Hong-Wu Chen

PRC2/EZH2 inhibitors (PRC2i/EZH2i) are promising for the treatment of advanced cancers including metastatic prostate cancer. Here, we show that PRC2i/EZH2i alone or in combination with androgen receptor (AR) inhibitors induced diverse cell state programs (CSPs) (e.g., response to stress or IFN, MYC targets, stem cells, EMT lineage plasticity, and multiple developmental programs), which led to increased tumor cell invasion, metastasis, and resistance to other drugs, in addition to modest suppression of tumor growth. In contrast to the current perception, our comprehensive, integrated genomics and epigenomics profiling of patient-derived xenografts (PDXs) and clinical tumors revealed that PRC2/EZH2 suppressed CSP genes by maintaining chromatin bivalency. Hyperactive Wnt/β-catenin signaling and inhibitors of polycomb-repressive complex 2/enhancer of zeste homolog 2 (PRC2/EZH2) and the AR alter chromatin bivalency through antagonism of PRC2 and stimulation of MLL2/KMT2B in a feed-forward manner. The circadian rhythm regulator REV-ERBα unexpectedly reprogrammed β-catenin in promoting bivalency resolution and CSP gene expression. Dual targeting of Wnt/β-catenin and EZH2 diminished diverse cell states by restoring bivalency and effectively blocked tumor growth. Our findings provide unexpected insights into chromatin bivalency and dysregulated circadian rhythms in the control of cell state diversity and identify alternative therapeutic strategies that target PRC2/EZH2 for advanced malignancies.

Enzalutamide resistance remains a significant challenge in the treatment of advanced prostate cancer. Identifying molecular drivers of enzalutamide resistance is crucial for developing effective therapeutic strategies. In this study, we identify insulin-like growth factor–binding protein 3 (IGFBP3) as a key driver of enzalutamide resistance in castration-resistant prostate cancer (CRPC). We demonstrate that IGFBP3 expression is significantly upregulated in enzalutamide-resistant C4-2B MDVR cells compared with parental C4-2B cells. This upregulation was consistently observed across multiple enzalutamide-resistant CRPC models, including LNCaP-derived 42D and 42F cells, as well as long-term enzalutamide-resistant cell lines derived from LNCaP, VCaP, LAPC-4, and CWR-R1 cells. Additionally, enzalutamide treatment directly induced IGFBP3 expression in sensitive cells. Elevated IGFBP3 expression was also observed in CRPC patient samples after enzalutamide treatment and was associated with higher Gleason scores and reduced disease-free survival. Mechanistically, IGFBP3 activates the sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway, which promotes cell survival and resistance to enzalutamide. IGFBP3 knockdown decreased SphK1 expression, reduced S1P secretion, and enhanced enzalutamide sensitivity, whereas IGFBP3 overexpression induced SphK1 expression and S1P production, conferring enzalutamide resistance. Inhibition of IGFBP3 via siRNA reduced cell viability, induced apoptosis, and resensitized resistant models to enzalutamide. Similarly, targeting SphK1 with the inhibitor SKI-II suppressed SphK1 activity, reduced S1P production, enhanced enzalutamide sensitivity, and significantly inhibited resistant tumor growth while enhancing enzalutamide sensitivity. Collectively, these findings highlight IGFBP3-mediated SphK1 signaling as a critical mediator of enzalutamide resistance and suggest that targeting the IGFBP3/SphK1/S1P axis represents a promising therapeutic strategy to overcome resistance in advanced prostate cancer.

