaI and SpeI and gel purified. The promoter sequences of genes Zeb1, Serpine1, CDH1, CD24, CD44, CD166 were amplified from human genomic DNA using pre-designed primers containing ClaI and SpeI restriction cleavage sites using AccuPrime Taq DNA polymerase (Thermo Fisher Scientific). 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 TB medium with ampicillin. Plasmids were then isolated using a Plasmid Miniprep Kit (Qiagen) and sent for sequencing (1stBASE Laboratories). After confirming the plasmid sequence, transfection into HEK293 cells was performed to generate lentivirus. Transfection was performed using FuGENE 6 (Promega). Viral supernatants were harvested at 24 and 48 hours, and NAMEC8 cells were then infected twice with the virus using polybrene (8 μg / μl), and good transduction efficiency was confirmed using the Dual-Glo luciferase system (Promega).

[0144] Compound library screening Stock plates of the compound library were prepared at 1 mM concentration in DMSO, courtesy of the GIS CHiP facility. NAMEC8-SERPINE1prom-Luc cells were seeded in 384-well plates at a density of 700 cells / well in a total volume of 50 μl using an automated pipetting device (BioTek). 0.5 μl of compound was then added to the wells using robotic pin transfer with a Bravo workstation (Agilent Technologies). Cells were then grown for 3 days and promoter-luciferase activity was measured using the Dual-Glo Luciferase System (Promega).

[0145] Dual-Glo luciferase assay The assay was performed according to the manufacturer's instructions. To measure luciferase activity in 384-well plates, the medium was first removed from the plates. Firefly luciferase activity was determined by adding 30 μl of Dual-Glo luciferase substrate (Promega) to the wells, incubating for 10 minutes, and measuring luminescence using a TECAN M1000 Pro plate reader. Renilla luciferase activity was then measured by adding 15 μl of Dual-Glo Stop & Glo substrate to the wells, incubating for 10 minutes, and measuring luminescence on a plate reader. Promoter luciferase activity was then calculated by taking the ratio of firefly luciferase to Renilla luciferase.

[0146] ERE luciferase activity assay ERE-luciferase construct was kindly provided by Mr. Alan Prem Kumar, CSI, Singapore. To measure ERE-luciferase activity, cells were seeded 24 h before transfection. ERE-luciferase (1 μg) and NSV-Renilla (0.1 μg) were packaged using Lipofectamine 2000 and transfected into NAMEC8. 24 h after transfection, cells were re-seeded in 96-well plates for drug treatment. Firefly and Renilla luciferase activities were measured 4 h after treatment with the Dual-Glo Luciferase System (Promega).

[0147] Lentiviral shRNA and overexpression constructs Lentiviral shRNAs were obtained from the Broad Institute RNAi Consortium (TRC) collection. The clone IDs of the shRNAs used are TRCN0000072181 (GFP control), TRCN0000330783 (RXRα shRNA1), TRCN0000021616 (RXRα shRNA2), TRCN0000021618 (RXRα shRNA3), and TRCN0000203516 (AGPAT9 shRNA). Each shRNA was cloned into the lentiviral plasmid pLKO.1 (Addgene).

[0148] To overexpress CPT1A, the ORF of CPT1A was excised from pCMV6-CPT1A (Origene catalog number RC200485) and cloned into pMN-GFP vector (kindly provided by Yu Qiang lab at GIS). Positive clones were confirmed by sequencing and used for retrovirus production. To overexpress Zeb1, the ORF of Zeb1 was cloned into FUW-LPT2 vector. Positive clones were confirmed by sequencing and used for lentivirus production.

[0149] For lentivirus production, plasmids were packaged in HEK293T cells using FuGENE 6 (Promega) with packaging plasmids. For retrovirus production, plasmids were packaged in PlatA cells using FuGENE 6 (Promega) with packaging plasmids. Viruses were harvested at 48 hours and transduced into target cells. Infected cells were selected with 2 μg / ml puromycin for 7 days.

[0150] RNA isolation, reverse transcription, and real-time PCR analysis Cells were rinsed twice with ice-cold PBS. Total RNA was extracted using Trizol (Invitrogen) and column purified using the RNeasy kit (Qiagen). cDNA synthesis was performed using the SuperScript III First Strand Synthesis System (Thermo Fisher Scientific) with 500 ng total RNA for 2 hours at 37°C, followed by 5-fold dilution. mRNA levels were measured using an ABI Prism 7900HT Sequence Detection Instrument 2.2 (Applied Biosystems) with gene-specific primers listed below. Results were normalized to GAPDH and analyzed using SDS2.2 software.

[0151] Proliferation assay To measure cell growth rates, 1000 cells were seeded in triplicate in 96-well plates. Cell viability was measured using Cell Titre Glo reagent (Promega) or CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) (Promega) according to the manufacturer's instructions.

[0152] Protein extraction and immunoblotting Cells were harvested and scraped off the culture dishes in ice-cold PBS. Cells were then centrifuged at 12000 rpm for 5 min at 4°C. Pellets were then either flash frozen in liquid nitrogen or lysed in ice-cold RIPA lysis buffer (Thermo Scientific) and left on ice for 30 min. Lysates were then centrifuged at 12000 rpm for 10 min at 4°C. Supernatants were then aspirated and placed into new tubes. Protein concentrations were measured using Coomaisse Blue. 50-80 μg of total protein was mixed with NuPAGE™ LDS sample buffer and heated at 70°C for 10 min. Samples were loaded onto NuPAGE™ 4-12% Bis-Tris gels (precast polyacrylamide gels; Invitrogen™). Gel electrophoresis was performed at 200V for 1.5 hours and then blotted onto nitrocellulose membrane (Trans-Blot Turbo™ Transfer Pack; Bio-Rad). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS; 1st BASE) with 0.1% Tween-20 (Santa Cruz) and then incubated with primary antibodies overnight at 4°C. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Finally, immunoreactive proteins were visualized using a Chemidoc™ Imager (Bio-Rad, 17001401) with enhanced chemiluminescence (ECL) reagents (Thermo Scientific, 34075).

[0153] Fluorescence-activated cell sorting (FACS) Cells were trypsinized until they detached from the cell culture plate. Neutralizing medium (DMEM containing 20% ​​FBS) was then added to inactivate the trypsin. Cells were then centrifuged at 1200 rpm for 3 min and the cell pellet was resuspended in PBS / 2% FBS. Cells were stained with DAPI (1:2000) and filtered through a 40 μm strainer before sorting. FACS was performed on a BD Aria Fusion Cell Analyzer.

[0154] CD24 and CD44 staining Cells were harvested by FACS and resuspended in 100 μl of a solution containing CD24-PE and CD44-APC (BD-Biosciences) antibodies diluted in 2% FBS in PBS (1:40 dilution). Cells were stained for 45 minutes with vortexing every 10 minutes to ensure uniform staining. After antibody incubation, cells were washed once and resuspended in 300 μl of 2% FBS in PBS. Cells were then filtered through a 40 μm strainer and analyzed on a BD LSR Fortessa X-20 Cell Analyzer.

[0155] Mammosphere assay For mammosphere culture, cells were seeded at 700 cells / well in 96-well ultra-low attachment 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.

[0156] Immunofluorescence (IF) staining of cells Cell cultures were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking with 1% bovine serum albumin in PBST. Cells were incubated with specific primary antibodies followed by appropriate secondary antibodies conjugated with Alexa Fluor-488 or Alexa Fluor-594 (Molecular Probes). Cell nuclei were then stained with DAPI and then observed and imaged on an Axio Observer D1 epifluorescence microscope with a built-in AxioCamMR3 camera (Zeiss) using optimal filters and 100-200x magnification.

[0157] Generation of SUM159 PACR cells SUM159 cells were exposed to stepwise increases in PAC starting at 10 nM. Medium with freshly prepared drug was replaced every 2 days and cells were split whenever they reached 80-90% confluency. The concentration of PAC was increased by 10 nM with each split until cells were able to survive at 100 nM. Cells were then subjected to cell viability assays to determine the IC 50 The IC 50 Values ​​were determined using GraphPad Prism software. IC 50 Cells were considered resistant to PAC if values ​​were increased at least 10-fold compared to the parental cell line.

