LSD1 promotes prostate cancer reprogramming by repressing TP53 signaling independently of its demethylase function.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that promotes stemness and cell survival in cancers such as prostate cancer. Most prostate malignancies are adenocarcinomas with luminal differentiation. However, some tumors undergo cellular reprogramming to a more lethal subset termed neuroendocrine prostate cancer (NEPC) with neuronal differentiation. The frequency of NEPC is increasing since the widespread use of potent androgen receptor signaling inhibitors. Currently, there are no effective treatments for NEPC. We previously determined that LSD1 promotes survival of prostate adenocarcinoma tumors. However, the role of LSD1 in NEPC is unknown. Here, we determined that LSD1 is highly upregulated in NEPC versus adenocarcinoma patient tumors. LSD1 suppression with RNAi or allosteric LSD1 inhibitors - but not catalytic inhibitors - reduced NEPC cell survival. RNA-Seq analysis revealed that LSD1 represses pathways linked to luminal differentiation, and TP53 was the top reactivated pathway. We confirmed that LSD1 suppressed the TP53 pathway by reducing TP53 occupancy at target genes while LSD1's catalytic function was dispensable for this effect. Mechanistically, LSD1 inhibition disrupted LSD1-HDAC interactions, increasing histone acetylation at TP53 targets. Finally, LSD1 inhibition suppressed NEPC tumor growth in vivo. These findings suggest that blocking LSD1's noncatalytic function may be a promising treatment strategy for NEPC.

Androgen receptor activity in T cells limits checkpoint blockade efficacy.

Immune checkpoint blockade has revolutionized the field of oncology, inducing durable anti-tumour immunity in solid tumours. In patients with advanced prostate cancer, immunotherapy treatments have largely failed1-5. Androgen deprivation therapy is classically administered in these patients to inhibit tumour cell growth, and we postulated that this therapy also affects tumour-associated T cells. Here we demonstrate that androgen receptor (AR) blockade sensitizes tumour-bearing hosts to effective checkpoint blockade by directly enhancing CD8 T cell function. Inhibition of AR activity in CD8 T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy via increased IFNγ expression. AR bound directly to Ifng and eviction of AR with a small molecule significantly increased cytokine production in CD8 T cells. Together, our findings establish that T cell intrinsic AR activity represses IFNγ expression and represents a novel mechanism of immunotherapy resistance.

Alternative splicing of LSD1+8a in neuroendocrine prostate cancer is mediated by SRRM4.

Neuroendocrine prostate cancer (NEPC) is the most virulent form of prostate cancer. Importantly, our recent work examining metastatic biopsy samples demonstrates NEPC is increasing in frequency. In contrast to prostate adenocarcinomas that express a luminal gene expression program, NEPC tumors express a neuronal gene expression program. Despite this distinction, the diagnosis of NEPC is often challenging, demonstrating an urgent need to identify new biomarkers and therapeutic targets. Our prior work demonstrated that the histone demethylase LSD1 (KDM1A) is important for survival of prostate adenocarcinomas, but little was known about LSD1's role in NEPC. Recently, a neural-specific transcript variant of LSD1-LSD1+8a-was discovered and demonstrated to activate neuronal gene expression in neural cells. The splicing factor SRRM4 was previously shown to promote LSD1+8a splicing in neuronal cells, and SRRM4 promotes NEPC differentiation and cell survival. Therefore, we sought to determine if LSD1+8a might play a role in NEPC and whether LSD1+8a splicing was linked to SRRM4. To investigate a potential role for LSD1+8a in NEPC, we examined a panel of prostate adenocarcinoma and NEPC patient-derived xenografts and metastatic biopsies. LSD1+8a was expressed exclusively in NEPC samples and correlated significantly with elevated expression of SRRM4. Using SRRM4-overexpressing cell lines, we determined that SRRM4 mediates alternative splicing of LSD1+8a. Finally, using gain of function studies, we confirmed that LSD1+8a and SRRM4 co-regulate target genes distinct from canonical LSD1. Our findings suggest further study of the interplay between SRRM4 and LSD1+8a and mechanisms by which LSD1+8a regulates gene expression in NEPC is warranted.

