ABSTRACT
Polo-like kinase 1 (PLK1) inhibitors have had limited antitumor efficacy as single agents, and a focus of current efforts is on combination therapies. We initially confirmed that the PLK1 specific inhibitor onvansertib (ONV) could enhance responses to a PARP inhibitor (olaparib) in prostate cancer xenografts. To identify more effective combinations we screened a library of bioactive compounds for efficacy in combination with ONV in LNCaP prostate cancer cells, which identified a series of compounds including multiple AKT inhibitors. We confirmed in vitro synergy between ONV and the AKT inhibitor ipatasertib (IPA) and found that the combination increased apoptosis. Mechanistic studies showed that ONV increased expression of the anti-apoptotic protein SURVIVIN, and that this was mitigated by IPA. Studies in three PTEN deficient prostate cancer xenograft models showed that co-treatment with IPA and ONV led to significant tumor growth inhibition compared to monotherapies. Together these in vitro and in vivo studies demonstrate that the efficacy of PLK1 antagonists can be enhanced by PARP or AKT inhibition, and support further development of these combination therapies.
ABSTRACT
Degradation of unliganded androgen receptor (AR) in prostate cancer cells can be prevented by proteasome inhibition, but this is associated with only modest increases in polyubiquitylated AR. An inhibitor (VLX1570) of the deubiquitylases associated with the proteasome did not increase ubiquitylation of unliganded AR, indicating that AR is not targeted by these deubiquitylases. We then identified a series of AR ubiquitylation sites, including a not previously identified site at K911, as well as methylation sites and previously identified phosphorylation sites. Mutagenesis of K911 increases AR stability, chromatin binding, and transcriptional activity. We further found that K313, a previously reported ubiquitylation site, could also be methylated and acetylated. Mutagenesis of K313, in combination with K318, increases AR transcriptional activity, indicating that distinct posttranslational modifications at K313 differentially regulate AR activity. Together these studies expand the spectrum of AR posttranslational modifications, and indicate that the K911 site may regulate AR turnover on chromatin.
Subject(s)
Proteasome Endopeptidase Complex , Receptors, Androgen , Male , Humans , Receptors, Androgen/genetics , Ubiquitination , Protein Processing, Post-Translational , Chromatin/geneticsABSTRACT
Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which these variants drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full-length AR (ARfl), despite its continued high-level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in VCaP16 cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression in VCaP cells increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and NFIX knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.
ABSTRACT
Abiraterone is a standard treatment for metastatic castrate-resistant prostate cancer (mCRPC) that slows disease progression by abrogating androgen synthesis and antagonizing the androgen receptor (AR). Here we report that inhibitors of the mitotic regulator polo-like kinase-1 (Plk1), including the clinically active third-generation Plk1 inhibitor onvansertib, synergizes with abiraterone in vitro and in vivo to kill a subset of cancer cells from a wide variety of tumor types in an androgen-independent manner. Gene-expression analysis identified an AR-independent synergy-specific gene set signature upregulated upon abiraterone treatment that is dominated by pathways related to mitosis and the mitotic spindle. Abiraterone treatment alone caused defects in mitotic spindle orientation, failure of complete chromosome condensation, and improper cell division independently of its effects on AR signaling. These effects, although mild following abiraterone monotherapy, resulted in profound sensitization to the antimitotic effects of Plk1 inhibition, leading to spindle assembly checkpoint-dependent mitotic cancer cell death and entosis. In a murine patient-derived xenograft model of abiraterone-resistant metastatic castration-resistant prostate cancer (mCRPC), combined onvansertib and abiraterone resulted in enhanced mitotic arrest and dramatic inhibition of tumor cell growth compared with either agent alone. Overall, this work establishes a mechanistic basis for the phase II clinical trial (NCT03414034) testing combined onvansertib and abiraterone in mCRPC patients and indicates this combination may have broad utility for cancer treatment. SIGNIFICANCE: Abiraterone treatment induces mitotic defects that sensitize cancer cells to Plk1 inhibition, revealing an AR-independent mechanism for this synergistic combination that is applicable to a variety of cancer types.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Male , Humans , Animals , Mice , Receptors, Androgen/metabolism , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Androgens , MitosisABSTRACT
Androgen receptor (AR) in prostate cancer (PCa) can drive transcriptional repression of multiple genes including MYC, and supraphysiological androgen is effective in some patients. Here, we show that this repression is independent of AR chromatin binding and driven by coactivator redistribution, and through chromatin conformation capture methods show disruption of the interaction between the MYC super-enhancer within the PCAT1 gene and the MYC promoter. Conversely, androgen deprivation in vitro and in vivo increases MYC expression. In parallel, global AR activity is suppressed by MYC overexpression, consistent with coactivator redistribution. These suppressive effects of AR and MYC are mitigated at shared AR/MYC binding sites, which also have markedly higher levels of H3K27 acetylation, indicating enrichment for functional enhancers. These findings demonstrate an intricate balance between AR and MYC, and indicate that increased MYC in response to androgen deprivation contributes to castration-resistant PCa, while decreased MYC may contribute to responses to supraphysiological androgen therapy.
