Paul G. Hynes

MiR-1 and miR-200 inhibit EMT via Slug-dependent and tumorigenesis via Slug-independent mechanisms.

Epithelial–mesenchymal transition (EMT) is a developmental program of signaling pathways that determine commitment to epithelial and mesenchymal phenotypes. In the prostate, EMT processes have been implicated in benign prostatic hyperplasia and prostate cancer progression. In a model of Pten- and TP53-null prostate adenocarcinoma that progresses via transforming growth factor β-induced EMT, mesenchymal transformation is characterized by plasticity, leading to various mesenchymal lineages and the production of bone. Here we show that SLUG is a major regulator of mesenchymal differentiation. As microRNAs (miRs) are pleiotropic regulators of differentiation and tumorigenesis, we evaluated miR expression associated with tumorigenesis and EMT. Mir-1 and miR-200 were reduced with progression of prostate adenocarcinoma, and we identify Slug as one of the phylogenetically conserved targets of these miRs. We demonstrate that SLUG is a direct repressor of miR-1 and miR-200 transcription. Thus, SLUG and miR-1/miR-200 act in a self-reinforcing regulatory loop, leading to amplification of EMT. Depletion of Slug inhibited EMT during tumorigenesis, whereas forced expression of miR-1 or miR-200 inhibited both EMT and tumorigenesis in human and mouse model systems. Various miR targets were analyzed, and our findings suggest that miR-1 has roles in regulating EMT and mesenchymal differentiation through Slug and functions in tumor-suppressive programs by regulating additional targets.

Critical and reciprocal regulation of KLF4 and SLUG in transforming growth factor β-initiated prostate cancer epithelial-mesenchymal transition.

ABSTRACT Epithelial-mesenchymal transition (EMT) is implicated in various pathological processes within the prostate, including benign prostate hyperplasia (BPH) and prostate cancer progression. However, an ordered sequence of signaling events initiating carcinoma-associated EMT has not been established. In a model of transforming growth factor β (TGFβ)-induced prostatic EMT, SLUG is the dominant regulator of EMT initiation in vitro and in vivo, as demonstrated by the inhibition of EMT following Slug depletion. In contrast, SNAIL depletion was significantly less rate limiting. TGFβ-stimulated KLF4 degradation is required for SLUG induction. Expression of a degradation-resistant KLF4 mutant inhibited EMT, and furthermore, depletion of Klf4 was sufficient to initiate SLUG-dependent EMT. We show that KLF4 and another epithelial determinant, FOXA1, are direct transcriptional inhibitors of SLUG expression in mouse and human prostate cancer cells. Furthermore, self-reinforcing regulatory loops for SLUG-KLF4 and SLUG-FOXA1 lead to SLUG-dependent binding of polycomb repressive complexes to the Klf4 and Foxa1 promoters, silencing transcription and consolidating mesenchymal commitment. Analysis of tissue arrays demonstrated decreased KLF4 and increased SLUG expression in advanced-stage primary prostate cancer, substantiating the involvement of the EMT signaling events described in model systems.

TMPRSS2- Driven ERG Expression In Vivo Increases Self-Renewal and Maintains Expression in a Castration Resistant Subpopulation

Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1hi/EpCAM+ basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.

Characterizing the Contribution of Stem/Progenitor Cells to Tumorigenesis in the Pten-/-TP53-/- Prostate Cancer Model

Loss of PTEN is one of the most common mutations in prostate cancer, and loss of wild‐type TP53 is associated with prostate cancer progression and castrate resistance. Modeling prostate cancer in the mouse has shown that while Pten deletion in prostate epithelial cells leads to adenocarcinoma, combined loss of Pten and TP53 results in rapidly developing disease with greater tumor burden and early death. TP53 contributes significantly to the regulation of stem cell self‐renewal, and we hypothesized that loss of Pten/TP53 would result in measurable changes in prostate cancer stem/progenitor cell properties. Clonogenic assays that isolate progenitor function in primary prostate epithelial cells were used to measure self‐renewal, differentiation, and tumorigenic potential. Pten/TP53 null as compared with wild‐type protospheres showed increased self‐renewal activity and modified lineage commitment. Orthotopic transplantation of Pten/TP53 null cells derived from protospheres produced invasive Prostatic Intraepithelial Neoplasia (PIN)/adenocarcinoma, recapitulating the pathology seen in primary tumors. Pten/TP53 null progenitors relative to wild type also demonstrated increased dependence on the AKT/mammalian target of rapamycin complex 1 (mTORC1) and androgen receptor (AR) pathways for clonogenic and tumorigenic growth. These data demonstrate roles for Pten/TP53 in prostate epithelial stem/progenitor cell function, and moreover, as seen in patients with castrate‐resistant prostate cancer, suggest for the involvement of an AR‐dependent axis in the clonogenic expansion of prostate cancer stem cells. STEM CELLS 2010;28:2129–2140

