Search

High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background ('synergies'). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding sites of androgen-driven transcription factors and the promoters of androgen-regulated genes. Concatemers of high-order contacts in highly expressed genes were demethylated relative to pairwise contacts at the same loci. Synergies in breast cancer cells were associated with tyfonas, a class of complex DNA amplicons. These results rigorously link genome-wide high-order 3D interactions to lineage-defining transcriptional programs and establish Pore-C and Chromunity as scalable approaches to assess high-order genome structure.

PDF file - 410K, Dose responses, transcriptional, biological, biochemical, and in vivo supplemental data

PDF file - 91K

PDF file - 464K, Mimicking tumor-derived BAF57 elevation in vitro.

Supplementary Data from Cyclin D1 Splice Variants: Polymorphism, Risk, and Isoform-Specific Regulation in Prostate Cancer

LNCaP GSEA analyses RNAseq data.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1–S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon.Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer.Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor–resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion.Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201–14. ©2018 AACR.

LAPC4 phosphoproteomic data

Cbioportal analyses

LAPC4 aggressive phenotypes

Purpose: BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression have remained undefined in advanced disease such as castration-resistant prostate cancer and/or metastasis.Experimental Design: Clinical human specimens of primary and metastatic prostate cancer were immunohistochemically examined for tumor-grade association of BAF57 expression. Global gene expression analyses were conducted in models mimicking tumor-associated BAF57 expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal remodelers such as integrins were validated. Cell migration assays were used to profile the biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression.Results: Immunohistochemical quantitation of primary human specimens revealed that BAF57 was significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage, which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura.Conclusion: The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57 upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of metastatic potential that could be leveraged for therapeutic intervention. Clin Cancer Res; 19(10); 2657–67. ©2013 AACR.

PDF file - 130K

PDF file - 2249K, BAF57 aberrations govern pro-metastatic phenotypes.

Purpose: Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged.Experimental Design: First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies.Results: Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism.Conclusions: These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. (Clin Cancer Res 2009;15(17):5338–49)

LAPC4 GSEA analyses RNAseq data

LAPC4 CDK4/6 inhibitor resistance

PDF file - 2354K, Microarray analyses of BAF57 deregulation.

PDF file - 681K, BAF57 dependent modulation of integrin signaling.

PDF file - 91K, Oligonucleotide Sequences and Kegg Pathway Summary.

MAPK reliance in LAPC4

PDF file - 554K, Metastatic loci from individual patients display differences in BAF57 elevation.

Supplementary Figure 2 from Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

Supplementary Figure 4 from Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

Supplementary Figure 1 from Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

Supplementary Figure 5 from Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

The cyclin D1b oncogene arises from alternative splicing of the CCND1 transcript, and harbors markedly enhanced oncogenic functions not shared by full-length cyclin D1 (cyclin D1a). Recent studies showed that cyclin D1b is selectively induced in a subset of tissues as a function of tumorigenesis; however, the underlying mechanism(s) that control tumor-specific cyclin D1b induction remain unsolved. Here, we identify the RNA-binding protein ASF/SF2 as a critical, allele-specific, disease-relevant effector of cyclin D1b production. Initially, it was observed that SF2 associates with cyclin D1b mRNA (transcript-b) in minigene analyses and with endogenous transcript in prostate cancer (PCa) cells. SF2 association was altered by the CCND1 G/A870 polymorphism, which resides in the splice donor site controlling transcript-b production. This finding was significant, as the A870 allele promotes cyclin D1b in benign prostate tissue, but in primary PCa, cyclin D1b production is independent of A870 status. Data herein provide a basis for this disparity, as tumor-associated induction of SF2 predominantly results in binding to and accumulation of G870-derived transcript-b. Finally, the relevance of SF2 function was established, as SF2 strongly correlated with cyclin D1b (but not cyclin D1a) in human PCa. Together, these studies identify a novel mechanism by which cyclin D1b is induced in cancer, and reveal significant evidence of a factor that cooperates with a risk-associated polymorphism to alter cyclin D1 isoform production. Identification of SF2 as a disease-relevant effector of cyclin D1b provides a basis for future studies designed to suppress the oncogenic alternative splicing event. Cancer Res; 70(10); 3975–84. ©2010 AACR.