Subject(s)
Enhancer Elements, Genetic , Oncogene Protein p55(v-myc)/genetics , Receptors, Androgen/genetics , Androgens/metabolism , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , Gene Expression Regulation, Neoplastic , Humans , Male , Oncogene Protein p55(v-myc)/metabolism , Promoter Regions, Genetic , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Signal TransductionABSTRACT
Neuroendocrine (NE) prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (PCa) arising either de novo or from transdifferentiated prostate adenocarcinoma following androgen deprivation therapy (ADT). Extensive computational analysis has identified a high degree of association between the long noncoding RNA (lncRNA) H19 and NEPC, with the longest isoform highly expressed in NEPC. H19 regulates PCa lineage plasticity by driving a bidirectional cell identity of NE phenotype (H19 overexpression) or luminal phenotype (H19 knockdown). It contributes to treatment resistance, with the knockdown of H19 re-sensitizing PCa to ADT. It is also essential for the proliferation and invasion of NEPC. H19 levels are negatively regulated by androgen signaling via androgen receptor (AR). When androgen is absent SOX2 levels increase, driving H19 transcription and facilitating transdifferentiation. H19 facilitates the PRC2 complex in regulating methylation changes at H3K27me3/H3K4me3 histone sites of AR-driven and NEPC-related genes. Additionally, this lncRNA induces alterations in genome-wide DNA methylation on CpG sites, further regulating genes associated with the NEPC phenotype. Our clinical data identify H19 as a candidate diagnostic marker and predictive marker of NEPC with elevated H19 levels associated with an increased probability of biochemical recurrence and metastatic disease in patients receiving ADT. Here we report H19 as an early upstream regulator of cell fate, plasticity, and treatment resistance in NEPC that can reverse/transform cells to a treatable form of PCa once therapeutically deactivated.