Loss of Androgen-Regulated MicroRNA 1 Activates SRC and Promotes Prostate Cancer Bone Metastasis

ABSTRACT Bone metastasis is the hallmark of progressive and castration-resistant prostate cancers. MicroRNA 1 (miR-1) levels are decreased in clinical samples of primary prostate cancer and further reduced in metastases. SRC has been implicated as a critical factor in bone metastasis, and here we show that SRC is a direct target of miR-1. In prostate cancer patient samples, miR-1 levels are inversely correlated with SRC expression and a SRC-dependent gene signature. Ectopic miR-1 expression inhibited extracellular signal-regulated kinase (ERK) signaling and bone metastasis in a xenograft model. In contrast, SRC overexpression was sufficient to reconstitute bone metastasis and ERK signaling in cells expressing high levels of miR-1. Androgen receptor (AR) activity, defined by an AR output signature, is low in a portion of castration-resistant prostate cancer. We show that AR binds to the miR-1-2 regulatory region and regulates miR-1 transcription. Patients with low miR-1 levels displayed correlated low canonical AR gene signatures. Our data support the existence of an AR–miR-1–SRC regulatory network. We propose that loss of miR-1 is one mechanistic link between low canonical AR output and SRC-promoted metastatic phenotypes.

Identification of Different Classes of Luminal Progenitor Cells within Prostate Tumors

Primary prostate cancer almost always has a luminal phenotype. However, little is known about the stem/progenitor properties of transformed cells within tumors. Using the aggressive Pten/Tp53-null mouse model of prostate cancer, we show that two classes of luminal progenitors exist within a tumor. Not only did tumors contain previously described multipotent progenitors, but also a major population of committed luminal progenitors. Luminal cells, sorted directly from tumors or grown as organoids, initiated tumors of adenocarcinoma or multilineage histological phenotypes, which is consistent with luminal and multipotent differentiation potentials, respectively. Moreover, using organoids we show that the ability of luminal-committed progenitors to self-renew is a tumor-specific property, absent in benign luminal cells. Finally, a significant fraction of luminal progenitors survived in vivo castration. In all, these data reveal two luminal tumor populations with different stem/progenitor cell capacities, providing insight into prostate cancer cells that initiate tumors and can influence treatment response.

Self-Renewing Pten-/-TP53-/- Protospheres Produce Metastatic Adenocarcinoma Cell Lines with Multipotent Progenitor Activity

Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.

Prostate cancer stem cells: The case for model systems.

Advanced prostate cancers are treated with androgen deprivation therapy, which usually leads to a rapid and significant reduction in tumor burden but subsequent development of castration-resistant and metastatic disease almost always occurs. The source of tumor heterogeneity and the accompanying mechanisms leading to treatment resistance are major areas of prostate cancer research. Although our understanding of tumor heterogeneity is evolving, the functional isolation of tumor propagating populations, also known as cancer stem cells (CSCs), is fundamental to the identification and molecular characterization of castration-resistant prostate cancer cells. Of clinical importance, knowledge of prostate CSCs has implications for design of next generation-targeted therapies aimed at both eradicating primary tumor mass and preventing castration-resistant disease. The inability to routinely transplant fractionated primary human prostate tumors has prevented progress in analyzing the source of heterogeneous and treatment-resistant populations in prostate cancer. Here, we briefly overview the mechanisms of castration resistance, including the hypothesis for the existence of androgen-independent prostate CSCs. Finally, we discuss the interpretation of preclinical models and their utility for characterizing prostate CSCs in androgen-replete and androgen-deprived conditions.

Abstract 3240: A transgenic mouse model of a common genetic aberration in prostate cancer: Chromosomal rearrangement of TMPRSS2:ERG

