Orla Casey

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.

Prostate epithelial PTEN/TP53 loss leads to transformation of multipotential progenitors and epithelial to mesenchymal transition

Loss of PTEN and loss of TP53 are common genetic aberrations occurring in prostate cancer. PTEN and TP53 contribute to the regulation of self-renewal and differentiation in prostate progenitors, presumptive tumor initiating cells for prostate cancer. Here we characterize the transformed phenotypes resulting from deletion of the Pten and TP53 tumor suppressors in prostate epithelium. Using the PB-Cre4(+)Pten(fl/fl)TP53(fl/fl) model of prostate cancer, we describe the histological and metastatic properties of primary tumors, transplanted primary tumor cells, and clonal cell lines established from tumors. Adenocarcinoma was the major primary tumor type that developed, which progressed to lethal sarcomatoid carcinoma at approximately 6 months of age. In addition, basal carcinomas and prostatic urothelial carcinomas were observed. We show that tumor heterogeneity resulted, at least in part, from the transformation of multipotential progenitors. CK8+ luminal epithelial cells were capable of undergoing epithelial to mesenchymal transition in vivo to sarcomatoid carcinomas containing osseous metaplasia. Metastasis rarely was observed from primary tumors, but metastasis to lung and lymph nodes occurred frequently from orthotopic tumors initiated from a biphenotypic clonal cell line. Androgen deprivation influenced the differentiated phenotypes of metastases. These data show that one functional consequence of Pten/TP53 loss in prostate epithelium is lineage plasticity of transformed cells.

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.

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.

Androgen deprivation leads to increased carbohydrate metabolism and hexokinase 2-mediated survival in Pten/Tp53 -deficient prostate cancer

Prostate cancer is characterized by a dependence upon androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) is the accepted treatment for progressive prostate cancer. Although ADT is usually initially effective, acquired resistance termed castrate-resistant prostate cancer (CRPC) develops. PTEN and TP53 are two of the most commonly deleted or mutated genes in prostate cancer, the compound loss of which is enriched in CRPC. To interrogate the metabolic alterations associated with survival following ADT, we used an orthotopic model of Pten/Tp53 null prostate cancer. Metabolite profiles and associated regulators were compared in tumors from androgen-intact mice and in tumors surviving castration. AR inhibition led to changes in the levels of glycolysis and tricarboxylic acid (TCA) cycle pathway intermediates. As anticipated for inhibitory reciprocal feedback between AR and PI3K/AKT signaling pathways, pAKT levels were increased in androgen-deprived tumors. Elevated mitochondrial hexokinase 2 (HK2) levels and enzyme activities also were observed in androgen-deprived tumors, consistent with pAKT-dependent HK2 protein induction and mitochondrial association. Competitive inhibition of HK2-mitochondrial binding in prostate cancer cells led to decreased viability. These data argue for AKT-associated HK2-mediated metabolic reprogramming and mitochondrial association in PI3K-driven prostate cancer as one survival mechanism downstream of AR inhibition.

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 A059: Lifestyle and health-related quality of life in men with metastatic prostate cancer

Background: Patients with metastatic prostate cancer live with a considerable disease burden that may have a profound impact on physical activity and quality of life. This patient group may survive for many years after disease onset; however, there is little evidence on their habitual levels of physical activity. The aim of this study was to investigate physical activity levels and associated health-related quality of life in prostate cancer patients with bone metastases. Methods: ExPeCT (Exercise, Prostate Cancer and Circulating Tumour Cells: CTRIAL-IE 15-21) (ClincalTrials.gov identifier NCT02453139) is an ongoing multicenter trial examining the effect of a 6-month structured exercise intervention for patients with metastatic prostate cancer. Participants complete questionnaires examining self-reported health-related quality of life (FACT-P), sleep (Pittsburgh Sleep Index), depression (PHQ-9), and physical activity (Harvard Health Professionals Study Questionnaire) at baseline, three, and six months. Analysis of 3- and 6-month data is ongoing. Result: An interim analysis of the baseline outcome measures of 64 patients with bone metastases was completed (mean age 69.4 (SD 7.35) years and mean BMI 29.2 (SD 5.8) kg/m2). Median time since diagnosis was 34 months (IQR 7-54) and 55% (n=35) of participants had >1 region affected by metastatic disease. Preliminary data demonstrated that 38% (n=24) of participants did not meet the current aerobic exercise guidelines for cancer survivors. In total, 20% (n=13) of participants reported engaging in vigorous activity and 16% (n=11) reported completing resistance exercise. There was no correlation found between physical activity levels and quality of life (r=0.01), sleep (r=0.02), or depression (r=-0.15) scores. The majority of participants (61% (n=41)) had sleep scores of >7, indicative of poor sleep quality. Sleep scores correlated negatively with global quality of life (r =-0.55, p Discussion: Findings highlight the association between reduced quality of life and poor sleep quality among men with metastatic prostate cancer. In addition, data suggest a high prevalence of suboptimal physical activity levels in this population. Patients with metastatic disease may benefit from lifestyle interventions that aim to increase physical activity levels. Citation Format: Grainne Sheill, Lauren Brady, Emer Guinan, Juliette Hussey, David Hevey, Tatjana Vlajnic, Orla Casey, Anne-Marie Baird, Fidelma Cahill, Mieke Van Hemelrijck, Nicola Peat, Sarah Rudman, Thomas Lynch, Rustom P. Manecksha, Brian Hayes, Moya Cunningham, Liam Grogan, John McCaffrey, Dearbhaile M. O’Donnell, Ray McDermott, John O Leary, Stephen P. Finn. Lifestyle and health-related quality of life in men with metastatic 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 A059.