BET bromodomain inhibition blocks the function of a critical AR-independent master regulator network in lethal prostate cancer.

BET bromodomain inhibitors block prostate cancer cell growth at least in part through c-Myc and androgen receptor (AR) suppression. However, little is known about other transcriptional regulators whose suppression contributes to BET bromodomain inhibitor anti-tumor activity. Moreover, the anti-tumor activity of BET bromodomain inhibition in AR-independent castration-resistant prostate cancers (CRPC), whose frequency is increasing, is also unknown. Herein, we demonstrate that BET bromodomain inhibition blocks growth of a diverse set of CRPC cell models, including those that are AR-independent or in which c-Myc is not suppressed. To identify transcriptional regulators whose suppression accounts for these effects, we treated multiple CRPC cell lines with the BET bromodomain inhibitor JQ1 and then performed RNA-sequencing followed by Master Regulator computational analysis. This approach identified several previously unappreciated transcriptional regulators that are highly expressed in CRPC and whose suppression, via both transcriptional or post-translational mechanisms, contributes to the anti-tumor activity of BET bromodomain inhibitors.

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

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.

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.

Role of SMC1 in overcoming drug resistance in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the hardest subtypes of breast cancer to treat due to the heterogeneity of the disease and absence of well-defined molecular targets. Emerging evidence has shown the role of cohesin in the formation and progression of various cancers including colon and lung cancer but the role of cohesin in breast cancer remains elusive. Our data showed that structural maintenance of chromosome 1 (SMC1), a subunit of the cohesin protein complex, is differentially overexpressed both at RNA and protein level in a panel of TNBC cell lines as compared to normal epithelial or luminal breast cancer cells, suggesting that the amplified product of this normal gene may play role in tumorigenesis in TNBC. In addition, our results show that induced overexpression of SMC1 through transient transfection enhanced cell migration and anchorage independent growth while its suppression with targeted small interfering RNA (siRNA) reduced the migration ability of TNBC cells. Increased expression of SMC1 also lead to increase in the mesenchymal marker vimentin and decrease in the normal epithelial marker, E-cadherin. Immunocytochemical studies along with flow cytometry and cell fractionation showed the localization of SMC1 in the nucleus, cytoplasm and also in the plasma membrane. The knockdown of SMC1 by siRNA sensitized the TNBC cells towards a PARP inhibitor (ABT-888) and IC50 was approximately three fold less than ABT-888 alone. The cytotoxic effect of combination of SMC1 suppression and ABT-888 was also confirmed by the colony propagation assay. Taken together, these studies report for the first time that SMC1 is overexpressed in TNBC cells where it plays a role in cell migration and drug sensitivity, and thus provides a potential therapeutic target for this highly invasive breast cancer subtype.

P300 regulates the human RLIP76 promoter activity and gene expression.

A 76-kDa Ral-interacting protein (RLIP76) has been implicated in the pathogenesis of cancer and diabetes. It is often over expressed in human malignant cell lines and human tumor samples and has been associated with metastasis and chemoresistance. RLIP76 homozygous knockout mice exhibit increased insulin sensitivity, hypoglycemia, and hypolipidemia, and resist cancer development. Little is known about the mechanism by which the expression of RLIP76 is regulated. In the present study, we functionally characterized the RLIP76 promoter using deletion mapping and mutational analysis to investigate the regulation of RLIP76 transcription. We have identified the promoter regions important for RLIP76 transcription, including a strong cis-activating element in the proximal promoter containing overlapping consensus cMYB and cETS binding sites. Transcription factor cMYB and the coactivator p300 associated with RLIP76 gene promoter as shown by CHIP assay. Knockdown of p300 in HEK293 cells reduced the activity of the promoter fragment containing wild type cMYB/cETS binding site in comparison to that with deleted or mutated cMYB/cETS binding site. Knockdown of p300 also decreased the RLIP76 expression as indicated by immunoblotting, immunocytochemistry and flow cytometry analysis. Thus, we report for the first time that p300 associates with the RLIP76 promoter via an overlapping cMYB and cETS binding site and regulates RLIP76 promoter activity and its expression.

