Lina Gao

Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation

The androgen receptor (AR) is the principal therapeutic target in prostate cancer. For the past 70 years, androgen deprivation therapy (ADT) has been the major therapeutic focus. However, some patients do not benefit, and those tumors that do initially respond to ADT eventually progress. One recently described mechanism of such an effect is growth and survival-promoting effects of the AR that are exerted independently of the AR ligands, testosterone and dihydrotestosterone. However, specific ligand-independent AR target genes that account for this effect were not well characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer growth, is a key ligand-independent AR target gene. Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-growth effects of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer – AR and c-Myc – and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control.

Epigenetic regulation of androgen receptor signaling in prostate cancer.

Prostate cancer is most common cancer in men in the United States, and it is the second leading cause of cancer-related death in American men. The Androgen receptor (AR), a nuclear hormone and transcription factor, is the most therapeutically relevant target in this disease. While most efforts in the clinic are still directed at lowering levels of androgens that activate AR, resistance to androgen deprivation eventually develops, and most prostate cancer deaths are attributable to this castration-resistant form of this disease. Recent work has shed light on the importance of epigenetic events including facilitation of AR signaling by histone-modifying enzymes and also on the role that enzymes such as HDAC6 play in stabilizing AR in prostate cancer cells. Herein, we summarize recent findings on the role of epigenetic enzymes in AR signaling and highlight examples on how interdiction of critical epigenetic enzymes may attenuate AR action in prostate cancer.

A HIF-Regulated VHL-PTP1B-Src Signaling Axis Identifies a Therapeutic Target in Renal Cell Carcinoma

Signaling through the VHL-PTP1B-Src pathway in renal cell carcinomas may determine sensitivity to Src inhibitors and provide a basis for treatment planning. Detecting Sensitivity to Src Inhibitors Typically, tumor suppressors are welcome tenants in cells, protecting them from becoming cancerous. But in a twist of fate, 40% of patients with renal cell carcinoma (RCC) regard the presence of a functional tumor suppressor—the von Hippel-Lindau (VHL) protein—as bad news. And for good reason. In contrast to the other 60% of RCC patients whose tumors are driven by the loss of intact VHL, the tumor-suppressor positive cancers are more likely to be resistant to immunotherapy and chemotherapy, and because researchers do not know what drives tumorigenesis, no rational targeted therapies exist. Not content to wait for another twist of fate, Suwaki et al. have delved deeply into these kidney cancers and found that VHL functions as part of an activated signaling pathway that renders the cells sensitive to anticancer agents that target the Src oncoprotein. The presence of VHL and other markers of this pathway can flag those RCC patients who may benefit from drugs that block Src kinase activity. Enhanced activity of the Src tyrosine kinase has been implicated in cancer development and is the target of the anticancer drug dasatinib. By measuring the extent of phosphorylation of critical proteins in a pair of cell lines with and without functional VHL, the authors saw that Src and its substrates were activated only when VHL was present. And in 215 RCCs, the presence of VHL tended to be associated with highly active Src kinase. Dasatinib inhibited DNA synthesis and cell growth only in cells with VHL, whether they were grown in culture or as xenografts in mice. The authors also replicated the well-known ability of VHL to negatively regulate the hypoxia-sensitive transcription factor HIF, which is activated indirectly by Src. As expected, expression of HIF in VHL-containing cancer cells conferred resistance to dasatinib. An independent analysis of this pathway in tumor samples from an additional 131 patients with RCC confirmed the positive correlation between VHL and Src and its associated pathway proteins. Here, the authors used quantitative phosphoproteomics and immunohistochemical profiling to show the correlation. Because they used automated digital image analysis and an unsupervised hierarchical clustering of the tumors on the basis of the expression of VHL, Src, pFAK and PTP1B, this approach has the potential to be used in the clinic for tumor characterization. Personalized approaches to cancer treatment require an armamentarium of matched pairs of drugs and validated biomarkers that predict response to therapy. The markers for an activated Src pathway discerned by the authors could in theory be assessed in any solid cancer; in fact, a test in bladder carcinomas showed that, as in RCCs, the Src pathway proteins were activated. If such a test can be applied to many solid cancers, physicians could use it to predict whether a patient is likely to respond to anti-Src agents. Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG’s predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