Subject(s)
Carcinoma, Neuroendocrine/genetics , Carcinoma, Neuroendocrine/pathology , Cell Plasticity/genetics , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Long Noncoding/metabolism , Androgen Antagonists/therapeutic use , Animals , Benzamides/pharmacology , Benzamides/therapeutic use , Biomarkers, Tumor/metabolism , Carcinoma, Neuroendocrine/diagnosis , Carcinoma, Neuroendocrine/drug therapy , Cell Line, Tumor , Cell Lineage/genetics , Cell Nucleus/metabolism , Cell Proliferation/genetics , Cohort Studies , DNA Methylation/genetics , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Epigenesis, Genetic/drug effects , Genome, Human , Histones/metabolism , Humans , Male , Neoplasm Grading , Neoplasm Invasiveness , Neoplastic Stem Cells/metabolism , Nitriles/pharmacology , Nitriles/therapeutic use , Organoids/metabolism , Organoids/pathology , Phenylthiohydantoin/pharmacology , Phenylthiohydantoin/therapeutic use , Phylogeny , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic/genetics , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/drug therapy , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Long Noncoding/genetics , Receptors, Androgen/metabolism , SOXB1 Transcription Factors/metabolism , Transcription, Genetic/drug effectsABSTRACT
One mechanism for reactivation of androgen receptor (AR) activity after androgen deprivation therapy in castration-resistant prostate cancer (CRPC) is expression of splice variants such as ARv7 that delete the ligand binding domain and have constitutive activity. Exogenous overexpressed ARv7 can function as a homodimer or heterodimer with full length AR (ARfl), which is highly expressed with ARv7 in CRPC. However, the extent to which endogenous ARv7 function is dependent on heterodimerization with ARfl remains to be determined. We used double-crosslinking to stabilize AR complexes on chromatin in a CRPC cell line expressing endogenous ARfl and ARv7 (LN95 cells), and established that only trace levels of ARfl were associated with ARv7 on chromatin. Consistent with this result, depletion of ARfl with an AR degrader targeting the AR ligand binding domain did not decrease ARv7 binding to chromatin or its association with HOXB13, but did decrease overall AR transcriptional activity. Comparable results were obtained in CWR22RV1 cells, another CRPC cell line expressing ARfl and ARv7. These results indicate that ARv7 function in CRPC is not dependent on ARfl, and that both contribute independently to overall AR activity.
Subject(s)
Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , Cell Line , Cell Line, Tumor , Chromatin/genetics , HEK293 Cells , Homeodomain Proteins/genetics , Humans , Ligands , Male , Prostate/metabolism , Protein Domains/geneticsABSTRACT
BACKGROUND: Gli is an oncogenic transcription factor family thought to be involved in breast cancer (BrCa) cell growth. Gli activity is regulated by a post-translational proteolytic process that is suppressed by Hedgehog signaling. In prostate cancer cells, however, Gli activation is mediated by an interaction of active androgen receptor proteins with Gli3 that stabilizes Gli3 in its un-proteolyzed form. Here we show that the estrogen receptor (ER), ERα, also binds Gli3 and activates Gli in BrCa cells. Moreover, we show that ER + BrCa cells are dependent on Gli3 for cancer cell growth. METHODS: Transfection with Gli-luciferase reporter was used to report Gli activity in 293FT or BrCa cells (MCF7, T47D, MDA-MB-453) with or without steroid ligands. Co-immunoprecipitation and proximity ligation were used to show association of Gli3 with ERα. Gli3 stability was determined by western blots of BrCa cell extracts. ERα knockdown or destabilization (by fulvestrant) was used to assess how loss of ERα affects estradiol-induced Gli reporter activity, formation of intranuclear ERα-Gli3 complexes and Gli3 stability. Expression of Gli1 and/or other endogenous Gli-target genes in BrCa cells were measured by qPCR in the presence or absence of estradiol. Gli3 knockdown was assessed for effects on BrCa cell growth using the Cyquant assay. RESULTS: ERα co-transfection increased Gli reporter activity in 293FT cells that was further increased by estradiol. Gli3 co-precipitated in ERα immunoprecipitates. Acute (2 h) estradiol increased Gli reporter activity and the formation of intranuclear ERα-Gli3 complexes in ER + BrCa cells but more chronic estradiol (48 h) reduced ERα-Gli complexes commensurate with reduced ERα levels. Gli3 stability and endogenous activity was only increased by more chronic estradiol treatment. Fulvestrant or ERα knockdown suppressed E2-induction of Gli activity, intranuclear ERα-Gli3 complexes and stabilization of Gli3. Gli3 knockdown significantly reduced the growth of BrCa cells. CONCLUSIONS: ERα interacts with Gli3 in BrCa cells and estradiol treatment leads to Gli3 stabilization and increased expression of Gli-target genes. Furthermore, we found tthat Gli3 is necessary for BrCa cell growth. These results support the idea that the ERα-Gli interaction and Gli3 may be novel targets for effective control of BrCa growth.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Receptors, Estrogen/metabolism , Zinc Finger Protein Gli3/metabolism , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Estradiol/pharmacology , Female , HEK293 Cells , Humans , Protein Stability/drug effectsABSTRACT
MCL1 has critical antiapoptotic functions and its levels are tightly regulated by ubiquitylation and degradation, but mechanisms that drive this degradation, particularly in solid tumors, remain to be established. We show here in prostate cancer cells that increased NOXA, mediated by kinase inhibitor activation of an integrated stress response, drives the degradation of MCL1, and identify the mitochondria-associated ubiquitin ligase MARCH5 as the primary mediator of this NOXA-dependent MCL1 degradation. Therapies that enhance MARCH5-mediated MCL1 degradation markedly enhance apoptosis in response to a BH3 mimetic agent targeting BCLXL, which may provide for a broadly effective therapy in solid tumors. Conversely, increased MCL1 in response to MARCH5 loss does not strongly sensitize to BH3 mimetic drugs targeting MCL1, but instead also sensitizes to BCLXL inhibition, revealing a codependence between MARCH5 and MCL1 that may also be exploited in tumors with MARCH5 genomic loss.