Supplementary Figure 3 from Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

Supplementary Figure 6 from Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

Despite recent therapeutic advances, prostate cancer remains a leading cause of cancer-related death. A subset of castration-resistant prostate cancers become androgen receptor (AR) signaling independent and develop neuroendocrine prostate cancer (NEPC) features through lineage plasticity. These NEPC tumors, associated with aggressive disease and poor prognosis, are driven, in part, by aberrant expression of N-Myc, through mechanisms that remain unclear. Integrative analysis of the N-Myc transcriptome, cistrome, and interactome using in vivo, in vitro, and ex vivo models (including patient-derived organoids) identified a lineage switch towards a neural identity associated with epigenetic reprogramming. N-Myc and known AR cofactors (e.g., FOXA1 and HOXB13) overlapped, independently of AR, at genomic loci implicated in neural lineage specification. Moreover, histone marks specifically associated with lineage-defining genes were reprogrammed by N-Myc. We also demonstrated that the N-Myc–induced molecular program accurately classifies our cohort of patients with advanced prostate cancer. Finally, we revealed the potential for enhancer of zeste homolog 2 (EZH2) inhibition to reverse the N-Myc–induced suppression of epithelial lineage genes. Altogether, our data provide insights into how N-Myc regulates lineage plasticity and epigenetic reprogramming associated with lineage specification. The N-Myc signature we defined could also help predict the evolution of prostate cancer and thus better guide the choice of future therapeutic strategies.

The normal androgen receptor (AR) cistrome and transcriptional program are fundamentally altered in prostate cancer (PCa). Here, we show that SPOP mutations, an early event in prostate tumorigenesis, reshape the chromatin landscape and AR-directed transcriptional program in normal prostate cells. Induction of SPOP mutation results in DNA accessibility and AR binding patterns found in human PCa. Consistent with dependency on this AR reprogramming, castration of SPOP mutant mouse models results in the loss of neoplastic phenotypes. Finally, human SPOP mutant PCa show improved response to AR-targeted therapies. Together, these results show that a single genomic alteration may be sufficient to reprogram the chromatin of normal prostate cells toward oncogenic phenotypes and that SPOP mutant tumors may be preferentially dependent on AR signaling through this mechanism.

PURPOSE: Biobanking tissue of high quality and fidelity is imperative for cancer genomics research. Since it is a challenging process, we sought to develop a protocol that improves the fidelity and quantity of biobanked primary prostate cancer (PCa) tissue. MATERIALS AND METHODS: We conducted a pilot study evaluating pathologic concordance of biobanked tissue and the radical prostatectomy specimen using either standard protocol (SP) vs next-generation protocol (NGP). RESULTS: There were no significant differences in clinical and pathologic characteristics (age, BMI, pre-operative PSA, prostate weight, race, final prostatectomy Gleason score, or pathologic tumor and nodal stages) between the two protocol arms. Utilization of the NGP compared to the SP resulted in a significantly higher rate of pathologic concordance between the biobanked and RP specimens (61.8% vs 37.9%, p=0.0231) as well as a nearly two-fold increase in the amount of biobanked tumor tissue (330 mm(3) vs 174 mm(3), p