Epigenetic and transcriptional dysregulation plays a fundamental role in tumor lineage plasticity (LP). However, the underlying mechanisms, especially for the initial events of LP development, are still poorly understood. Here, we report that in progression of prostate cancer from adenocarcinoma to treatment-induced neuroendocrine prostate cancer (t-NEPC), anti-androgen receptor (AR) signaling inhibitors (ARSIs) reprogram the function of circadian regulator/nuclear receptor REV-ERBα by switching its target gene programs from kinase signaling and metabolic programs to programs of LP, which includes neurogenesis, stem cell, and epithelial–mesenchymal transition as well as over fifteen LP drivers including POU3F2/BRN2, ASCL1, FOXA2, ONECUT2, and MYCN. Unexpectedly, REV-ERBα facilitates the chromatin occupancy of BRN2, ASCL1, and FOXA1 in their activation of LP programs, thus functioning as a master regulator of ARSI-induced LP driver network. Mechanistically, REV-ERBα induces chromatin accessibility and H3K27ac modification at promoters of LP genes through its recruitment of BRD4 and p300. Overexpression of REV-ERBα alone is sufficient to induce LP and neuroendocrine phenotype and confers resistance to ARSI in adenocarcinoma cells. Loss of REV-ERBα potently inhibits NEPC cell growth and abolishes the expression of LP drivers and gene programs. Pharmacological inhibition of REV-ERBα exhibits high potency in blocking the growth of NEPC tumors including patient-derived xenografts. Our findings reveal that therapy-induced LP development entails a coordinated induction of a network of LP drivers and that REV-ERBα is an unexpected master regulator of the network and a promising therapeutic target for treatment of advanced prostate cancer such as NEPC.

Clinical treatment options for triple-negative breast cancer (TNBC) are currently limited to chemotherapy because of a lack of effective therapeutic targets. Recent evidence suggests that long noncoding RNAs (lncRNAs) encode bioactive peptides or proteins, thereby playing noncanonical yet significant roles in regulating cellular processes. However, the potential of lncRNA-translated products in cancer progression remains largely unknown. In this study, we identified a previously undocumented small protein encoded by the lncRNA LINC02870. This protein is localized at the endoplasmic reticulum (ER) and participates in ER stress, thus, we named it the endoplasmic reticulum stress protein (ERSP). ERSP was highly expressed in TNBC tissues, and elevated LINC02870 content was correlated with poor prognosis in TNBC patients. Loss of ERSP inhibited TNBC growth and metastasis both in vitro and in vivo. The pro-oncogenic effects of ERSP could be attributed to its selective activation of the IRE1α/XBP1s branch. ERSP enhances the unfolded protein response (UPR) by interacting with XBP1s, facilitating the nuclear accumulation of XBP1s, thereby promoting the expression of ER stress-related genes. These findings highlight the regulatory role of the lncRNA-encoded protein ERSP in ER stress and suggest that it is a potential therapeutic target for TNBC.

Profound functional switch of key regulatory factors may play a major role in homeostasis and disease. Dysregulation of circadian rhythm (CR) is strongly implicated in cancer with mechanisms poorly understood. We report here that the function of REV-ERBα, a major CR regulator of the orphan nuclear receptor subfamily, is dramatically altered in tumors in both its genome binding and functional mode. Loss of CR is linked to a functional inversion of REV-ERBα from a repressor in control of CR and metabolic gene programs in normal tissues to a strong activator in different cancers. Through changing its association from NCoR/HDAC3 corepressor complex to BRD4/p300 coactivators, REV-ERBα directly activates thousands of genes including tumorigenic programs such as MAPK and PI3K-Akt signaling. Functioning as a master transcriptional activator, REV-ERBα partners with pioneer factor FOXA1 and directly stimulates a large number of signaling genes, including multiple growth factors, receptor tyrosine kinases, RASs, AKTs, and MAPKs. Moreover, elevated REV-ERBα reprograms FOXA1 to bind new targets through a BRD4-mediated increase in local chromatin accessibility. Pharmacological targeting with SR8278 diminishes the function of both REV-ERBα and FOXA1 and synergizes with BRD4 inhibitor in effective suppression of tumorigenic programs and tumor growth. Thus, our study revealed a functional inversion by a CR regulator in driving gene reprogramming as an unexpected paradigm of tumorigenesis mechanism and demonstrated a high effectiveness of therapeutic targeting such switch.