Subject(s)
Antineoplastic Agents/pharmacology , Membrane Proteins/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Ubiquitin-Protein Ligases/metabolism , Apoptosis , Cell Line, Tumor , Humans , Membrane Proteins/genetics , Mitochondria/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Proteolysis , Proto-Oncogene Proteins c-bcl-2/genetics , Stress, Physiological , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Up-RegulationABSTRACT
PURPOSE: Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR-/Low) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression. EXPERIMENTAL DESIGN: Unique prostate cancer cell models that capture AR-/Low stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored. RESULTS: Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells. CONCLUSIONS: Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.
Subject(s)
Androgen Receptor Antagonists/pharmacology , Drug Resistance, Neoplasm , Neuroendocrine Cells/pathology , Prostatic Neoplasms, Castration-Resistant/pathology , Receptors, Androgen/metabolism , SOX9 Transcription Factor/metabolism , Stem Cells/pathology , Animals , Cell Line, Tumor , Humans , Male , Mice , NF-kappa B/metabolism , Neuroendocrine Cells/metabolism , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Receptors, Androgen/genetics , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , SOX9 Transcription Factor/genetics , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Signal Transduction , Stem Cells/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
In this issue of Cancer Discovery, Rasool and colleagues show that TF11H/CDK7 phosphorylates the MED1 component of the Mediator complex, which enhances its interaction with androgen receptor (AR), and that this phosphorylation is increased in prostate cancer that is resistant to castration and enzalutamide. A covalent CDK7-specific inhibitor (THZ1) impairs AR-mediated MED1 recruitment to chromatin, and can suppress enzalutamide resistance in vitro and induce tumor regression in a castration-resistant prostate cancer xenograft model, suggesting a novel therapeutic approach for advanced prostate cancer.See related article by Rasool et al., p. 1538.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Male , Mediator Complex , Mediator Complex Subunit 1 , Phosphorylation , Signal TransductionABSTRACT
CONTEXT: It is increasingly evident that non-protein-coding regions of the genome can give rise to transcripts that form functional layers of the cancer genome. One of most abundant classes in these regions is long noncoding RNAs (lncRNAs). They have gained increasing attention in prostate cancer (PCa) and paved the way for a greater understanding of these cryptic regulators in cancer. OBJECTIVE: To review current research exploring the functional biology of lncRNAs in PCa over the past three decades. EVIDENCE ACQUISITION: A systematic review was performed using PubMed to search for reports with terms "long noncoding RNA", "prostate", and "cancer" over the past 30â¯yr (1988-2018). EVIDENCE SYNTHESIS: We comprehensively surveyed the literature collected and summarise experiments leading to the characterisation of lncRNAs in PCa. A historical timeline of lncRNA identification is described, where each lncRNA is categorised mechanistically and within the primary areas of carcinogenesis: tumour risk and initiation, tumour promotion, tumour suppression, and tumour treatment resistance. We describe select lncRNAs that exemplify these areas. We also review whether these lncRNAs have a clinical utility in PCa diagnosis, prognosis, and prediction, and as therapeutic targets. CONCLUSIONS: The biology of lncRNA is multifaceted, demonstrating a complex array of molecular and cellular functions. These studies reveal that lncRNAs are involved in every stage of PCa. Their clinical utility for diagnosis, prognosis, and prediction of PCa is well supported, but further evaluation for their therapeutic candidacy is needed. We provide a detailed resource and view inside the lncRNA landscape for other cancer biologists, oncologists, and clinicians. PATIENT SUMMARY: In this study, we review current knowledge of the non-protein-coding genome in prostate cancer (PCa). We conclude that many of these regions are functional and a source of accurate biomarkers in PCa. With a strong research foundation, they hold promise as future therapeutic targets, yet clinical trials are necessary to determine their intrinsic value to PCa disease management.