[0158] I C 50 In vitro measurement of Cells were seeded in 96-well plates at a density of 2000 cells / well. The next day, drugs (5-FU / PAC) were added in a 10-point dose-response format. Cells were allowed to grow for 3 days and then cell viability was measured using Cell Titer Glo reagent (Promega). Data were normalized to vehicle control. Isobologram plots were generated and IC was calculated using GraphPad Prism software. 50 value was determined.

[0159] In vivo experiments using NSG mice All animal experiments were approved by the Agency for Science, Technology and Research, Singapore Biological Resources Centre IACUC (protocol number 140940). For pretreated SUM159 and MCF7-Ras cells, 1 × 10 6 The cells were suspended in 100 μl of medium containing 50% Matrigel and injected subcutaneously into both flanks of female NSG mice. Tumors in NSG mice were inspected weekly by palpation and harvested when they reached a diameter of approximately 15 mm. For treatment in NSG mice, 1 × 10 5NAMEC8-Ras cells were suspended in 100 μl of medium containing 50% Matrigel and injected subcutaneously into both flanks of NSG mice. NSG mice were inspected for tumor formation by palpation, and when tumors reached approximately 3 mm, mice were randomized into two groups: vehicle (5% DMSO in corn oil) and treatment (Bexarotene, 100 mg / Kg / day orally). Tumor size was measured weekly using calipers, and mice were sacrificed when tumors reached approximately 15 mm or after approximately 4 weeks of treatment. For patient-derived cells, 2 × 10 cells per site were injected. 6 The cells were injected subcutaneously into NSG mice.

[0160] Orthotopic tumor implantation of MCF7-Slug+Sox9 cells labeled with tdTomato fluorescent protein was performed as previously described. Briefly, 1 × 10 6 The cells were resuspended in 30 μl of medium containing 50% Matrigel and injected into the mammary fat pad of NSG mice. Mice were randomly divided into three groups (control, DOX, and etomoxir + DOX). Mice were administered vehicle control (0.9% w / v NaCl) or etomoxir (20 mg / kg) by daily ip injection for three weeks. Doxycycline was then administered via drinking water for two weeks. Mice were sacrificed 10 weeks after cessation of doxycycline treatment and lung metastases were analyzed under a fluorescent microscope.

[0161] Hematoxylin and eosin staining of tumor sections Tumor sections were fixed overnight in 4% paraformaldehyde, immersed in 70% ethanol, and sent to the Advanced Molecular Pathology laboratory at IMCB for paraffin processing and hematoxylin and eosin staining.

[0162] Immunofluorescence (IF) staining of tumor sections Tumor sections were fixed overnight in 4% paraformaldehyde, immersed in 70% ethanol, and sent to the Advanced Molecular Pathology laboratory at IMCB for paraffinization. For IF staining, slides were deparaffinized and rehydrated with PBS. The tissues were surrounded by a hydrophobic barrier using a barrier pen and blocked for 1 h with blocking buffer (1% bovine serum albumin in PBS solution; BSA, 1% horse serum, and 0.2% Triton X-100). The tissues were then washed with PBS and incubated overnight at 4°C in specific primary antibodies. The slides were washed with PBS and incubated with appropriate secondary antibodies conjugated with Alexa Fluor-488 or Alexa Fluor-594 (Molecular Probes) for 1 h at room temperature. The tissues were then mounted on slides using VECTASHIELD® mounting medium with DAPI (Vector Laboratories) and a cover glass. For long-term storage, the coverslips were permanently sealed around the perimeter with nail polish. Slides were observed and imaged with an Axio Observer D1 epifluorescence microscope with a built-in AxioCamMR3 camera (Zeiss) using optimal filters and 100-200x magnification. Fluorescent image analysis and fluorescent overlay images were obtained with AxioVision Rel 4.8 imaging software.

[0163] ChIP-Seq ChIP assays were performed as previously described. Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature, and then the formaldehyde was inactivated by adding 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 decrosslinked and used for library preparation using the Illumina TruSeq ChIP kit, followed by analysis using Illumina sequencing.

[0164] RNA-Seq 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 subsequently analyzed using Illumina sequencing.

[0165] Genomic Data Analysis For ChIP-Seq data processing, reads were aligned to the human genome (hg19) using the BWA aligner. Aligned reads were de-duplicated using Picard Markduplicates.jar. Peak calling was performed using MACS2 peak-caller with default parameters. Differential binding was performed using DiffBind. H3K27ac super-enhancers were identified using the ROSE algorithm as described. ChIP-Seq data have been deposited in GEO (accession number: GSE119824).

[0166] For RNA-Seq data processing, reads were aligned to the human reference genome GRCh37.p13 using TopHat2 and read counting was performed using featureCounts. Lowly expressed genes (i.e. genes with less than 10 counts in less than 60% of samples) were excluded from further analysis. Library size was then scaled by the trimmed mean of M-values ​​(TMM) method. Differentially expressed genes were identified using the R package limma and input into GREAT software for gene ontology pathway analysis. RNA-Seq data have been deposited in the SRA database (accession number: SRP152713).

[0167] lipid extraction Lipids from the cell pellets were extracted using butanol:methanol (1:1) as the extraction solvent. The extraction solvent contained an internal standard mixture consisting of the following: 17:0 LPC (lysophosphocholine), C14 LPE (lysophosphoethanolamine), C17 ceramide, C17 phosphocholine, C17 phosphoserine, C17:0 PG, C8-ceramide, DMPE (1,2-dimyristoyl-glycero-3-phosphoethanolamine), DMPG (1,2-dimyristoyl-glycero-3-phospho-(1'rac-glycerol), DMPS (dimyristoyl-glycero-phosphoserine), 12:0 DG (1,2-dilauroyl-sn-glycerol), C8 GluCer (D-glucosyl-β1-1'-N-octanoyl-D-erythro-sphingosine), 24:0 SM (N-lignoceroyl-D-erythro-sphingosylphosphorylcholine), 24:1 SM (N-Nervonoyl-D-erythro-sphingosylphosphorylcholine), d5-TAG 48:0. All standards were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA) except for d5-TAG 48:0, which was obtained from CDN isotopes. All standards were added at a final concentration of 100 ng / ml. 100 μl of extraction solvent containing the standard was added to 1 × 10 cells in the vial containing the cell pellet. 7 The mixture was vortexed for 15 seconds. The sample was then sonicated in a sonicator bath for 60 minutes at room temperature. The mixture was then centrifuged at 14000g for 5 minutes. The supernatant was transferred to a new tube and kept at -80°C until analysis.

[0168] Lipidomic analysis Samples were analyzed on a liquid chromatography tandem mass spectrometry (LC-MS / MS) instrument (Agilent 1290 UHPLC connected to an Agilent 6490 QQQ). A reversed-phase column (Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 × 50 mm, 1.8 μm) was used for separation. Gradient elution was performed using mobile phase A (40% acetonitrile / 60% water with 10 mM ammonium formate) and mobile phase B (90% isopropanol / 10% acetonitrile with 10 mM ammonium formate). Quality control (QC) samples were generated by pooling aliquots of lipid extracts from each sample. To monitor the performance of the experimental procedure, QC samples were injected every 10 samples during the analysis. 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 was extracted using MassHunter Quantitative Analysis software (Agilent). Lipids were normalized using both internal standards and cell number. Graphs and statistical analysis were performed using GraphPad Prism and the Mann-Whitney U test.

[0169] lipid droplet staining Intracellular lipid droplets were visualized with fluorescent Bodipy dye. Cells in culture were fixed with 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 overnight at 4 °C with Bodipy diluted in PBS (final concentration 1 μg / ml). Cells were washed with PBS and stained with DAPI. Fluorescent images were obtained with an Axio Observer D1 epifluorescence microscope with a built-in AxioCamMR3 camera (Zeiss) using optimal filters and 100-200× magnification.