BACKGROUND: Molecular characterization of prostate cancer (PCa) has revealed distinct subclasses based on underlying genomic alterations occurring early in the natural history of the disease. However, how these early alterations influence subsequent molecular events and the course of the disease over its long natural history remains unclear. METHODS: We explored the molecular and clinical progression of different genomic subtypes of PCa using distinct tumor lineage models based on human genomic and transcriptomic data. We developed transcriptional classifiers, and defined “early” and “late” categories of molecular subclasses from 8,158 PCa patients. Molecular subclasses were correlated with clinical outcomes and pathologic characteristics using Kaplan-Meier and logistic regression analyses. RESULTS: We identified PTEN and CHD1 alterations as subtype-specific late progression events specifically in ERG-overexpressing (ERG+) and SPOP-mutant tumors, respectively, and 2 distinct progression models consisting of ERG/PTEN (normal to ERG+ to PTEN-deleted) and SPOP/CHD1 (normal to SPOP-mutated to CHD1-deleted) with shared early tumorigenesis but distinct pathways toward progression. We found that within ERG+ and SPOP-mutant subtypes, late events were associated with worse prognosis. Importantly, the clinical and pathologic features associated with distinct late events at radical prostatectomy were strikingly different; PTEN deletions were associated with increased locoregional stage, while CHD1 deletions were only associated with increased grade, despite equivalent metastatic potential. CONCLUSION: These findings suggest a paradigm in which specific subtypes of PCa follow distinct pathways of progression, at both the molecular and clinical levels. Therefore, the interpretation of common clinical parameters such as locoregional tumor stage may be influenced by the underlying tumor lineage, and potentially influence management decisions. FUNDING: Prostate Cancer Foundation, National Cancer Institute, Urology Care Foundation, Damon Runyon Cancer Research Foundation, US Department of Defense, and the AIRC Foundation.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1–S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon. Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer. Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor–resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion. Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201–14. ©2018 AACR.

SUMMARY The normal androgen receptor (AR) cistrome and transcriptional program are fundamentally altered in prostate cancer (PCa). Here, we profile the chromatin landscape and AR-directed transcriptional program in normal prostate cells and show the impact of SPOP mutations, an early event in prostate tumorigenesis. In genetically normal mouse prostate organoids, SPOP mutation results in accessibility and AR binding patterns similar to that of human PCa. Consistent with dependence on AR signaling, castration of SPOP mutant mouse models results in the loss of neoplastic phenotypes, and human SPOP mutant PCa shows a favorable response to AR-targeted therapies. Together, these data validate mouse prostate organoids as a robust model for studying epigenomic and transcriptional alterations in normal prostate, provide valuable datasets for further studies, and show that a single genomic alteration may be sufficient to reprogram the chromatin of normal prostate cells toward oncogenic phenotypes, with potential therapeutic implications for AR-targeting therapies., Graphical abstract, In brief Grbesa et al. use multilevel epigenomic and transcriptional profiling to characterize genetically normal prostate organoids and show the impact of an early cancer driver (SPOP mutation). Integrative analysis identifies transcriptional programs that are AR regulated and reprogrammed by SPOP mutation, suggesting a basis for enhanced sensitivity to AR-targeting therapies.

Recurrent point mutations in SPOP define a distinct molecular subclass of prostate cancer. Here, we describe a mouse model showing that mutant SPOP drives prostate tumorigenesis in vivo. Conditional expression of mutant SPOP in the prostate dramatically altered phenotypes in the setting of Pten loss, with early neoplastic lesions (high-grade prostatic intraepithelial neoplasia) with striking nuclear atypia, and invasive poorly differentiated carcinoma. In mouse prostate organoids, mutant SPOP drove increased proliferation and a transcriptional signature consistent with human prostate cancer. Using these models and human prostate cancer samples, we show that SPOP mutation activates both PI3K/mTOR and androgen receptor (AR) signaling, effectively uncoupling the normal negative feedback between these two pathways.

Deletion of the gene encoding the chromatin remodeler CHD1 is among the most common alterations in prostate cancer (PCa); however, the tumor-suppressive functions of CHD1 and reasons for its tissue-specific loss remain undefined. We demonstrated that CHD1 occupied prostate-specific enhancers enriched for the androgen receptor (AR) and lineage-specific cofactors. Upon CHD1 loss, the AR cistrome was redistributed in patterns consistent with the oncogenic AR cistrome in PCa samples and drove tumor formation in the murine prostate. Notably, this cistrome shift was associated with a unique AR transcriptional signature enriched for pro-oncogenic pathways unique to this tumor subclass. Collectively, these data credential CHD1 as a tumor suppressor in the prostate that constrains AR binding/function to limit tumor progression.