Subject(s)
Drug Discovery , Prostatic Neoplasms , RNA, Long Noncoding , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Humans , Male , Pharmacogenetics , Procedures and Techniques Utilization , Prognosis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/therapy , RNA, Long Noncoding/analysis , RNA, Long Noncoding/geneticsABSTRACT
Background: Treatment-induced neuroendocrine prostate cancer (tNEPC) is an aggressive variant of late-stage metastatic castrate-resistant prostate cancer that commonly arises through neuroendocrine transdifferentiation (NEtD). Treatment options are limited, ineffective, and, for most patients, result in death in less than a year. We previously developed a first-in-field patient-derived xenograft (PDX) model of NEtD. Longitudinal deep transcriptome profiling of this model enabled monitoring of dynamic transcriptional changes during NEtD and in the context of androgen deprivation. Long non-coding RNA (lncRNA) are implicated in cancer where they can control gene regulation. Until now, the expression of lncRNAs during NEtD and their clinical associations were unexplored. Results: We implemented a next-generation sequence analysis pipeline that can detect transcripts at low expression levels and built a genome-wide catalogue (n = 37,749) of lncRNAs. We applied this pipeline to 927 clinical samples and our high-fidelity NEtD model LTL331 and identified 821 lncRNAs in NEPC. Among these are 122 lncRNAs that robustly distinguish NEPC from prostate adenocarcinoma (AD) patient tumours. The highest expressed lncRNAs within this signature are H19, LINC00617, and SSTR5-AS1. Another 742 are associated with the NEtD process and fall into four distinct patterns of expression (NEtD lncRNA Class I, II, III, and IV) in our PDX model and clinical samples. Each class has significant (z-scores >2) and unique enrichment for transcription factor binding site (TFBS) motifs in their sequences. Enriched TFBS include (1) TP53 and BRN1 in Class I, (2) ELF5, SPIC, and HOXD1 in Class II, (3) SPDEF in Class III, (4) HSF1 and FOXA1 in Class IV, and (5) TWIST1 when merging Class III with IV. Common TFBS in all NEtD lncRNA were also identified and include E2F, REST, PAX5, PAX9, and STAF. Interrogation of the top deregulated candidates (n = 100) in radical prostatectomy adenocarcinoma samples with long-term follow-up (median 18 years) revealed significant clinicopathological associations. Specifically, we identified 25 that are associated with rapid metastasis following androgen deprivation therapy (ADT). Two of these lncRNAs (SSTR5-AS1 and LINC00514) stratified patients undergoing ADT based on patient outcome. Discussion: To date, a comprehensive characterization of the dynamic landscape of lncRNAs during the NEtD process has not been performed. A temporal analysis of the PDX-based NEtD model has for the first time provided this dynamic landscape. TFBS analysis identified NEPC-related TF motifs present within the NEtD lncRNA sequences, suggesting functional roles for these lncRNAs in NEPC pathogenesis. Furthermore, select NEtD lncRNAs appear to be associated with metastasis and patients receiving ADT. Treatment-related metastasis is a clinical consequence of NEPC tumours. Top candidate lncRNAs FENDRR, H19, LINC00514, LINC00617, and SSTR5-AS1 identified in this study are implicated in the development of NEPC. We present here for the first time a genome-wide catalogue of NEtD lncRNAs that characterize the transdifferentiation process and a robust NEPC lncRNA patient expression signature. To accomplish this, we carried out the largest integrative study that applied a PDX NEtD model to clinical samples. These NEtD and NEPC lncRNAs are strong candidates for clinical biomarkers and therapeutic targets and warrant further investigation.