Rlip76 transports sunitinib and sorafenib and mediates drug resistance in kidney cancer.

RLIP76 is a stress-responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It represents the predominant glutathione-conjugate (GS-E) transporter in cells. We have shown that RLIP76 plays a crucial role in defending cancer cells from radiation and chemotherapeutic toxin-mediated apoptosis, and that its inhibition by antibodies or depletion by siRNA or antisense causes apoptosis in a number of cancer cell types. We demonstrated for the first time that the striking anti-neoplastic effects with no evident toxicity in terms of either weight loss or metabolic effects are also demonstrable for the antibody, antisense and siRNA in a renal cell xenografts model of Caki-2 cells (Singhal et al., Cancer Res., 2009, 69: 4244). Present studies were performed to determine if RLIP76 targeting is more broadly applicable in other kidney cancer cell lines, to compare the signaling effects of RLIP76 antisense with kinase inhibitors used in treatment of renal cell carcinoma, and to determine whether kinase inhibitors were substrates for transport by RLIP76. Results of these studies show that sorafenib as well as sunitinib are substrates for transport by RLIP76 thus are competitive inhibitors of GS-E transport. Furthermore, kinase inhibition in the ERK as well as PI3K pathways by RLIP76 depletion is more profound and consistent and is more widely apparent in a number of renal carcinoma cell lines. These studies offer strong support for our overall hypothesis that RLIP76 is an overarching anti-apoptosis mechanism that, if inhibited, can be more broadly effective in the treatment of renal cell carcinoma.

Functional reconstitution of RLIP76 catalyzing ATP-dependent transport of glutathione-conjugates.

RLIP76, a stress-responsive, multi-functional protein with multi-specific transport activity towards glutathione-conjugates (GS-E) and chemotherapeutic agents is frequently overexpressed in malignant cells. Our recent studies suggest that it plays a prominent anti-apoptotic role selectively in cancer cells. The present studies were performed to compare RLIP76 activity towards glutathione-conjugates in recombinant and K562 human erythroleukemia cells. The purity and identity of recombinant and K562 RLIP76 was established by SDS-PAGE and Western blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Comparison of ATPase activity and transport kinetics for DNP-SG and GS-HNE between recombinant vs. K562 RLIP76 revealed higher specific activity of ATPase and transport for recombinant purified RLIP76, indicating that additional factors present in recombinant purified RLIP76 can modulate its transport activity.

Diminished drug transport and augmented radiation sensitivity caused by loss of RLIP76.

This study was undertaken to characterize the consequences of Ral-interacting protein (RLIP76)-loss with respect to drug resistance, transport, radiation resistance, and alternative transport mechanisms in mouse embryonic fibroblasts (MEFs). MEFs were derived from RLIP76+/+, RLIP76+/- and RLIP76-/- mice. The transport of doxorubicin (DOX), colchicine (COL), leukotriene C4 and dinitrophenyl S-glutathione (DNP-SG) was analyzed in inside-out vesicles (IOVs) prepared from MEFs. We used immuno-titration of transport activity to determine the contribution of RLIP76, MRP1, and p-glycoprotein (Pgp) towards total transport activity. Loss of RLIP76 alleles resulted in significant sensitization to radiation, DOX, cisplatin, and vinorelbine (VRL). In IOVs prepared from MEFs, we observed a stepwise loss of transport activity. Loss of RLIP76 confers sensitivity to xenobiotics and radiation due to the loss of a common transport mechanism for glutathione-electrophile conjugates and xenobiotics.