HDAC inhibition impedes epithelial-mesenchymal plasticity and suppresses metastatic, castration-resistant prostate cancer

Abstract

PI3K (phosphoinositide 3-kinase)/AKT and RAS/MAPK (mitogen-activated protein kinase) pathway coactivation in the prostate epithelium promotes both epithelial-mesenchymal transition (EMT) and metastatic castration-resistant prostate cancer (mCRPC), which is currently incurable. To study the dynamic regulation of the EMT process, we developed novel genetically defined cellular and in vivo model systems from which epithelial, EMT and mesenchymal-like tumor cells with Pten deletion and Kras activation can be isolated. When cultured individually, each population has the capacity to regenerate all three tumor cell populations, indicative of epithelial-mesenchymal plasticity. Despite harboring the same genetic alterations, mesenchymal-like tumor cells are resistant to PI3K and MAPK pathway inhibitors, suggesting that epigenetic mechanisms may regulate the EMT process, as well as dictate the heterogeneous responses of cancer cells to therapy. Among differentially expressed epigenetic regulators, the chromatin remodeling protein HMGA2 is significantly upregulated in EMT and mesenchymal-like tumors cells, as well as in human mCRPC. Knockdown of HMGA2, or suppressing HMGA2 expression with the histone deacetylase inhibitor LBH589, inhibits epithelial-mesenchymal plasticity and stemness activities in vitro and markedly reduces tumor growth and metastasis in vivo through successful targeting of EMT and mesenchymal-like tumor cells. Importantly, LBH589 treatment in combination with castration prevents mCRPC development and significantly prolongs survival following castration by enhancing p53 and androgen receptor acetylation and in turn sensitizing castration-resistant mesenchymal-like tumor cells to androgen deprivation therapy. Taken together, these findings demonstrate that cellular plasticity is regulated epigenetically, and that mesenchymal-like tumor cell populations in mCRPC that are resistant to conventional and targeted therapies can be effectively treated with the epigenetic inhibitor LBH589.

Figure 1. Prostate tumor cells with PI3K/AKT and RAS/MAPK co-activation display epithelial-mesenchymal plasticity in vitro

(a) Schematic outlining the generation of the PKV cell line from EpCAM⁺/GFP^- epithelial cells FACS sorted from 10-12 week old CPKV prostates. (b) EpCAM⁺/GFP^- epithelial cells plated in culture spontaneously undergo EMT and express GFP. Scale bar, 50 μm; BF, brightfield. (c) The PKV cell line contains heterogenous epithelial (E), EMT, and MES-like (M) tumor cell populations as assessed by FACS analysis. (d) qPCR analysis confirms that EMT and MES-like (M) tumor cells from the PKV cell line have upregulated EMT signature gene expression compared to epithelial tumor cells. Expression is relative to gene expression values found in epithelial (E) tumor cells. (e) Matrigel invasion assay reveals that EMT and MES-like (M) tumor cells are significantly more invasive than epithelial (E) tumor cells isolated from the PKV cell line. (f) Each tumor cell population within the PKV cell line was isolated by FACS and cultured separately in vitro. Representative FACS plots of each cell population 14 days after plating are shown. Each tumor cell population has the plasticity to generate all 3 tumor cell populations. (g) Each tumor cell population within the PKV cell line was isolated by FACS and cultured separately in vitro. The percentage of each tumor cell population (E, EMT, M) within each individually plated cell type (Epithelial, EMT, MES-like) was assessed by FACS 1, 3, 7, 10, and 14 days post-sort. Data in d, e, and g are represented as mean ± SEM from 2-3 independent experiments done in triplicate. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2. Epithelial-mesenchymal transition states dictate response to PI3K and MAPK pathway inhibition and differential gene expression profile

(a) PKV cells were treated with vehicle alone (DMSO), the PI3K/mTOR inhibitor PKI-587 (100nM), the MEK inhibitor PD0325901 (100nM), or both (10nM for each) for 7 days. MES-like (M) tumor cell growth is unaffected by treatment with PI3K and MAPK pathway inhibitors. % growth is relative to vehicle-treated cells (DMSO). (b) The gene transcription profiles of epithelial (E), EMT, and MES-like (M) tumor cells isolated from the prostates of 10-12 week old CPKV mice as assessed by RNA-seq. Heatmap displays mean-centered gene transcription levels of 2190 genes with average FPKM no less than 0.01 and coefficients of variation higher than 0.5. (c) Venn diagram showing the overlap of differentially expressed genes (DEGs) between epithelial vs. EMT (E vs. EMT) and EMT vs. MES-like populations (EMT vs. M). The MES-like tumor cell population has a large number of DEGs compared to the EMT (4234) tumor cell population. (d) Significantly enriched Gene Ontology (GO) items for differentially transcribed genes between epithelial (E) and EMT tumor cell populations. (e) Significantly enriched GO items for differentially transcribed genes between EMT and MES-like (M) tumor cell populations. Items in bold are solely enriched in the EMT-M transition. Data in a is represented as mean ± SEM from 3 independent experiments done in triplicate. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 3. HMGA2 is highly expressed in human mCRPC and in murine EMT and mesenchymal-like tumor cells