Subject(s)
Neuroendocrine Tumors/genetics , Prostatic Neoplasms/genetics , RNA, Long Noncoding/genetics , Animals , Binding Sites , Cell Transdifferentiation/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Male , Mice , Neoplasm Metastasis , Neuroendocrine Tumors/pathology , Nucleotide Motifs/genetics , Phenotype , Prostatic Neoplasms/pathology , RNA, Long Noncoding/metabolism , Transcription Factors/metabolism , Transcriptome/genetics , Xenograft Model Antitumor AssaysABSTRACT
Hedgehog (Hh) is an oncogenic signaling pathway that regulates the activity of Gli transcription factors. Canonical Hh is a Smoothened- (Smo-) driven process that alters the post-translational processing of Gli2/Gli3 proteins. Though evidence supports a role for Gli action in prostate cancer (PCa) cell growth and progression, there is little indication that Smo is involved. Here we describe a non-canonical means for activation of Gli transcription in PCa cells mediated by the binding of transcriptionally-active androgen receptors (ARs) to Gli3. Androgens stimulated reporter expression from a Gli-dependent promoter in a variety of AR + PCa cells and this activity was suppressed by an anti-androgen, Enz, or by AR knockdown. Androgens also upregulated expression of endogenous Gli-dependent genes. This activity was associated with increased intranuclear binding of Gli3 to AR that was antagonized by Enz. Fine mapping of the AR binding domain on Gli2 showed that AR recognizes the Gli protein processing domain (PPD) in the C-terminus. Mutations in the arginine-/serine repeat elements of the Gli2 PPD involved in phosphorylation and ubiquitinylation blocked the binding to AR. ß-TrCP, a ubiquitin ligase that recognizes the Gli PPD, competed with AR for binding to this site. AR binding to Gli3 suppressed its proteolytic processing to the Gli3 repressor form (Gli3R) whereas AR knockdown increased Gli3R. Both full-length and truncated ARs were able to activate Gli transcription. Finally, we found that an ARbinding decoy polypeptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR. Exogenous overexpression of this decoy suppressed Gli transcriptional activity in PCa cells. Collectively, this work identifies a novel pathway for non-canonical activation of Hh signaling in PCa cells and identifies a means for interference that may have clinical relevance for PCa patients.
Subject(s)
Hedgehog Proteins/genetics , Nerve Tissue Proteins/metabolism , Prostatic Neoplasms/genetics , Receptors, Androgen/metabolism , Zinc Finger Protein Gli3/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Male , Promoter Regions, Genetic , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Binding , Signal Transduction/physiology , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Discovery , ETS Motif , Prostatic Neoplasms/metabolism , Protein Interaction Domains and Motifs , Transcriptional Regulator ERG/chemistry , Transcriptional Regulator ERG/metabolism , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Drug Discovery/methods , Gene Expression Regulation, Neoplastic , Humans , Magnetic Resonance Spectroscopy , Male , Models, Molecular , Molecular Conformation , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Protein Binding , Structure-Activity Relationship , Transcriptional Regulator ERG/genetics , ZebrafishABSTRACT
Treatment-induced neuroendocrine transdifferentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR+/PSA+ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem medium. Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells and tumor-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or return back to an AR+ prostate-like state. Once returned, the AR+ cells were resistant to androgen signaling inhibition. Acute androgen deprivation or anti-androgen treatment in serum-containing medium led to the transient appearance of a sub-population of cells with similar characteristics. Finally, a 132 gene signature derived from reprogrammed PCa cell lines distinguished tumors from PCa patients with adverse outcomes. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumors from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumor behaviors.