[0170] Fatty acid β-oxidation assay FAO rates were measured using a mitochondrial stress test using an XF96 analyzer (Seahorse Bioscience) according to the manufacturer's instructions. Cells were seeded at a density of 9000 cells / well in complete medium in 96-well XF96 cell culture plates. The following day, cells were then incubated for 18 hours in XF modified DMEM assay medium (Seahorse Bioscience) supplemented with 1 mM glutamine, 0.5 mM glucose, 1% FBS, and 0.5 mM carnitine. On the day of the assay, cells were incubated for 45 min prior to the assay in FAO assay medium (KHB: 2.5 mM glucose, 0.5 mM carnitine, 5 mM HEPES supplemented with 111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl, adjusted to pH 7.4). 2 , 2 mM MgSO 4 , 1.2 mM NaH 2 PO 4 ) and non-CO 2 Plates were incubated in an incubator. Seahorse XF cell Mito stress test compounds (2 μM oligomycin, 2 μM FCCP, 2 μM rotenone). Etomoxir (40 μM) was added 15 min prior to the assay for inhibition of FAO. XF palmitate-BSA FAO substrate or BSA was added to the cells immediately prior to the start of the assay. Plates were then placed in the XF96 analyzer and the XF cell Mito stress test was run using the recommended protocol. The rate of FAO was calculated by the difference in maximum respiration when supplemented with palmitate-BSA FAO substrate compared to the BSA control.

[0171] statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc.) v7.0. All data are expressed as mean ± SEM unless otherwise specified. Student's t-test (two-tailed) was used to compare two groups and p-values ​​were calculated. P<0.05 was considered statistically significant. Significance levels are indicated in the relevant figure legends.

[0172] Example 1 To broaden our understanding of the molecular basis and therapeutic utility of the MET process, we performed a high-throughput small molecule library screen to identify a class of agents with MET-promoting properties in an unbiased manner. This revealed retinoids as the most potent MET promoters, but this potency and durability could be further enhanced through epigenetic reinforcement with HDAC inhibitors. Surprisingly, beyond classical MET marker genes and phenotypic changes, treatment with retinoids led to a basal-to-luminal-like transition accompanied by a switch in cytokeratin expression, as well as re-expression of ER in several patient-derived tumor cell models. Administration of retinoids to tumor-bearing animals induced changes in tumor architecture that resembled MET and halted tumor growth. Furthermore, we revealed for the first time that cell state transitions in breast cancer are defined by reprogramming of lipid metabolism, specifically the involvement of retinoids and their regulation of the switch between β-oxidation and lipid storage. Retinoids, via retinoic acid receptors (RARs) and retinoid X receptors (RXRs), bind and target key lipid metabolism genes as revealed by genome-wide analysis. This resulted in a diversion of fatty acid utilization towards lipid droplet storage in epithelial cell states and towards β-oxidation in mesenchymal cell states. Disruption of key metabolic enzymes mediating this flux resulted in inhibition of the MET process. Conversely, perturbation to β-oxidation using the CPT1 inhibitor etomoxir was able to rechannel fatty acid flux, promote a more epithelial cell phenotype, and further block EMT-driven breast cancer metastasis in animal models.

[0173] High-throughput small molecule library screen identifies retinoids as promoters of mesenchymal-epithelial transition Acquisition of a mesenchymal cell state in breast cancer is often associated with a more aggressive and invasive phenotype that is both chemoresistant and cancer stem cell-like. We have determined that conversion of mesenchymal to a more epithelial cell state through the process of mesenchymal-epithelial transition (MET) can reduce the tumorigenicity and chemoresistance of cancer cells in the form of combination therapy in breast cancer. To identify small molecule compounds capable of promoting MET, we utilized NAMEC8, a mesenchymal breast cell line that responds to perturbations in cell state coupled with a promoter-firefly luciferase reporter as a readout for cell state changes (Figure 1A). We tested several gene promoters, including CD24, CD44, CD166, CDH1, ZEB1, and SERPINE1, that have previously been shown to be associated with either epithelial or mesenchymal cell phenotypes. Other studies have shown that TGFβ promotes EMT in some cancer cell lines, while inhibition of the TGFβ pathway either blocks EMT or moderately induces 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 conditions (Figure 1B).In these SERPINE1prom-Luc, they were downregulated by at least 50%, so they showed the most dynamic response when treated with A83-01.

[0174] We screened four chemical compound libraries (LOPAC, anticancer, kinase inhibitors, bioactive lipids), containing more than 2700 compounds, to identify classes of molecules with MET-promoting properties (Figure 1C). Of note, in mesenchymal NAMEC8 SERPINE1prom-Luc reporter cells, we further integrated a Renilla luciferase reporter, under the control of a constitutive CMV promoter, as a readout for cell number. The aim was to identify compounds that alter 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 screens and dose-response measurements, showing a concordance rate of 83.6% (51 compounds), thereby highlighting the robustness of the reporter-based screen (Figure 1D, Figure 1E, and Table 1). At least eight 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, have not previously been implicated in cell state transitions but were identified as potential regulators. Among the classes, retinoids appeared to induce the most dramatic decrease in the SERPINE1prom-Luc reporter, as they did so in a dose-dependent manner (Figure 1E).

[0175] [Table 1A]

[0176] [Table 1B]

[0177] Retinoids induce the transformation of basal-like breast cancer cells into luminal cells and enhance their sensitivity to chemotherapy We chose to focus on retinoids as MET inducers for several reasons. First, they represent the most potent class of MET inducer compounds in the screen. Second, they are clinically viable agents used in acute promyelocytic leukemia as differentiation agents, although their utility and efficacy towards other cancers remains unclear. Finally, the manner and mechanism by which retinoids induce changes in cellular state or promote differentiation is less clear. From the screen, we selected the strongest RARα (TTNPB) and RXRα (bexarotene) agonists for validation studies and biochemical and pathway characterization. Consistent with the downregulation of SERPINE1prom-Luc reporter activity, which marked the mesenchymal cell state, treatment of NAMEC8 and another mesenchymal TNBC cell line, SUM159, with either bexarotene or TTNPB caused a dramatic change in morphology from dispersed spindle and fibroblast-like cells to an epithelial-like morphology characterized by dense clusters of epithelial islands (Figures 2A and 9A). This morphological change was associated with the downregulation of mesenchymal signature markers (ZEB1, SERPINE1, FN1, SLUG) and the concomitant acquisition of epithelial marker CDH1 (Figures 2B, 2C, 2D, and 9B, 9C). For the first time, we noted a switch in cytokeratin expression from KRT5, typically associated with basal-like triple-negative breast cancer, to KRT5, which is expressed in the luminal ER+ subtype of breast cancer. Mucin 1 (MUC1), a hallmark marker of luminal breast cancer that is not commonly seen in TNBC, was similarly upregulated. These data provided the first indication that, beyond MET, these cancer cells, thought to represent basal-like triple-negative breast cancer, had acquired luminal-like characteristics. As a mesenchymal phenotype has been shown to be associated with a cancer stem-like state, we examined the expression of CD44 and the mammosphere-forming capacity of retinoid-treated cells. Both NAMEC8 and SUM159 cells showed a marked reduction in surface CD44 expression via flow cytometry and a dramatic reduction in mammosphere-forming capacity (Figures 2E, 2F, and 9D, 9E).