Prostate cancer (PCa) remains a leading cause of cancer-related death in the USA. While localized lesions are effectively treated through radical prostatectomy and/or radiation therapy, treatment for metastatic disease leverages the addiction of these tumors on the androgen receptor (AR) signaling axis for growth and disease progression. Though initially effective, tumors resistant to AR-directed therapeutics ultimately arise (a stage of the disease known as castration-resistant prostate cancer) and are responsible for PCa-specific mortality. Importantly, an abundance of clinical and preclinical evidence strongly implicates AR signaling cascades in the development of metastatic disease in both early and late stages, and thus a concerted effort has been made to delineate the AR-specific programs that facilitate progression to metastatic PCa. A multitude of downstream AR targets as well as critical AR cofactors have been identified which impinge upon both the AR pathway as well as associated metastatic phenotypes. This review will highlight the functional significance of these pathways to disseminated disease and define the molecular underpinnings behind these unique, AR-driven, metastatic signatures.

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.

Purpose: BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression have remained undefined in advanced disease such as castration-resistant prostate cancer and/or metastasis. Experimental Design: Clinical human specimens of primary and metastatic prostate cancer were immunohistochemically examined for tumor-grade association of BAF57 expression. Global gene expression analyses were conducted in models mimicking tumor-associated BAF57 expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal remodelers such as integrins were validated. Cell migration assays were used to profile the biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression. Results: Immunohistochemical quantitation of primary human specimens revealed that BAF57 was significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage, which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura. Conclusion: The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57 upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of metastatic potential that could be leveraged for therapeutic intervention. Clin Cancer Res; 19(10); 2657–67. ©2013 AACR.

Precision medicine is an approach that takes into account the influence of individuals' genes, environment, and lifestyle exposures to tailor interventions. Here, we describe the development of a robust precision cancer care platform that integrates whole-exome sequencing with a living biobank that enables high-throughput drug screens on patient-derived tumor organoids. To date, 56 tumor-derived organoid cultures and 19 patient-derived xenograft (PDX) models have been established from the 769 patients enrolled in an Institutional Review Board–approved clinical trial. Because genomics alone was insufficient to identify therapeutic options for the majority of patients with advanced disease, we used high-throughput drug screening to discover effective treatment strategies. Analysis of tumor-derived cells from four cases, two uterine malignancies and two colon cancers, identified effective drugs and drug combinations that were subsequently validated using 3-D cultures and PDX models. This platform thereby promotes the discovery of novel therapeutic approaches that can be assessed in clinical trials and provides personalized therapeutic options for individual patients where standard clinical options have been exhausted. Significance: Integration of genomic data with drug screening from personalized in vitro and in vivo cancer models guides precision cancer care and fuels next-generation research. Cancer Discov; 7(5); 462–77. ©2017 AACR. See related commentary by Picco and Garnett, p. 456. This article is highlighted in the In This Issue feature, p. 443

The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle (“Cell Cycle Common”), versus those that were specifically enriched in a subset of cell cycle phases (“Phase Restricted”). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide-desaturase 1 (DEGS1) was identified as an AR regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention, and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

PARP-1 is an abundant nuclear enzyme that modifies substrates by poly(ADP-ribose)-ylation. PARP-1 has well-described functions in DNA damage repair and also functions as a context-specific regulator of transcription factors. With multiple models, data show that PARP-1 elicits protumorigenic effects in androgen receptor (AR)–positive prostate cancer cells, in both the presence and absence of genotoxic insult. Mechanistically, PARP-1 is recruited to sites of AR function, therein promoting AR occupancy and AR function. It was further confirmed in genetically defined systems that PARP-1 supports AR transcriptional function, and that in models of advanced prostate cancer, PARP-1 enzymatic activity is enhanced, further linking PARP-1 to AR activity and disease progression. In vivo analyses show that PARP-1 activity is required for AR function in xenograft tumors, as well as tumor cell growth in vivo and generation and maintenance of castration resistance. Finally, in a novel explant system of primary human tumors, targeting PARP-1 potently suppresses tumor cell proliferation. Collectively, these studies identify novel functions of PARP-1 in promoting disease progression, and ultimately suggest that the dual functions of PARP-1 can be targeted in human prostate cancer to suppress tumor growth and progression to castration resistance. Significance: These studies introduce a paradigm shift with regard to PARP-1 function in human malignancy, and suggest that the dual functions of PARP-1 in DNA damage repair and transcription factor regulation can be leveraged to suppress pathways critical for promalignant phenotypes in prostate cancer cells by modulation of the DNA damage response and hormone signaling pathways. The combined studies highlight the importance of dual PARP-1 function in malignancy and provide the basis for therapeutic targeting. Cancer Discov; 2(12); 1134–49. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 1065