Subject(s)
Cell Transdifferentiation/physiology , Cellular Reprogramming/physiology , Drug Resistance, Neoplasm/physiology , Neoplastic Stem Cells/pathology , Prostatic Neoplasms/pathology , Animals , Blotting, Western , Disease Progression , Flow Cytometry , Fluorescent Antibody Technique , Heterografts , Humans , Male , Mice , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , ZebrafishABSTRACT
Despite the substantial benefit of androgen deprivation therapy (ADT) for metastatic prostate cancer, patients often progress to castration-resistant disease (CRPC) that is more difficult to treat. CRPC is associated with renewed androgen receptor activity in tumor cells and restoration of tumor androgen levels through acquired intratumoral steroidogenesis (AIS). Although prostate cancer (PCa) cells have been shown to have steroidogenic capability in vitro, we previously found that benign prostate stromal cells (PrSCs) can also synthesize testosterone (T) from an adrenal precursor, DHEA, when stimulated with a hedgehog (Hh) pathway agonist, SAG. Here, we show exposure of PrSCs to a different Smoothened (Smo) agonist, Ag1.5, or to conditioned medium from sonic hedgehog overexpressing LNCaP cells induces steroidogenic enzyme expression in PrSCs and significantly increases production of T and its precursor steroids in a Smo-dependent manner from 22-OH-cholesterol substrate. Hh agonist-/ligand-treated PrSCs produced androgens at a rate similar to or greater than that of PCa cell lines. Likewise, primary bone marrow stromal cells became more steroidogenic and produced T under the influence of Smo agonist. Treatment of mice bearing LNCaP xenografts with a Smo antagonist, TAK-441, delayed the onset of CRPC after castration and substantially reduced androgen levels in residual tumors. These outcomes support the idea that stromal cells in ADT-treated primary or metastatic prostate tumors can contribute to AIS as a consequence of a paracrine Hh signaling microenvironment. As such, Smo antagonists may be useful for targeting prostate tumor stromal cell-derived AIS and delaying the onset of CRPC after ADT.
Subject(s)
Hedgehog Proteins/metabolism , Paracrine Communication/physiology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Tumor Microenvironment/physiology , Androgens/metabolism , Animals , Bone Marrow/metabolism , Castration/methods , Cell Line, Tumor , Humans , Male , Mice , Mice, Nude , Receptors, Androgen/metabolism , Signal Transduction/physiology , Stromal Cells/metabolism , Testosterone/metabolismABSTRACT
Androgens regulate biological pathways to promote proliferation, differentiation, and survival of benign and malignant prostate tissue. Androgen receptor (AR) targeted therapies exploit this dependence and are used in advanced prostate cancer to control disease progression. Contemporary treatment regimens involve sequential use of inhibitors of androgen synthesis or AR function. Although targeting the androgen axis has clear therapeutic benefit, its effectiveness is temporary, as prostate tumor cells adapt to survive and grow. The removal of androgens (androgen deprivation) has been shown to activate both epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD) programs. EMT has established roles in promoting biological phenotypes associated with tumor progression (migration/invasion, tumor cell survival, cancer stem cell-like properties, resistance to radiation and chemotherapy) in multiple human cancer types. NEtD in prostate cancer is associated with resistance to therapy, visceral metastasis, and aggressive disease. Thus, activation of these programs via inhibition of the androgen axis provides a mechanism by which tumor cells can adapt to promote disease recurrence and progression. Brachyury, Axl, MEK, and Aurora kinase A are molecular drivers of these programs, and inhibitors are currently in clinical trials to determine therapeutic applications. Understanding tumor cell plasticity will be important in further defining the rational use of androgen-targeted therapies clinically and provides an opportunity for intervention to prolong survival of men with metastatic prostate cancer.