[0178] To further evaluate the effect of retinoids beyond classical cancer cell lines, which tend to be homogenous, distinct from their parental tumors, or altered in plasticity, we generated a panel of patient-derived tumor cell lines from biopsied or resected triple-negative and ER+ breast tumors (Table 2). Consistent with previous findings, bexarotene treatment of patient-derived triple-negative breast cancer cell lines (BC2.2, BC22.1, BC29.1, MetBR 007-DR) enhanced E-cadherin expression, whereas SLUG expression was reduced to various degrees. More notably, however, we continued to observe a switch in cytokeratin expression from KRT5 to KRT8, as well as the acquisition of MUC1 expression (Figures 2G, 2H, and 9F, 9G). The ability of patient-derived cancer cells to form mammospheres was also reduced upon retinoid treatment (Figures 2I and 9H). Surprisingly, in three of these tumor cell lines, we noted upregulation of estrogen receptor (ER) expression, a defining feature of ER+, luminal breast cancer. Estrogen response element (ERE) luciferase reporter was also increased upon treatment with bexarotene or TTNPB (Figure 9I). In support of this, ER target genes such as ABCA3, NRIP1, TFF1, and CREB1 were strongly elevated by bexarotene treatment in both cancer cell lines (NAMEC8 and BC2.2) (Figure 9J). As other basal-like cancer cells have acquired ER expression to date, we wondered whether they also acquired sensitivity to anti-ER therapy administered to breast cancer patients, including ER+ tumors. Interestingly, bexarotene-treated cancer cells appeared to acquire sensitivity to low doses of the selective ER modulator, raloxifene hydrochloride (Figure 9K). Nevertheless, we noted that the difference in drug sensitivity was a modest two-fold, suggesting that the conversion from a basal to a true luminal phenotype may be incomplete.Interestingly enough, when bexarotene was administered to patient-derived tumor cell lines (BC24.1 and BC33.1) generated from ER+, luminal-like breast tumors, there was no dramatic change in the expression of epithelial / mesenchymal or luminal / basal markers (Figure 2G and Figure S9L), suggesting that retinoids exert their effects in mesenchymal-like, but not epithelial-like, breast cancer cells.

[0179] Next, we wanted to establish that retinoids were indeed acting and signaling through retinoic acid receptors (RXRα or RARα). Using shRNA targeting RXRα, stable knockdown was achieved in NAMEC8 (Figure 9M). Bexarotene-induced acquisition of epithelial luminal markers and loss of mesenchymal basal markers was suppressed in RXRα knockdown cells to date (Figure 9N, Figure 9O). These confirmed that retinoid-induced cell state changes were dependent on the RXRα receptor and its downstream activity.

[0180] Acquisition of a mesenchymal phenotype via EMT has been shown to confer resistance to a wide range of chemotherapy. We have demonstrated that the reversal phenomenon, MET, can overcome resistance by sensitizing other mesenchymal cancer cells to chemotherapy drugs. Not surprisingly, in both classical mesenchymal-like cancer cell lines (NAMEC8 and SUM159) and two patient-derived TNBC cell lines (BC2.2 and BC22.1), exposure to bexarotene, which induces MET, was able to confer sensitivity to 5-FU or paclitaxel, the standard of care for TNBC, by at least 4-6 fold (Figures 2J, 2K, and 9P, 9Q, 9R).

[0181] 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 resistant to chemotherapy. To test this, we generated paclitaxel-resistant (PACR) SUM159 cells, which exhibited a >20-fold increase in paclitaxel resistance compared to parental (PT) cells (Figure 2L). SUM159 PACR cells changed from a mesenchymal-like to an epithelial-like phenotype, adopted a clustered morphology, and showed increased expression of epithelial and luminal markers following treatment with bexarotene (Figure 9S, Figure 9T, Figure 9U). Bexarotene-treated PACR cells also exhibited reduced mammosphere formation capacity (Figure 9V), suggesting a decrease in cancer stem cell-like properties. More importantly, they acquired sensitivity to an alternative chemotherapeutic drug, 5-fluorouracil (5-FU), thereby demonstrating that drug-resistant cancer cells can respond to such therapeutic strategies (Figure 2M).

[0182] [Table 2]

[0183] Retinoid treatment promotes MET in vivo and reduces tumorigenicity of TNBC cells in animals We have determined that the conversion from mesenchymal to epithelial cell state can cause a decrease in tumorigenicity in other basal, mesenchymal-like breast cancer cells. Ex vivo treatment of SUM159 cells with bexarotene or TTNPB indeed drastically reduced their tumorigenic potential by 7 weeks after implantation into immunodeficient NSG mice, although tumor growth was subsequently photographed (Figure 3A and Figure 10A), indicating that a short period (14 days) of ex vivo treatment was not sufficient to mediate a permanent or durable change in cell state. It was also suggested that continuous treatment with bexarotene may be necessary to stop tumor growth. Interestingly, when the luminal epithelial-like breast cancer cell line, MCF7-Ras, was similarly exposed to bexarotene or all-trans-retinoic acid (ATRA), no effect on tumor cell growth was observed (Figure 3B and Figure 10B). Of note, the reduction in tumorigenicity was not due to the effect of retinoids to arrest cell proliferation, as control cells maintained similar proliferative capacity ( Fig. 10C ), clearly highlighting the utility of retinoid-induced cell state changes in basal, but not luminal, breast cancer.

[0184] To evaluate the efficacy of retinoids in tumor-bearing animals, we first implanted 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 approximately 3 mm by the fourth week. One group received bexarotene and the other group received a vehicle control via oral gavage daily for the duration of the study. Bexarotene-fed tumor-bearing mice showed a significant reduction in tumor growth rate and mass compared to vehicle-treated mice (Figure 3C, and Figure 10D, Figure 10E). To understand the cause of this growth reduction, we collected the remaining tumors for analysis. Tumor immunohistochemistry showed that the control tumors were grade 3 carcinomas with intraductal features, while tumors from bexarotene-treated mice were noncohesive and spread outward. The latter also showed the presence of spindle cells with focal necrosis, vascular cores suggesting possible papillary or pseudopapillary components (Figure 3D), thereby suggesting a transition in cellular state. To confirm this, we performed immunofluorescence staining of EMT markers on tumor sections. Tumors from bexarotene-treated mice to date showed expression of E-cadherin and loss of fibronectin, thereby exhibiting a MET phenotype, which occurred in vivo as a result of the initial therapeutic intervention. Furthermore, in these tumors, there was a switch in cytokeratin expression from basal-associated KRT5 to luminal-associated KRT8 (Figures 3E and 10F). As retinoids activate target genes such as STRA6, CRAPBP2, DHRS3, ANGPTL4, IP6K3, and ACSL5 through RXR and RAR receptors that bind DNA, we analyzed their expression in tumors from treated animals. All target gene expression was upregulated at least two-fold (FIG. 3F), thereby confirming that the cell state switch was mediated through retinoid target gene activity.

[0185] Example 2 Epigenetic perturbations enhance and increase the durability of retinoid-induced cell state changes Although retinoids were able to promote MET in mesenchymal breast cancer cells, the phenotype appeared to be at least partially reversible in the absence of sustained retinoid exposure. This pointed to the inherent plasticity of cancer cells, which may be due to dramatic epigenetic remodeling. We hypothesized that in combination with retinoid-induced cell state transitions, the epigenetic landscape could be modified to reinforce an epithelial cell state. First, we tested whether epigenetically modifying compounds themselves could induce MET. In a similar manner to the screen for MET compounds as described above, we screened 35 epigenetically modifying compounds, six of which were able to downregulate SERPINE1prom-Luc activity in mesenchymal NAMEC8 reporter cells. Although these reduced activity to 15-53%, the majority did not achieve the same extent by bexarotene treatment (Figure 4A). We next evaluated whether the combination of epigenetic compounds with bexarotene could have a greater effect on MET. Interestingly, some epigenetic compounds could provide additional benefits together with bexarotene, thus inducing a more dramatic downregulation of SERPINE1prom-Luc activity (Figure 4B). We focused on Cl-994 and SBHA, both of which are class I HDAC inhibitors (HDACi).

[0186] Combining HDACi with bexarotene resulted in more robust changes in epithelial cell morphology and related EMT markers (Figure 4C, Figure 4D, and Figure 11A). For example, ZEB1 and SLUG were more strongly reduced, while the acquisition of KRT8 and MUC1 was more pronounced. Since retinoids alone did not result in a stable MET phenotype, we evaluated whether the combination could prolong the maintenance of the epithelial cell state following withdrawal of both agents. Indeed, the reduction in SERPINE1prom-Luc activity in NAMEC8 cells treated with retinoids alone was restored 3 days after withdrawal, whereas it persisted in cells treated with the combination of retinoids and HDACi (Figure 11B, Figure 11C). More importantly, when the compounds were withdrawn following 7 days of treatment, the cancer cells that had undergone MET were able to maintain the epithelial phenotype for at least another 7 days (Figure 4E, Figure 4F, Figure 4G). Moreover, mammosphere formation capacity was also frequently suppressed (Figure 4H, and Figures S11D, S11E). In contrast, cells treated with bexarotene alone gradually reverted to a more mesenchymal phenotype, with partial recovery of mammosphere formation capacity. In the absence of retinoid ligands, RAR and RXR bind DNA, whereas they associate with transcriptional corepressors, including NCoR and HDAC, leading to gene silencing. When exposed to retinoids, they bind to the receptor, leading to the recruitment of coactivators such as histone acetyltransferase, p300. At the same time, inhibition of HDAC may have allowed the maintenance of an epigenetic context that promotes gene transcription, thereby resulting in a stronger and more durable MET (Figure 4I).