Natural compounds ursolic acid (UA) and digoxin isolated from fruits and other plants display potent anti-cancer effects in preclinical studies. UA and digoxin have been at clinical trials for treatment of different cancers including prostate cancer, pancreatic cancer and breast cancer. However, they displayed limited benefit to patients. Currently, a poor understanding of their direct targets and mechanisms of action (MOA) severely hinders their further development. We previously identified nuclear receptor RORγ as a novel therapeutic target for castration-resistant prostate cancer (CRPC) and triple-negative breast cancer (TNBC) and demonstrated that tumor cell RORγ directly activates gene programs such as androgen receptor (AR) signaling and cholesterol metabolism. Previous studies also demonstrated that UA and digoxin are potential RORγt antagonists in modulating the functions of immune cells such as Th17 cells. Here we showed that UA displays a strong activity in inhibition of RORγ-dependent transactivation function in cancer cells, while digoxin exhibits no effect at clinically relevant concentrations. In prostate cancer cells, UA downregulates RORγ-stimulated AR expression and AR signaling, whereas digoxin upregulates AR signaling pathway. In TNBC cells, UA but not digoxin alters RORγ-controlled gene programs of cell proliferation, apoptosis and cholesterol biosynthesis. Together, our study reveals for the first-time that UA, but not digoxin, acts as a natural antagonist of RORγ in the cancer cells. Our finding that RORγ is a direct target of UA in cancer cells will help select patients with tumors that likely respond to UA treatment.

Background: Treatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies is an aggressive variant of prostate cancer with an unfavorable prognosis. The underlying mechanisms for early neuroendocrine differentiation are poorly defined and diagnostic and prognostic biomarkers are needed. Methods: We performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell line C4-2B MDVR and NEPC patient databases to identify neural lineage signature (NLS) genes. Correlation of NLS genes with clinicopathologic features was determined. Cell viability was determined in C4-2B MDVR and H660 cells after knocking down ARHGEF2 using siRNA. Organoid viability of patient-derived xenografts was measured after knocking down ARHGEF2. Results: We identify a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency, and neural stem cell differentiation, which may indicate an early or intermediate stage of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes in advanced prostate cancer. Differentially expressed NLS genes stratify small-cell NEPC from prostate adenocarcinoma, which are closely associated with clinicopathologic features such as Gleason Score and metastasis status. Higher ARGHEF2, LHX2, and EPHB2 levels among the 95 NLS genes correlate with a shortened survival time in NEPC patients. Furthermore, downregulation of ARHGEF2 gene expression suppresses cell viability and markers of neuroendocrine differentiation in enzalutamide-resistant and neuroendocrine cells. Conclusions: The 95 neural lineage gene signatures capture an early molecular shift toward neuroendocrine differentiation, which could stratify advanced prostate cancer patients to optimize clinical treatment and serve as a source of potential therapeutic targets in advanced prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) features high intratumoral cholesterol levels, due to aberrant regulation of cholesterol homeostasis. However, the underlying mechanisms are still poorly understood. The retinoid acid receptor-related orphan receptor gamma (RORγ), an attractive therapeutic target for cancer and autoimmune diseases, is strongly implicated in prostate cancer progression. We demonstrate in this study that in mCRPC cells and tumors, RORγ plays a crucial role in deregulation of cholesterol homeostasis. First, we found that RORγ activates the expression of key cholesterol biosynthesis proteins, including HMGCS1, HMGCR, and SQLE. Interestingly, we also found that RORγ inhibition induces cholesterol efflux gene program including ABCA1, ABCG1 and ApoA1. Our further studies revealed that liver X receptors (LXRα and LXRβ), the master regulators of cholesterol efflux pathway, mediate the function of RORγ in repression of cholesterol efflux. Finally, we demonstrated that RORγ antagonist in combination with statins has synergistic effect in killing mCRPC cells through blocking statin-induced feedback induction of cholesterol biosynthesis program and that the combination treatment also elicits stronger anti-tumor effects than either alone. Altogether, our work revealed that in mCRPC, RORγ contributes to aberrant cholesterol homeostasis by induction of cholesterol biosynthesis program and suppression of cholesterol efflux genes. Our findings support a therapeutic strategy of targeting RORγ alone or in combination with statin for effective treatment of mCRPC.