(a) Heat map comparing the overlap of differentially expressed genes in primary/localized and metastatic (met) prostate tumor specimens from the Taylor et al. and Grasso et al. human prostate cancer datasets based on rank-rank hypergeometric overlap (RRHO) analysis used to measure and visualize the degree of statistically significant overlap between two expression datasets (Left Panel). The color bar indicates the transformed log10 hypergeometric enrichment p-value between two ordered gene sets. The high heat area along the diagonal indicates significant overlap in differentially expressed genes between the two human prostate cancer datasets. Right Panel, selected epithelial and EMT signature genes were plotted corresponding to their signed and logged p-value ranks based on differential expression between metastatic and primary cancer samples. The depth of the blue color represents the density of all genes. A number of EMT signature genes (large red circle) were consistently ranked at similar positions in both human prostate cancer datasets. HMGA2 (small yellow circle) was among the group of genes differentially expressed in metastatic prostate cancer in both human datasets. (b) EMT genes upregulated in human metastatic prostate cancer were differentially expressed in murine datasets obtained from 1) laser capture microdissection microarray analysis of prostate tissue samples (p<0.05) and 2) RNA-seq analysis of FACS sorted cell populations (FDR<0.05). EMT genes were often upregulated in poorly differentiated prostate tissue and in EMT and MES-like (M) tumor cells. Up, upregulated; n.s., not significant. (c) HMGA2 expression levels in benign, localized (local), and metastatic CRPC (met) patient samples from Grasso et al. reveals that HMGA2 expression is significantly upregulated in mCRPC compared to localized disease. (d) Hmga2 expression is significantly upregulated in EMT and MES-like (M) tumor cells compared to epithelial tumor cells (Left Panel). Data was combined from 3 independent experiments done in triplicate. Right Panel, Fisher’s exact test was used to assess enrichment of HMGA2-regulated genes in differentially expressed genes between the EMT vs. epithelial (EMT v. E) and MES-like vs. epithelial (M v. E) tumor cell populations. Dotted line, p-value= 0.05; n.s, not significant. (e) HMGA2 protein expression is highly induced in both the stroma and epithelium (arrows) of CPKV prostates compared to CPV and V prostates. Scale bar, 50 μm. Data in c and d are represented as mean ± SEM. *, p<0.05; **, p<0.01.

Figure 4. HMGA2 regulates stemness and epithelial-mesenchymal plasticity in prostate tumor cells with PI3K/AKT and RAS/MAPK co-activation

(a) ShRNA-targeted knockdown of HMGA2 protein expression in PKV cells. Mouse embryonic stem cells (M. ESC) were used as a positive control for HMGA2 expression. β-actin was used as a loading control. Control, PKV cells. Scramble, shScramble. (b) The proliferation of the PKV-shScramble (Scramble) and PKV-shHmga2 (shHmga2) cell lines was measured by MTT assay and is presented as % growth compared to control PKV cells. (c) PKV cells stably expressing shHmga2 have significantly reduced matrigel sphere-forming capacity compared to control PKV-shScramble (Scramble) cells. (d) HMGA2 knockdown reduces the expression of a number pluripotency and self-renewal factors. Expression is relative to gene expression values found in PKV-shScramble (Scramble) cells. (e) FACS analysis of the PKV-shScramble (Scramble) and PKV-shHmga2 cell lines revealed that throughout passaging, PKV-shHmga2 cells maintained a lower percentage of MES-like and higher percentage of EMT tumor cells compared to PKV-shScramble cells, indicative of a blockade in the transition of EMT tumor cells into fully MES-like tumor cells. (f) FACS sorted MES-like tumor cell populations from PKV-shHmga2 cells have reduced mesenchymal content and increased epithelial and EMT tumor cell numbers compared to control PKV-shScramble (Scramble) cells after 7 days in culture. Data in b-f are represented as mean ± SEM from 2-3 independent experiments done in triplicate. *, p<0.05; **, p<0.01.