[0187] Lipid metabolism genes are regulated by retinoid receptors RAR and RXR are DNA-binding transcription factors that exert their effects by regulating the expression of target genes in the presence or absence of retinoids. Reported pathways involved in retinoid signaling include TGFβ, NFkB, and Nrf2. 27-29To understand how RAR and RXR program cell state transitions, we performed ChIP-seq to identify a profile of 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 treatments and then performed genome-wide analysis of target gene binding sites for RXR, RAR, and p300. Exposure to bexarotene, an RXR ligand, resulted in a subset of genes bound to RXR as well as RAR, as both could form heterodimers. These genes further showed an increase in p300 binding and H3K27ac (histone 3, lysine 27 acetylation) modification, which marks gene enhancers (Figures 5A, 5B, and 12A, 12B). For example, well-known retinoid target genes such as STRA6 and DHRS3, among others, which are not naturally bound by RXR in mesenchymal cell conditions, were bound by MET (FIGS. 5C and 12C).

[0188] We next sought to identify functional pathways regulated by RXR by mapping the top RXR-bound genes using gene ontology analysis. This revealed lipid metabolism as a major pathway perturbed during MET, a phenomenon not previously investigated (Figure 5D). To further test whether RXR-bound genes were related to transcriptional changes caused by RXR activation, we performed RNA-seq of cancer cell lines and cancer cells from patients treated with bexarotene to detect gene expression changes. Consistent with the above analysis, genes upregulated at least more than 2-fold showed clear enrichment in pathways related to lipid metabolism (Figure 12D, Figure 12E). Super-enhancers are a hallmark of keystone genes that help define cell states and cell lineages. Analysis of H3K27ac marks revealed critical genes that contained super-enhancers during MET. Among these were retinoid target genes such as DHRS3, RXR, and GDF15, and 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 clear RXR recruitment and enhanced H3K27ac modification upon bexarotene treatment (Figures 5C and 12C); this further highlights their putative role in MET.

[0189] To clarify which specific aspects of lipid metabolism may be altered during cell state change, we examined in more detail the list of RXR target genes that showed consistent up- or down-regulation across many cancer cell lines and cancer cells from patients treated with bexarotene (Figure 5F). Interestingly, many of the lipid-related genes form the pathway that produces triacylglycerides (TAG) from fatty acids. Genes involved in TAG synthesis (ACSL4, ACSL5, AGPAT4, AGPAT9, GPAM, DGAT2, PLIN1) were up-regulated, while MGLL, a lipase involved in TAG degradation, was strongly down-regulated (Figure 5G and Figure 12G). These results therefore strongly pointed to the reprogramming of lipid metabolism during MET.

[0190] Mesenchymal-epithelial transition limits β-oxidation by channeling fatty acids towards lipid stores The main end result of fatty acids is their conversion and storage as TAG. The key lipid metabolism enzymes long-chain fatty acid CoA ligase (ACSL), glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), prostatic acid phosphatase (PPAP), and diglyceride acyltransferase (DGAT) were all upregulated upon retinoid treatment, which could help drive the conversion of fatty acids to intermediates such as diacylglycerides (DAG) and TAG (Figure 4a). To demonstrate this, we used lipid-based mass spectrometry-based lipidomics to determine the end result of fatty acids and their downstream intermediates upon altered cellular conditions. Indeed, following bexarotene treatment, lipid species such as phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), which are downstream metabolites of fatty acids, were dramatically increased. Accumulation of DAG and TAG species was also observed (Fig. 6a). TAG, when accumulated, may be housed in lipid droplets. Interestingly, clearly abundant lipid droplets were observed in mesenchymal cancer cells undergoing 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 be different in mesenchymal and epithelial cell conditions.

[0191] We sought to functionally test whether intermediate lipid enzymes involved in the TAG synthesis pathway were indeed required for the regulation of MET. Pharmacological inhibition of GPAT (using FSG67) and DGAT (using amidepsin A), as well as shRNA knockdown of AGPAT9, all resulted in a clear reduction in lipid droplet accumulation during retinoid treatment when compared to vehicle treatment or control shRNA knockdown (Figure 14b). This was due to blockade at MET, as acquisition of 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 (Figure 14c). Associated with blockade at MET was a corresponding rescue in the mammosphere-forming ability of these mesenchymal NAMEC8 cells, clearly highlighting the importance of the TAG synthesis pathway to the epithelial cell state (Figures 6c and 14d).

[0192] Since the more epithelial cell state seemed to be associated with greater lipid storage, we deduced that in the mesenchymal cell state, fatty acids could be utilized as an energy source for alternative biochemical pathways, such as fatty acid β-oxidation (FAO). β-oxidation is a catabolic process in which fatty acids are broken down by mitochondria to produce acetyl-CoA for fueling 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 residing in the mesenchymal state clearly preferred palmitate for energy production. On the other hand, bexarotene-treated cells, which had transitioned to the epithelial state, showed a lower OCR when supplemented with palmitate (Figure 6d, and Figure 14e, Figure 14f), thereby suggesting that fatty acid utilization and fate differ between cancer cells residing in different cell states. Cancer cells of mesenchymal origin tend to rely on β-oxidation, whereas those of epithelial origin appear to shun fatty acids for storage in lipid droplets.

[0193] Observing this switch in the end result of fatty acid utilization, from β-oxidation to lipid storage, in retinoid-induced MET, led us to consider whether cancer cells in their native cellular state also manifested such metabolic choices. Comparing two isogenic cell lines stably residing in either epithelial (HMLE) or mesenchymal (NAMEC8) cellular states, the former indeed showed a greater accumulation of lipid species favorable for TAG production and ultimately lipid droplet formation (Fig. 6e, 6f, and 14g). The higher reliance on β-oxidation was reflected in the faster FAO rate in mesenchymal NAMEC8 compared to epithelial HMLE, and mesenchymal cancer cell lines (BT549, MDA-MB-468, and SUM159) similarly had faster FAO rates compared to epithelial cancer cell lines (MCF7 and T47D) (Fig. 6g). Importantly, the epithelial cancer cell line MCF7 did not exhibit such a metabolic preference, whereas a growth advantage was observed in the mesenchymal cancer cell line BT549 when supplemented with the fatty acids palmitate or lipoic acid under nutrient-depleted conditions (Figure 6h).

[0194] Induction of mesenchymal-epithelial transition depends on the fatty acid modifier carnitine palmitoyltransferase If cancer cells are indeed dependent on β-oxidation to maintain the mesenchymal phenotype, we hypothesized that inhibiting β-oxidation could be useful to promote or stabilize the epithelial cell phenotype by limiting the metabolic pathways on which cancer cells depend. Carnitine palmitoyltransferase I (CPT1) is a key mitochondrial enzyme involved in catalyzing the transfer of acyl groups from long-chain fatty acyl-CoAs to form acylcarnitines. In essence, it can transport fatty acids into mitochondria to fuel the TCA cycle and utilize fatty acids as a source of energy. We first showed that exposing mesenchymal cancer cells to the CPT1 inhibitor, etomoxir, alone or in combination with bexarotene, completely inhibited FAO activity (Figure 15a). We showed that bexarotene alone can cause MET, but etomoxir cannot do so by itself. Nevertheless, the combination of bexarotene and etomoxir exerted a more robust MET phenotype that was more durable and sustained even when the drugs were removed for at least 7 days (Fig. 7a, and Fig. 15b, Fig. 15c). Cells treated with the combination therapy also exhibited a sustained inhibitory effect on mammosphere formation after drug withdrawal (Fig. 7b, Fig. 15d).