Ionizing radiation (IR) is used frequently in the management of multiple tumor types, including both organ-confined and locally advanced prostate cancer (PCa). Enhancing tumor radiosensitivity could both reduce the amount of radiation required for definitive treatment and improve clinical outcome. Androgen suppression therapy improves clinical outcomes when combined with radiation therapy but is associated with significant acute and chronic toxicities; hence, there is a clear need for alternative means to increase the therapeutic window of radiotherapy. Herein, it is demonstrated that the mammalian target of rapamycin (mTOR) inhibitors rapamycin (sirolimus) and temsirolimus limit both hormone therapy (HT)-sensitive and castration-resistant PCa (CRPC) cell proliferation as single agents and have a profound radiosensitization effect when used in combination with IR. Importantly, the observed radiosensitization was influenced by the treatment schedule, in which adjuvant administration of mTOR inhibitors was most effective in limiting PCa cell population doubling. This schedule-dependent influence onin vitrotreatment outcome was determined to be the result of relative effects on the cell cycle kinetics. Finally, adjuvant administration of either mTOR inhibitor tested after IR significantly decreased clonogenic cell survival of both HT-sensitive and CRPC cells compared with IR alone. Taken together, these data demonstrate that inhibition of mTOR confers a radiosensitization phenotype that is dependent on relative cell cycle kinetics and provide a foundation for clinical assessment.

FOXA transcription factors are potent, context-specific mediators of development that hold specialized functions in hormone-dependent tissues. Over the last several years, FOXA1 has emerged as a critical mediator of nuclear steroid receptor signalling, manifest at least in part through regulation of androgen receptor and oestrogen receptor activity. Recent findings point towards a major role for FOXA1 in modulating nuclear steroid receptor activity in breast and prostate cancer, and suggest that FOXA1 may significantly contribute to pro-tumourigenic phenotypes. The present review article will focus on the mechanisms, consequence, and clinical relevance of FOXA1-mediated steroid nuclear receptor signalling in human malignancy.

Cyclin dependent kinase-4/6 (CDK4/6) kinase inhibitors have shown clinical benefit in treatment of solid tumor types, including breast cancer. However, resistance is common, and the underpinning mechanisms of action are not well understood. Given the dependence of CDK4/6 inhibitors on retinoblastoma tumor suppressor (RB) function for activity, this class of agents may be particularly effective in tumor types for which RB loss is infrequent or occurs late in tumor progression. Here, models of acquired palbociclib resistance were generated in early-stage, RB-positive cancers, wherein it was shown that acquired palbociclib resistance resulted in cross-resistance to other CDK4/6 inhibitors under clinical testing. Furthermore, cells showing acquired resistance exhibited aggressive in vitro and in vivo phenotypes without genetic loss of RB or RB pathway members, including enhanced proliferative capacity, migratory potential, and characteristics of epithelial-to-mesenchymal transition. Further analyses through integration of RNA sequencing and phospho-proteomics identified activation of the MAPK signaling pathway as a mediator of CDK4/6 inhibitor resistance, capable of bypassing CDK4/6 activity. However, this altered kinase dependence resulted in sensitization to MEK inhibitors, suggestive of new clinical opportunities in CDK4/6-resistant tumors. In sum, the studies herein not only identify activation of the MAPK pathway as capable of bypassing the CDK4/6 requirement and promoting aggressive tumor characteristics, but also nominate MEK inhibitors as potential mechanisms to treat or prevent CDK4/6 inhibitor resistance. Citation Format: Renee de Leeuw, Christopher M. McNair, Matthew J. Schiewer, Neermala P. Neupane, Michael A. Augello, Zhen Li, Larry Cheng, Akihiro Yoshida, J. Alan Diehl, E Starr Hazard, Sean M. McCourtney, Gary Hardiman, Maha H. Hussain, Justin M. Drake, Wm. Kevin Kelly, Karen E. Knudsen. Bypass kinase pathways lead to acquired CDK4/6 inhibitor resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A043.