Figure 5. HDACi treatment effectively targets EMT and mesenchymal-like tumor cells through inhibition of HMGA2 activity and induction of p53-mediated apoptosis

(a) LBH589 treatment (24 hr) of PKV cells reduces Hmga2 expression in a dose-dependent manner. Expression is relative to gene expression values found in vehicle-treated cells (DMSO). (b) LBH589 treatment (7 day) preferentially reduces MES-like (M) tumor cell numbers at low doses (1nM), and successfully targets all tumor cell populations at higher doses (10nM). % growth is relative to vehicle-treated cells (DMSO). (c) 7 day treatment of the PKV cell line with 10nM LBH589 induces significantly increased levels of apoptosis, as measured by the percentage of 7AAD⁺ cells, in all epithelial (E), EMT, and MES-like (M) tumor cell populations. (d) Low doses of LBH589 (1nM) significantly reduce the sphere-forming capacity of PKV cells after 7 days in Matrigel culture, similar to effects of HMGA2 knockdown (See Fig. 4C). (e) LBH589 treatment (24 hr) reduces the expression of various stemness factors in PKV cells compared to vehicle alone (DMSO), similar to the effects of HMGA2 knockdown (See Fig. 4D). (f) LBH589 treatment (6 hr) induces p53 expression and increases H3K27 and p53 acetylation levels in PKV cells. β-actin was used as a loading control. IP, immunoprecipitation; WCL, whole cell lystate; WB, Western Blot; Ac-Lys, acetylated lysine antibody. Data in a-f are represented as mean ± SEM. Data in b-e were collected from 2-3 independent experiments done in triplicate. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 6. HDACi treatment inhibits prostate tumor growth, tumor cell dissemination, and metastasis in vivo

(a) 10 wk old CPKV mice treated with LBH589 for 2 weeks had dramatically reduced tumor burden, particularly in the anterior lobes (arrows), compared to vehicle-treated mice (Top Panel). Scale Bar, 5 mm. Bottom Panel, the Ki67 proliferation index is significantly reduced in LBH589-treated CPKV mice (n=4) compared to CPKV mice receiving vehicle alone (n=3) in both the anterior and dorsolateral lobes of the prostate. (b) LBH589-treated CPKV mice (n=9) have a significant reduction in the EMT and MES-like tumor cell populations compared to vehicle-treated mice (n=6). Data were combined from 2 independent experiments. (c) LBH589 treatment significantly reduces HMGA2 expression (Top Panel) and induces p53 expression (Bottom Panel) in the prostates of CPKV mice. Scale bar, top panel, low magnification, 100 μm; top panel, high magnification, 25 μm; bottom panel, 25 μm. (d) LBH589 treatment inhibits the formation of lung macrometastases in NSG mice transplanted with PKV cells by tail vein injection. (e) Representative lung histology of NOD/SCID/IL2Rγ-null (NSG) mice 8 weeks after transplantation of PKV cells by tail vein injection. While LBH589-treated mice did not develop macrometases (0/4), they did develop small micrometastases that were non-proliferative (Ki67^-) and GFP⁺ (Top Panel). Bottom Panel, vehicle-treated mice, on the other hand, formed large, proliferating (Ki67⁺) macrometastases that are GFP^- and have strong expression of Pan-Cytokeratin (Pan-CK), indicative of an epithelial phenotype. Scale bar, low magnification, 500 μm; Scale bar, high magnification, 50 μm. Data in a and b are represented as mean ± SEM. *, p<0.05; **, p<0.01.

Figure 7. HDACi treatment can effectively inhibit the development of CRPC by targeting castration-resistant mesenchymal-like tumor cells

(a) While castration (Cx) of CPKV mice (n=12) at 6 weeks of age led to early lethality, LBH589 treatment in combination with Cx (n=7) significantly increased overall survival. Data were combined from 3 independent experiments. (b) LBH589 treatment in combination with Cx significantly lowered the Ki67 proliferation index in both the epithelium and stroma of CPKV prostates. n=3 for Intact and Cx samples; n=4 for Cx + LBH samples. (c) Castrated (Cx) mice (n=4) have no change in MES-like tumor cell numbers compared to intact, vehicle-treated mice (n=3). LBH589 treatment in combination with castration (n=6) successfully targets castration-resistant MES-like tumor cells and further reduces epithelial and EMT tumor cell numbers in vivo. Data were combined from 2 independent experiments. (d) AR target gene expression is significantly downregulated in MES-like (M) tumor cells compared to epithelial tumor cells isolated from the prostates of 10-12 week old CPKV mice as assessed by RNA-seq analysis. Expression is relative to gene expression values found in epithelial tumor cells. (e) 7 day treatment of PKV cells with media lacking androgens (CDT-FBS) significantly impeded the growth of epithelial (E) and EMT but not MES-like (M) tumor cells. Low doses of LBH589 (1nM) in combination with ADT sensitize MES-like (M) tumor cells to androgen withdrawal-induced growth inhibition. (f) LBH589 treatment reestablishes nuclear AR expression after Cx in the anterior lobes of CPKV prostates. Scale bar, low magnification, 50 μm; high magnification, 10 μm. (g) LBH589 treatment enhances AR target gene expression in CPKV prostates compared to vehicle alone. Expression is relative to gene expression values found in vehicle-treated CPKV mice. Data in b, c, d, e, and g are represented as mean ± SEM. *, Data in e and g were collected from 2-3 independent experiments done in triplicate. p<0.05; **, p<0.01; ***, p<0.001.