[0195] To demonstrate that the enhancement of MET by the addition of etomoxir is on-target, we performed genetic knockdown of CPT1A, the major isoform of CPT1, in NAMEC8. Knockdown of CPT1A led to increased accumulation of lipid droplets after bexarotene treatment (Fig. 7c), which corresponded to a stronger MET phenotype, as indicated by Stark reduction in mesenchymal / basal proteins and increase in luminal proteins (Fig. 7d). The enhanced MET phenotype was also manifested by a greater inhibition of mammosphere formation upon bexarotene treatment in CPT1A knockdown cells compared to control cells (Fig. 7e).

[0196] If mesenchymal cancer cells were indeed metabolizing fatty acids via β-oxidation, we deduced that the increased activity of CPT1 through its overexpression would overcome the effects of bexarotene and the induction of epithelial cell phenotype. We overexpressed CPT1A in NAMEC8 cells, which was remarkably sufficient to overcome the bexarotene-dependent inhibition of FAO (Fig. 15e). In the two cell lines overexpressing CPT1A, the cells clearly showed the absence of lipid droplets even upon bexarotene treatment (Fig. 7f and Fig. 15f), further demonstrating active FAO in CPT1A-overexpressing cells. This corresponded to the maintenance of the mesenchymal phenotype in CPT1A-overexpressing cells upon bexarotene treatment (Fig. 15g), as evidenced by cells growing in a dispersed manner, the maintenance of mesenchymal markers, and the suppressed induction of epithelial / luminal markers upon bexarotene treatment (Fig. 7g). Mammosphere formation assays performed on CPT1A overexpressing cell lines rescued mammosphere formation even in the presence of bexarotene treatment (Fig. 7h and Fig. 15h); this suggested that enhanced mitochondrial uptake of fatty acids for β-oxidation was sufficient to overcome the effect of the cell state change induced by bexarotene. Thus, although an upstream trigger of the cell state transition is required, reprogramming of metabolic requirements is essential to achieve the cell state and appears sufficient to overcome the effect of the MET inducer.

[0197] Impaired fatty acid oxidation rechannels lipids towards a supportive epithelial state and blocks EMT Since MET programs the end result of fatty acids towards lipid storage, we deduced that EMT, a reversal process, produced the opposite result. Using three different cell lines (HMLE-Twist-ER, HMLE-Zeb1, MCF7-Slug) containing inducible expression of EMT transcription factors, Twist, Zeb1, and Slug, respectively, we observed a remarkably large amount of lipid droplets when the cells were in an epithelial state, which disappeared as the cells transitioned to a mesenchymal state (Figure 8a). Using HMLE-Twist-ER cells, we also showed that activation of the EMT program was able to reprogram the metabolic requirements of cancer cells from lipid storage to β-oxidation, as measured by OCR (Figure 8b and Figure 16a).

[0198] Since the shift in metabolic reprogramming during EMT was translated into changes in FAO, we hypothesized that inhibition of CPT1 could result in EMT progression. Exposure of HMLE-Twist ER cells to 4-OHT induced EMT, while treatment with etomoxir could dramatically block this transition (Fig. 8c, d). This was recapitulated in yet another inducible EMT cell line model, MCF7-Slug+Sox9, whereby etomoxir similarly blocked Slug+Sox9-induced EMT in MCF7 breast cancer cells (Fig. 16b, 16c, d). The importance of CPT1 to the EMT program was also demonstrated by genetic knockdown of CPT1A, which resulted in failure to induce EMT progression and therefore maintenance of the epithelial phenotype (Fig. 8e, 8f, and 16e). This highlights the importance of fatty acid utilization in controlling and programming cell states. The effect of Etomoxir in blocking TGFβ-induced EMT is also shown in the lung cancer cell line A549 (FIGS. 16f and 16g).

[0199] Finally, we sought to functionally test whether direct perturbation of a metabolic pathway, in this case fatty acid oxidation, could alter the pathway of EMT-induced metastasis in an in vivo animal model. We utilized a cell line model, MCF7-Slug+Sox9, whereby doxycycline-inducible co-expression of EMT transcription factors, Slug and Sox9, in pre-established MCF7 tumor xenografts, was able to strongly induce EMT in vivo and promote metastasis of otherwise non-metastatic tumor cells from fat pads to the lungs of NSG mice (Fig. 8g). 32 Here, MCF7-Slug+Sox9 cells were implanted into the fat pads of NSG mice, and drug treatments were initiated one week after implantation. One group of mice was administered etomoxir (20 mg / kg per day) prior to doxycycline administration. Another group of mice was given doxycycline while sham-treated with vehicle control. After 10 weeks, immunofluorescence analysis showed that primary tumors in control-treated mice remained epithelial in nature, with expression of E-cadherin (Figure 8g). In contrast, tumors in doxycycline-treated mice showed defining features of EMT, including expression of the mesenchymal marker, vimentin, and concomitant loss of E-cadherin. However, tumors in doxycycline-treated mice that also received etomoxir showed retention of epithelial features, thereby indicating blockade of EMT in vivo. More importantly, extensive metastatic nodules were detected in the lungs of doxycycline-treated mice, while their counterparts receiving etomoxir had a similar number of metastatic lesions as control-treated mice (Figure 8g). This provided the first proof of concept that fatty acid oxidation is required for activation of the EMT program to generate metastasis, and that pharmacological inhibition of this critical metabolic pathway may be useful in controlling metastasis in breast cancer.

[0200] Epigenetic regulation determines cell state transitions Since EMT is a reversible process, epigenetic mechanisms such as histone modification and DNA methylation have been found to be important in regulating the expression of EMT-responsive genes. During the activation of the EMT program, epigenetic silencing of E-cadherin, for example, has become more evident. Furthermore, studies have also reported that various epigenetic regulators, such as histone deacetylase inhibitors (HDACi), can exert differentiation effects on cancer cells. Therefore, to clarify the molecular link between EMT and epigenetic regulation, the inventors carried out MET and EMT inhibition screening using a comprehensive epigenetic library consisting of a wide range of epigenetic compounds - the Structural Genomics Consortium (SGC) Epigenetic Chemical Probe Library (Cayman).

[0201] For MET screening, good hits correspond to those that can induce lower SERPINE1 promoter activity compared to A83-01. Primary screening revealed several hits that were able to induce MET without adversely affecting cell viability (Figure 17A). These hits include the proteins inhibitors of arginine methyltransferase 5 (PRMT5), G9a / GLP histone methyltransferase, and CREBBP / EP300 bromodomain. They were then tested at six different concentrations to evaluate their dose-dependent changes on SERPINE1 promoter activity at high drug concentrations and compared with the MET-inducing potential of each compound (Figure 17B). All four compounds showed different dose-dependent decreases in SERPINE1 promoter activity at high concentrations. Afterwards, they were then subjected to in vitro validation assays to test their effectiveness in promoting MET in mesenchymal TNBC cells.

[0202] Systematic validation of epigenetic hits identifies CREBBP / EP300 bromodomain inhibitors as potent MET inducers From the MET screen, four hits (Figure 17C, circled in red) that were the most potent in reducing SERPINE1 promoter activity were selected for in vitro validation studies. These compounds consisted of PRMT5 inhibitors - LLY-283 and GSK591, inhibitors of histone methyltransferases, G9a and GLP - UNC0642, and inhibitors of CREBBP and EP300 bromodomain - SGC-CBP30. These compounds were first treated with mesenchymal NAMEC8 cells to morphologically validate whether they could induce changes in cell state. As observed in Figure 3C, only treatment with SGC-CBP30 led to a clear change in cell morphology from dispersed spindle and fibroblast-like cells to dense clusters of epithelial islands, which is characteristic of epithelial-like morphology. This suggests that SGC-CBP30 is potentially a stronger agent to induce MET in mesenchymal cells compared to the other hits. In contrast, the other three compounds were not observed to induce an epithelial-like morphology in NAMEC8 (FIG. 17C).