Abstract A057: Examining the link between obesity, inflammation, and exercise in patients with metastatic prostate cancer—An interim analysis from the ExPeCT trial

Background: Globally, prostate cancer (PrCa) is the fourth most common cancer type. Obesity and inflammation have been shown to play significant roles in PrCa disease progression. Obesity and a high body mass index (BMI) are associated with increased PrCa-specific mortality in patients with advanced disease. Furthermore, proinflammatory cytokines can aid metastatic potential and promote angiogenesis. The ExPeCT (Exercise, Prostate Cancer and Circulating Tumor Cells) trial seeks to examine the effectiveness of a structured exercise program in modulating inflammatory mediators and obesity in patients with metastatic PrCa. Methods: ExPeCT (CTRIALIE 15-21 (ClincalTrials.gov identifier NCT02453139)) is a multicenter, randomized trial for patients with metastatic PrCa (n=67). Participants were randomized to either control or exercise arms. Participants in the exercise arm completed six months of prescribed aerobic exercise, which was monitored using percentage heart-rate reserve. Serum samples were collected for all participants at baseline (T0), three months (T3), and six months (T6), and assayed for 16 interlinked adipokines and cytokines using the Meso Scale Discovery platform. An interim statistical analysis was performed (n=26) comparing median change in serum analyte levels between control (n=13) and exercise (n=13) arms using non-parametric Wilcoxon rank-sum tests. Results: Among 26 patients included in our interim analysis, mean age at baseline was 71 years, median BMI was 29.1 kg/m2, and median waist circumference (WC) was 107 cm, with no significant differences between arms (all p>0.3). Between T0 and T6, WC decreased by a median of 3.8 cm in the exercise group and 2.6 cm in the control group (p=0.412), with a similar trend for BMI. Interim serum cytokine analysis showed a 3-fold increase in IL-10 levels in the exercise arm at T3 when compared to the control arm (p=0.036). No significant change in IL-10 levels was recorded at T6 between arms (p=0.776). Similarly, CXCL8 (IL-8) levels were increased by 1.8-fold at T3 in the exercise arm in comparison to the control arm (p=0.017), with no significant change reported at T6 (p=0.191). While changes were evident in serum TNFα, IL-6, VEGF, IL-17a, MMP9 and CCL5 (RANTES) levels, these did not reach significance. Differences in levels of adipokines leptin and resistin were also reported. A 1.5-fold increase in resistin expression was observed in the exercise arm at T6; however, it was not significant (p=0.293). A 2-fold decrease in leptin in the control arm relative to the exercise arm at T6 was also detected (p=0.676). Analysis of adiponectin, MMP2, and CCL2 is ongoing. Conclusion: Our interim analysis of ExPeCT trial participants demonstrated a significant increase in serum CXCL8 and IL-10 levels after three months of a supervised exercise intervention. These preliminary data suggest that a structured exercise program has the potential to modify inflammatory status in patients with metastatic PrCa. Citation Format: Lauren Brady, Grainne Sheill, Anne-Marie Baird, Emma H. Allott, Tatjana Vlajnic, John Greene, Orla Casey, Brian Hayes, Emer Guinan, Juliette Hussey, Fidelma Cahill, Mieke Van Hemelrijck, Nicola Peat, Sarah Rudman, Moya Cunningham, Liam Grogan, Thomas Lynch, Rustom P. Manecksha, John McCaffrey, Orla Sheils, Dearbhaile M. O’Donnell, John O’Leary, Ray McDermott, Stephen P. Finn. Examining the link between obesity, inflammation, and exercise in patients with metastatic prostate cancer—An interim analysis from the ExPeCT trial [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 A057.