379 Background: Cyclin Dependent Kinase-4/6 (CDK4/6) kinase inhibitors have shown clinical benefit in treatment of solid tumor types, including breast cancer. However, resistance is common, and the underpinning mechanisms of action are not well understood. Given the dependence of CDK4/6 inhibitors on retinoblastoma tumor suppressor (RB) function for activity, this class of agents may be particularly effective in tumor types for which RB loss is infrequent or occurs late in tumor progression. Methods: Here, models of acquired palbociclib resistance were generated in early stage, RB positive cancers, wherein it was shown acquired palbociclib resistance resulted in cross-resistance to other CDK4/6 inhibitors under clinical testing. Results: Cells showing acquired resistance exhibited aggressive in vitro and in vivo phenotypes without genetic loss of RB or RB pathway members, including enhanced proliferative capacity, migratory potential, and characteristics of epithelial to mesenchymal transition. Further analyses through integration of RNA sequencing and phospho-proteomics identified activation of the MAPK signaling pathway as a mediator of CDK4/6 inhibitor resistance, capable of bypassing CDK4/6 activity. However, this altered kinase dependence resulted in sensitization to MEK inhibitors, suggestive of new clinical opportunities in CDK4/6 resistant tumors. Conclusions: In sum, the studies herein not only identify activation of the MAPK pathway as capable of bypassing the CDK4/6 requirement and promoting aggressive tumor characteristics, but nominate MEK inhibitors as potential mechanisms to treat or prevent CDK4/6 inhibitor resistance.

Non-organ confined prostate cancer (PCa) is often effectively, but only transiently treated by targeting the androgen receptor (AR) signaling axis through androgen depletion strategies, often coupled with AR antagonists. Unfortunately, disease recurs within a median of 3-4 years, presenting as castration resistant PCa (CRPC), for which there are limited therapeutic options. This emphasizes the need for more efficacious drugs and a patient-tailored approach towards cancer therapy to improve disease outcome. One class of drugs currently tested clinically, Cdk4/6 kinase inhibitors, blocks phosphorylation of the retinoblastoma (RB) tumor suppressor, thereby boosting its function, and likely preventing castration resistance. As Cdk4/6 inhibitor resistance has already been reported in other cancers, some PCa patients are anticipated to develop drug resistance. Here, we created palbociclib-resistant PCa cell models by continuously culturing them in presence of the drug to unravel mechanisms of acquired resistance, and assessed them for cross-resistance to ribociclib and response to other therapeutics. While the parental PCa cell models, Cdk4/6 inhibitors efficiently induce a G1 cell cycle arrest, the resistant cell lines bypass this cell cycle checkpoint. Although loss of RB is a known mechanism for Cdk4/6i resistance, none of the models lost RB expression. Strikingly, these originally hormone-sensitive cell lines, upon developing Cdk4/6 inhibitor resistance display altered response to selected therapeutic regimens. Mechanisms of resistance, as informed by Whole Exome Sequencing and RNASeq, will be discussed. Citation Format: Renee de Leeuw, Matthew J. Schiewer, Christopher McNair, Michael A. Augello, Akihiro Yoshida, Edward S. Hazard, Sean Courtney, Gerard T. Hardiman, Justin Drake, Felix Y. Feng, Scott Tomlins, Maha H. Hussain, J Alan Diehl, William K. Kelly, Karen E. Knudsen. Cdk4/6 kinase inhibitor resistance in prostate cancer [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 5874. doi:10.1158/1538-7445.AM2017-5874