[0203] The hit-treated cells were then evaluated for transcriptional changes, EMT, and protein expression of basal / luminal markers (Figure 17D and Figure 17E). Similar to the morphological observations, SGC-CBP30 was shown to be the most potent 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. Conversely, no obvious changes were observed in cells treated with LLY-283, GSK591, and UNC0642. Furthermore, since the mesenchymal phenotype is often associated with basal-like cells and the current research interest is TNBC, basal and luminal markers were also evaluated upon drug treatment. SGC-CBP30 treatment was observed to increase mucin 1 (MUC1) - a glycoprotein commonly expressed in luminal breast cancer (Figure 17D and Figure 17E). This therefore suggests that SGC-CBP30 was not only able to induce MET but also to mediate the acquisition of luminal-like characteristics in basal-like mesenchymal cells.

[0204] When cells were resuspended in mammosphere medium, we observed that LLY-283 (p<0.01), GSK591 (p<0.01), and SGC-CBP30 (p<0.05) pretreatment significantly reduced the size and number of spheres formed. DMSO pretreated cells, in contrast, were able to effectively form spheres (Figure 17F). This therefore suggests that the in vitro stemness of mesenchymal NAMEC8 cells was reduced upon treatment with these compounds. In addition, mammary stem-like cells are typically characterized by the presence of CD44 high / CD24- / low cell surface markers. Therefore, the expression of these cell surface markers was analyzed by flow cytometry upon SGC-CBP30 treatment, as this compound exhibited stronger MET-inducing potential across various validation assays. Figure 3G shows that upon SGC-CBP30 treatment, a population of cells with low CD44 expression was observed, suggesting that the expression of this classical stemness marker was suppressed. This indicates a loss of stemness when NAMEC8 cells are treated with SGC-CBP30. As stem-like traits are associated with a mesenchymal phenotype, the loss of stem-like properties upon treatment strongly suggests a transition away from a mesenchymal cell state.

[0205] To ensure that the effects of these hits in promoting cell state transitions could be replicated in cancer cells, the compounds were further evaluated in the TNBC cell line, SUM159. Similar to what was shown in NAMEC8, SGC-CBP30 was observed to be a stronger MET inducer compared to the other hits, enhancing epithelial features and reducing stemness in SUM159 (Figure 18A-D). When SUM159 cells were analyzed by flow cytometry for expression of CD44 and CD24 cell surface markers upon SGC-CBP30 treatment, the cells showed a slight decrease in CD44 expression with the population of cells acquiring CD24 expression (Figure 3I). This suggests a transition from a stem-like state (CD44 high / CD24- / low) to a non-stem-like state (CD44 low / CD24 high) upon inhibition of the CREBBP / EP300 bromodomain in SUM159. These data thereby indicate that SGC-CBP30 was not only able to reduce mesenchymal and basal-like characteristics but also to induce loss of stemness in TNBC, potentially reducing tumorigenesis and invasiveness.

[0206] Inhibiting the histone acetyltransferase domain of the CREBBP / EP300 protein failed to induce robust MET conversion Since CREBBP and EP300 contain both bromo and HAT domains, we next explored whether inhibiting the HAT domains of these proteins would induce a similar phenotype as inhibiting the bromo domains. When NAMEC8 was treated with A-485, a potent and selective HAT inhibitor of CREBBP / EP300, dense clusters of epithelial islands were observed, displaying an epithelial-like cell morphology (Figure 19A). A-485 treatment also showed upregulation of E-cadherin protein expression with a slight decrease in mesenchymal markers (Figure 19B). These data suggest that, similar to SGC-CBP30, inhibiting the HAT domains of these proteins appeared to promote MET in mesenchymal cells. However, when A-485-treated cells were assessed for their in vitro stemness via mammosphere assay, there was no significant difference in sphere numbers from DMSO-treated cells (Figure 19C), indicating the limited potential of A-485 to reduce stemness of mesenchymal cells. Interestingly, when NAMEC8 cells were analyzed for expression of CD44 and CD24 cell surface markers upon A-485 treatment, a clear increase in CD44 expression was observed (Figure 19D). Thus, the data indicate that inhibiting the HAT domain of CREBBP / EP300 protein was able to induce an epithelial-like phenotype in NAMEC8 cells, but failed to reduce stemness in vitro.

[0207] Since CREBBP and EP300 are acetyltransferases, we next evaluated the three most common acetylated histone marks - lysines 14, 18, and 27 of histone H3 (H3K14ac, H3K18ac, and H3K27ac) after SGC-CBP30 and A-485 treatment. The data showed that upon SGC-CBP30 treatment, all of the acetylated marks were reduced, although to a lesser extent, compared to A-485 treatment (Figure 19E). As SGC-CBP30 was shown to be more potent than A-485 in MET conversion, this suggests that the cell state transition may not only be due to changes in acetylation status, but also that the involvement of bromodomains is essential. However, more work needs to be done to clarify whether bromodomains and HATs actually regulate different target genes and proteins and to explore the effect of bromodomain inhibition on MET conversion. By elucidating the mechanism by which inhibiting the bromodomain of CREBBP / EP300 can specifically induce cell state transition in mesenchymal cells, it can serve as a novel therapeutic approach against TNBC.

[0208] Inhibition of CREBBP / EP300 bromodomain reduces tumor burden in basal-like TNBC in vivo Having established that CREBBP / EP300-mediated epigenetic changes could induce MET and reduce stem-like properties of basal-like mesenchymal TNBC cells, we next investigated the tumorigenic potential of TNBC upon inhibition of CREBBP / EP300 bromodomain and HAT to determine whether they could reduce tumor burden in mice. When NAMEC8-Ras cells were pretreated with SGC-CBP30 and A-485 before subcutaneous injection into immunodeficient NOD scid gamma (NSG) mice, only SGC-CBP30 treatment resulted in significantly smaller tumors after 11 weeks compared to DMSO-treated cells. Tumor formation was still observed when pretreated with A-485 (Figure 19F). This captures the in vitro findings that SGC-CBP30 is more potent in MET transformation of mesenchymal cells compared to A-485. Therefore, this suggests that altering gene regulation in TNBC cells via CREBBP / EP300 bromodomain inhibition rendered the cells less tumorigenic. However, in vivo studies need to be repeated to ensure that this observation is consistent and reproducible. Furthermore, histological analysis needs to be performed on tumor tissues to determine if there are any morphological differences between tumors formed in DMSO and drug-treated cells. Nevertheless, initial in vivo data suggests that inhibiting the bromodomain of the CREBBP / EP300 protein reduced the tumorigenic potential of more aggressive TNBC.

[0209] Taken together, these data indicate that CREBBP and EP300 proteins potentially play a role in maintaining the mesenchymal cell state. Inhibiting the bromodomains of these two coactivator proteins potentially results in reduced transcriptional regulation and modification of the epigenetic landscape of genes essential for maintaining cells in a mesenchymal state. Thus, we hypothesize that the bromodomains of CREBBP and EP300 may regulate mesenchymal genes associated with super-enhancers. These genes were hyperacetylated, resulting in greater transcriptional activation mediated by the CREBBP / EP300 bromodomain (Figure 19G). Inhibiting the bromodomains prevented the binding of coactivators and transcription factors to the promoters of these mesenchymal genes, resulting in reduced acetylation and subsequent gene transcription. Thus, we hypothesized that the induction of MET is potentially due to reduced transcription of genes important for maintaining the mesenchymal cell state (Figure 19G).

[0210] Phenotypic screening of MET inducers against metabolic targets NAMEC8-SERPINE1 cells were used in screening a library of 303 compounds against metabolic targets (MedChem Express) to identify MET inducers. A list of 15 compounds was identified as potent inducers of MET, and we selected six compounds to proceed with in vitro orthogonal validation: Glutaminase C-IN-1, GSK256066, GSK2837808A, AGI-6780, SW033291, and NCT-503 (Figure 20A and Figure 20B). Dual-glo luciferase assays were performed on NAMEC8-SERPINE1 cells treated with the six selected compounds at concentrations ranging from 10 nM to 2 uM. A dose-dependent decrease in SERPINE1 promoter activity was observed for the compounds Glutaminase C-IN-1, GSK256066, and AGI-6780 (Figure 20C).

[0211] To explore any possible association between glutaminase and the mesenchymal phenotype, western blot analysis of glutaminase was performed on a panel of epithelial versus 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 is a determinant of the mesenchymal cell state and a potential biomarker for triple-negative breast cancer (Figure 20D).