The most prevalent gene fusion identified in prostate cancer (PC) to date is the recurrent fusion of the 5’ region of Transmembrane Serine Protease 2 (TMPRSS2) to the transcription factor Ets related gene (ERG). The fusion occurs in ∼50% of PC and has been identified in malignant cells preceding the development of prostate adenocarcinoma. This has led to the hypothesis that the rearrangement is an early and critical event in PC. The purpose of this study is to generate a novel mouse model to investigate the role of the transgene in development of PC, and to examine the biological and molecular properties of prostate epithelial cells carrying a translocation of the ERG oncogene. The fusion is a result of deletion in the genomic region between TMPRSS2 and ERG, which are both located on chromosome 21. Several fusion variants have been described and the focus of this study is the TMPRSS2 exon 2 and ERG exon 4 variant which has been associated with aggressive disease. Importantly, expression of the fusion gene initiates from the TMPRSS2 promoter which is primarily expressed in the prostate and regulated by androgen receptor (AR), which is central to PC development. The objectives of this study included the generation of a novel model that maintains regulation and processing of the translocation. This was achieved by using the complete promoter region, including androgen regulatory regions, and the human genomic sequence thus maintaining the intron/exon arrangement and facilitating alternative splicing. Transgenic mice in the FVB and C57/BL6 background were produced using a 230 Kb construct generated by recombineering technology, where ∼25Kb of the TMPRSS2 promoter region and its first two exons were fused to the entire genomic ERG locus following exon 4. Two splice variants of the transgene have been identified in mouse prostates corresponding to the presence and absence of a 72bp exon, replicating the pattern in human PC, where this has been associated with aggressive disease. No distinct histological phenotypic change has been observed in the murine prostate of transgenic animals at six to twelve months. Towards the aim of determining the role of this fusion in PC - further in vitro and in vivo assays are currently in progress with the objectives of 1) determining the role of AR in regulation of the promoter and 2) investigating alterations in gene expression profiles in distinct cell populations of the prostate harboring the transgene. Additional crosses with other mice carrying mutations significant to prostate cancer, e.g. Nkx 3.1 and PTEN mice are being performed thus allowing the investigation of the phenotypic effect of this fusion when combined with other common genetic events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3240.

Abstract B018: A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced prostate cancer

Androgen-deprivation therapy (ADT) remains the gold-standard therapy for prostate cancer (PrCa), and although ADT is initially effective, most men progress to castrate-resistant prostate cancer (CRPC) within 2-3 years. Advanced CRPC is challenging to treat because intrinsic tumor heterogeneity and phenotypic plasticity engender short-lived responses and underlie resistance to conventional therapies. Combined PTEN/TP53 alterations represent a major genotype of advanced CRPC (25-30%) and are associated with poor clinical outcomes. Established PrCa cell lines do not accurately represent the heterogeneity of advanced CRPC, and therefore, nonbiased pharmacogenomics screens have not been done. The development of clinically representative, tractable models suitable for high-throughput target identification and validation is crucial for advancing novel CRPC therapies to the clinic. A comprehensive nonbiased high-throughput screen performed on seven cell lines derived from a genetically engineered mouse model (GEMM) of Pten/Tp53 null PrCa identified strongly active compounds, including inhibitors of PI3K/AKT/mTOR signaling, the proteasome, cell cycle regulatory proteins, heat shock proteins, DNA repair signaling, NFKB signaling, MAPK signaling, and several types of epigenetic modifiers. HSP90 inhibitors were one of the most efficacious classes of compounds in the screen, and ganetespib, a clinically used second-generation HSP90 inhibitor with a favorable safety profile, was the most potent. Although HSP90 inhibitors have yet to be successful as single agents, they have not been thoroughly investigated in clinically representative models of advanced PrCa and have shown potential as “network drugs,” prompting our investigations into their utility in polytherapy. We first validated ganetespib as a single agent, where it displayed strong activity against several GEMM-derived and LuCaP PDX-derived organoid models encompassing genotypic, phenotypic, and lineage heterogeneity. These 10 novel LuCaP PDX-derived organoids are representative of the numerous categories of CRPC, including adenocarcinomas with wild-type AR, adenocarcinomas with altered AR, adenocarcinoma with neuroendocrine features, and neuroendocrine disease. Single-agent ganetespib was also strongly inhibitory in vivo, decreasing growth of Pten/Tp53 null endogenous GEMM tumors as well as a human PDX tumor. Mechanistic interrogation of cell lines, organoids, and tumors exposed to ganetespib revealed inhibition of targets from several inter-related networks including AR and pAKT, two central and mutually compensatory growth and survival pathways for PrCa. The efficacy of ganetespib against a diverse group of CRPC organoids and the simultaneous inhibition of PrCa survival signaling suggested it may work well in combination. We performed a proof-of-principle high-throughput matrix screen on organoids derived from a Pten/Tp53 null GEMM and identified docetaxel and etoposide to be synergistic when combined with ganetespib. Preclinical in vivo studies to validate these findings are ongoing. In all, comprehensive data from multiple near-patient models suggest novel contexts for second-generation HSP90-directed intervention against a variety of CRPC genotypes and phenotypes and expand upon the potential of HSP90 inhibitors to simultaneously inhibit oncogenic signaling and compensatory resistance mechanisms. Citation Format: Keith H. Jansson, John B. Tucker, Lauren E. Stahl, John K. Simmons, Caitlyn Fuller, Michael L. Beshiri, Supreet Agarwal, Yasmine Abbey, Lei Fang, Paul G. Hynes, Alilin Aian Neil, Jacob Cawley, Ross Lake, Crystal Tran, Caitlin M. Tice, JuanJuan Yin, Xiahu Zhang, Rajarshi Guha, Shelley Hoover, R. Mark Simpson, Holly Nguyen, Eva Corey, Craig J. Thomas, David Proia, Kathleen Kelly. A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced 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 B018.