LSD1 activates a lethal prostate cancer gene network independently of its demethylase function

Significance Medical castration or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine-specific demethylase 1 (LSD1) is an important regulator of gene expression, including in cancer. Here, we show that LSD1 is highly expressed in tumors of patients with lethal castration-resistant prostate cancer (CRPC) and that LSD1 promotes AR-independent survival in CRPC cells in a noncanonical, demethylase-independent manner. We determined that the drug SP-2509 acts as an allosteric inhibitor of LSD1–blocking demethylase-independent functions. Our demonstration of tumor suppression with this inhibitor in CRPC preclinical models provides the rationale for clinical trials with LSD1 inhibitors. Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine-specific demethylase 1 (LSD1) is a histone demethylase and an important regulator of gene expression. Here, we show that LSD1 promotes the survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with LSD1’s binding protein, ZNF217. Finally, that a small-molecule LSD1 inhibitor―SP-2509―blocks important demethylase-independent functions and suppresses castration-resistant prostate cancer cell viability demonstrates the potential of LSD1 inhibition in this disease.

Immunohistochemical expression of ERG in the molecular epidemiology of fatal prostate cancer study

Gene fusions between the ERG transcription factor and the androgen‐regulated gene TMPRSS2 occur in a subset of prostate cancers and contribute to transformation of prostatic epithelial cells. Prior reports have used fluorescence in situ hybridization (FISH) or quantitative PCR (QPCR) to determine the presence of TMPRSS2‐ERG fusions or ERG expression, respectively. Recently, several groups have reported on immunohistochemistry (IHC) to measure ERG expression, which is much more readily performed in clinical practice. However, the prior studies examining ERG expression by IHC had small sample sizes or they failed to clarify the association of ERG protein expression with important clinico‐pathological features or prostate cancer‐specific mortality. Methods: To address these deficits, we evaluated ERG expression by IHC in 208 radical prostatectomy samples from the Kaiser Permanente Molecular Epidemiology of Fatal Prostate Cancer (MEFPC) study, a case–control study of prostate cancer‐specific mortality.

Cellular androgen content influences enzalutamide agonism of F877L mutant androgen receptor.

Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation.

Maintenance and pharmacologic targeting of ROR1 protein levels via UHRF1 in t(1;19) pre-B-ALL.

Expression of the transmembrane pseudokinase ROR1 is required for survival of t(1;19)-pre-B-cell acute lymphoblastic leukemia (t(1;19) pre-B-ALL), chronic lymphocytic leukemia, and many solid tumors. However, targeting ROR1 with small-molecules has been challenging due to the absence of ROR1 kinase activity. To identify genes that regulate ROR1 expression and may, therefore, serve as surrogate drug targets, we employed an siRNA screening approach and determined that the epigenetic regulator and E3 ubiquitin ligase, UHRF1, is required for t(1;19) pre-B-ALL cell viability in a ROR1-dependent manner. Upon UHRF1 silencing, ROR1 protein is reduced without altering ROR1 mRNA, and ectopically expressed UHRF1 is sufficient to increase ROR1 levels. Additionally, proteasome inhibition rescues loss of ROR1 protein after UHRF1 silencing, suggesting a role for the proteasome in the UHRF1-ROR1 axis. Finally, we show that ROR1-positive cells are twice as sensitive to the UHRF1-targeting drug, naphthazarin, and undergo increased apoptosis compared to ROR1-negative cells. Naphthazarin elicits reduced expression of UHRF1 and ROR1, and combination of naphthazarin with inhibitors of pre-B cell receptor signaling results in further reduction of cell survival compared with either inhibitor alone. Therefore, our work reveals a mechanism by which UHRF1 stabilizes ROR1, suggesting a potential targeting strategy to inhibit ROR1 in t(1;19) pre-B-ALL and other malignancies.