[0212] To perform an in vitro validation of MET, NAMEC8 cells were treated with C-IN-1. Morphological changes from the dispersed distribution typically observed in mesenchymal cells to dense clusters of epithelial islands were induced upon C-IN-1 treatment (Figure 20E). Corresponding changes in molecular markers were observed by Western blotting, where mesenchymal proteins Zeb1, fibronectin, Slug, and Snail were decreased, epithelial E-cadherin was increased, basal cytokeratin 5 was decreased, and luminal markers cytokeratin 8, mucin 1, and ER were increased upon C-IN-1 treatment (Figure 20F). This provides molecular evidence for the induction of MET upon glutaminase inhibition.

[0213] Genetic knockdown of GLS1 was performed in NAMEC8 using three shRNAs (Figure 20G), followed by treatment with paclitaxel to investigate changes in chemotherapy sensitivity after MET induction. A decrease in IC50 for paclitaxel was observed in GLS1 sh2 cells compared to Ctrlsh cells (Figure 20H), indicating high chemotherapy sensitivity upon conversion to an epithelial phenotype. Mammosphere assay was also performed as an in vitro assay for stemness. A decrease in the number of spheres formed was observed in GLS1 sh2 and sh3 cells compared to Ctrlsh cells, suggesting a decrease in stemness (Figure 20I). Genetic knockdown of GLS1 was also performed in NAMEC8-HRas cells and subcutaneously injected into the flank of NSG mice. A decrease in tumor growth was observed in GLS1 sh2 cells compared to Ctrlsh cells (Figure 20J and Figure 20K), suggesting a lower possibility of tumor formation upon induction of MET.

[0214] Example 3 Consideration Altering cell state is an emerging approach to control the response of cancer cells to therapy. In acute promyelocytic leukemia and cutaneous T-cell lymphoma, retinoids have been applied as first-line adjuvants with curative intent. However, we have demonstrated that retinoids act as inducers of cell state transition in solid tumors, stimulating otherwise resistant breast cancer cells to respond better to standard treatment, which may be clinically useful in combination therapy. In addition, retinoid-induced MET transitions cells from a more aggressive mesenchymal state to a less metastatic epithelial state. In a small cohort study of metastatic breast cancer patients, retinoid therapy did not result in a response, possibly due to the lack of tumor molecular subtype stratification and patient selection. With new information arising from our study, we have shown that basal-like TNBC, which tend to be more mesenchymal in nature, are more likely to be responsive. This highlights the need for biomarkers that can stratify potential responders from non-responders.

[0215] Immune checkpoint blockade therapy has shown remarkable benefits against some cancers. The appeal of harnessing the immune system lies in its common denominator approach. The immune system has a natural advantage in that it has the innate ability to identify and attack cancer cells, no matter how diverse the molecular alterations of the cancer. Immune checkpoint (PD1, PD-L1) inhibitors have been effective in treating certain human cancers, e.g., melanoma and lung cancer, but have not had the same success in ER+ / PR+ breast cancer and are currently undergoing intense clinical investigation in metastatic TNBC. Keynote 012 reported a response rate of 18% using pembrolizumab monotherapy in a phase 1 study. Keynote 086, a phase 2 pembrolizumab monotherapy study, showed a response rate of 5%. Some studies have suggested a potential association between cancer cell status and expression of immune markers such as PD-L1 in cell-based models. Conversion of a mesenchymal cell state to an epithelial cell state induces intrinsic molecular changes that cause tumor cells to now be more susceptible to attack by immune cells, thereby improving the success of immunotherapy in breast cancer.

[0216] Mechanistically, there may be several routes to achieve MET phenotypic conversion. In our study, retinoids are the most potent class of such inducers identified in an unbiased manner. We first demonstrated that retinoids act by rewiring lipid metabolism during cell state conversion (Figure 7I). Whether lipid metabolism is in fact a driver or consequence of cell state change remains to be determined. Inhibition of beta-oxidation by etomoxir was insufficient for MET induction, suggesting that other pathways are also required or that several enzymes of the FAO pathway need to be inhibited simultaneously. Nevertheless, overexpression of CPT1A alone was sufficient to block bexarotene-induced MET, and the effect of bexarotene was prolonged by etomoxir treatment. This indicates that metabolic reprogramming is a necessary feature that allows cells to transition between cell states as well as maintain the transition. Beyond TNBC, MET conversion may also be broadly applicable to cancers harboring mesenchymal features and may also stimulate responses to other therapeutic agents.

[0217] Why do different cancer cell states impose unique metabolic requirements? In embryonic stem cells, glutamine and α-ketoglutarate (αKG) support pluripotency by increasing the αKG to succinate ratio that promotes DNA and histone demethylation. In cancer, the cell state controls proliferation, invasiveness, and stress tolerance, which necessitates stringent requirements for specific metabolites. For example, asparagine availability generally promotes EMT-driven metastasis or tumor growth, while cisplatin-resistant cancer cells appear to be glutamine-dependent for nucleotide biosynthesis. Our study suggests that channeling fatty acids towards β-oxidation for energy production may enable cancer cells in mesenchymal states to tolerate fluctuations in nutrient levels during invasion and metastasis, while the more proliferative epithelial state may preferentially employ aerobic glycolysis even under glucose-rich conditions typical for bulk tumor cells. Thus, the dependency on specific metabolic pathways during different stages of cancer progression can be exploited to target cellular states, potentially opening up new avenues for antimetabolic therapeutic strategies.

[0218] [Table 3A]

[0219] [Table 3B]

[0220] [Table 3C]

[0221] [Table 4A]

[0222] [Table 4B]

[0223]

Table 5

[0224]

Table 6

Claims

1. 1. An in vitro or ex vivo method for inducing mesenchymal-epithelial transition (MET) in a cancer cell, comprising contacting the cell with an inducer of mesenchymal-epithelial transition for a time and under conditions sufficient to induce MET in the cell, wherein the cancer cell is a basal-like (mesenchymal-like) cancer cell, and the inducer of MET is a retinoic acid receptor alpha (RAR-α) agonist, SGC-CBP30, or glutaminase C-IN-1; The method, wherein the RAR-α agonist is TTNPB, IRX5183, SY-1425, etretinate or fenretinide.

2. 2. The method of claim 1, wherein the cancer cells are selected from the group consisting of triple-negative breast cancer, head and neck cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, liver cancer, or colorectal cancer.

3. The method of claim 1, wherein the cancer cells are cancer cells that are resistant to standard treatment therapy.

4. 1. An inducer of mesenchymal-epithelial transition (MET) for use in inducing mesenchymal-epithelial transition in cancer cells in a patient, said inducer of MET being a retinoic acid receptor alpha (RAR-α) agonist, SGC-CBP30, or glutaminase C-IN-1; The RAR-α agonist is TTNPB, IRX5183, SY-1425, etretinate or fenretinide.

5. 1. Use of an inducer of mesenchymal-epithelial transition (MET) in the manufacture of a medicament for inducing mesenchymal-epithelial transition in cells of a patient, wherein the inducer of MET is a retinoic acid receptor alpha (RAR-α) agonist, SGC-CBP30, or glutaminase C-IN-1; The use, wherein the RAR-α agonist is TTNPB, IRX5183, SY-1425, etretinate or fenretinide.

6. 1. An inducer of mesenchymal-epithelial transition (MET) for use in sensitizing a cancer patient to an anti-cancer treatment, said inducer of MET being a retinoic acid receptor alpha (RAR-α) agonist, SGC-CBP30, or glutaminase C-IN-1; The RAR-α agonist is TTNPB, IRX5183, SY-1425, etretinate or fenretinide.

7. 1. Use of an inducer of mesenchymal-epithelial transition (MET) in the manufacture of a medicament for sensitizing a patient to an anti-cancer therapy, wherein the inducer of MET is a retinoic acid receptor alpha (RAR-α) agonist, SGC-CBP30, or glutaminase C-IN-1; The use, wherein the RAR-α agonist is TTNPB, IRX5183, SY-1425, etretinate or fenretinide.

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