Integrative molecular network analysis identifies emergent enzalutamide resistance mechanisms in prostate cancer

Recent work demonstrates that castration-resistant prostate cancer (CRPC) tumors harbor countless genomic aberrations that control many hallmarks of cancer. While some specific mutations in CRPC may be actionable, many others are not. We hypothesized that genomic aberrations in cancer may operate in concert to promote drug resistance and tumor progression, and that organization of these genomic aberrations into therapeutically targetable pathways may improve our ability to treat CRPC. To identify the molecular underpinnings of enzalutamide-resistant CRPC, we performed transcriptional and copy number profiling studies using paired enzalutamide-sensitive and resistant LNCaP prostate cancer cell lines. Gene networks associated with enzalutamide resistance were revealed by performing an integrative genomic analysis with the PAthway Representation and Analysis by Direct Reference on Graphical Models (PARADIGM) tool. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with MEK, EGFR, RAS, and NFKB. Functional validation studies of 64 genes identified 10 candidate genes whose suppression led to greater effects on cell viability in enzalutamide-resistant cells as compared to sensitive parental cells. Examination of a patient cohort demonstrated that several of our functionally-validated gene hits are deregulated in metastatic CRPC tumor samples, suggesting that they may be clinically relevant therapeutic targets for patients with enzalutamide-resistant CRPC. Altogether, our approach demonstrates the potential of integrative genomic analyses to clarify determinants of drug resistance and rational co-targeting strategies to overcome resistance.

Abstract 2406: LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation

Background: Androgen deprivation therapy (ADT) or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine specific demethylase 1 (LSD1) is a histone demethylase and a key regulator of gene expression in cancer. Prior work demonstrates that LSD1 may act as a cofactor of the AR in androgen-dependent prostate cancer cells. In this report, we describe a distinct role of LSD1 as a driver of proliferation and survival of castration-resistant prostate cancer (CRPC) cells independently of the AR and independently of histone demethylation. Methods: We used gain and loss of function studies to determine the importance of LSD1 for survival of prostate cancer cells. To identify transcriptional networks that contribute to cell survival, we suppressed LSD1 with RNAi and measured gene expression changes with microarrays. To determine the importance of histone demethylation in regulation of these gene networks, we suppressed LSD1 and measured levels of LSD1 canonical histone substrates (H3K4me2 and H3K9me2) genome-wide with chromatin immunoprecipitation-sequencing. Results: Cell viability assays demonstrated that LSD1 is important for proliferation and survival of CRPC cells independently of the AR. Microarray studies demonstrated that LSD1 activates androgen-independent genes that comprise cell cycle and embryonic stem cell maintenance gene sets that are enriched in lethal human tumors. Importantly, our global epigenomic studies after LSD1 suppression demonstrated that LSD1 activates these gene sets independently of demethylation of its canonical histone substrates. Conclusions: Our results demonstrate that LSD1 promotes CRPC cell survival independently of the AR and suggest that LSD1 regulates key pathways in CRPC through demethylation of non-histone substrates or via a scaffold function―mechanisms we are currently investigating. In summary, LSD1 contributes to CRPC cell survival through non-canonical mechanisms and represents an attractive therapeutic target in lethal prostate cancer. Citation Format: Archana Sehrawat, Lina Gao, Junior Tayou, Armand Bankhead, Laura M. Heiser, Carly J. King, Yuliang Wang, Jacob Schwartzman, Joshua Urrutia, Daniel J. Coleman, Sheila Weinmann, Bhaskar V. Kallakury, Deborah L. Berry, Reina Haque, Stephen K. Van Den Eeden, Tomasz M. Beer, George V. Thomas, Shannon McWeeney, Joshi J. Alumkal. LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2406. doi:10.1158/1538-7445.AM2017-2406