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.

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 ␤ (TGF␤)-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. TGF␤-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.

isrctn.org Identifier: ISRCTN78923965.

Prostate cancers display a range of clinical behavior, from slow-growing tumors of minor clinical significance to locally aggressive and ultimately metastatic disease. Human prostate adenocarcinoma has a mature luminal phenotype characterized by cytokeratin 8 (CK8) and androgen receptor (AR) expression and prostate-specific antigen (PSA) production. Progressive prostate cancer is almost always treated with androgen deprivation therapy; however, despite such treatment, approximately 10% of prostate cancers progress to metastatic disease.1 Defining mechanisms of resistance to androgen deprivation and progression to metastasis would be significantly aided by the availability of genetically defined models of prostate cancer progression.One of the most common genetic alterations in prostate cancer is deletion of at least one copy of the PTEN tumor suppressor, which occurs in approximately 70% of human prostate cancers. Biallelic deletion of PTEN and the associated increase in AKT phosphorylation, which occurs in roughly 25% of prostate cancers, is correlated with resistance to androgen deprivation therapy.2 A recent genomic profiling study of mostly primary prostate cancers demonstrated that 24% of cases had either a heterozygous or homozygous copy number loss of TP53.3 Other large-scale studies using combined immunohistochemistry (IHC) and sequencing approaches have shown that TP53 mutations occur in approximately 5% of primary tumors and at much higher frequencies in lymph node metastases (16%) and castrate-resistant (26%) tumors. 4,5 In addition, TP53 mutations were found to be independent predictors of tumor recurrence in low-and intermediate-grade cancers. Thus, loss of PTEN and aberrations of TP53 are implicated in aggressive forms of human prostate cancer.

Most forms of castration-resistant prostate cancer (CRPC) are dependent on the androgen receptor (AR) for survival. While, enzalutamide provides a substantial survival benefit, it is not curative and many patients develop resistance to therapy. Although not yet fully understood, resistance can develop through a number of mechanisms, such as AR copy number gain, the generation of splice variants such as AR-V7 and mutations within the ligand binding domain (LBD) of the AR. circular RNAs (circRNAs) are a novel type of non-coding RNA, which can regulate the function of miRNA, and may play a key role in the development of drug resistance. circRNAs are highly resistant to degradation, are detectable in plasma and, therefore may serve a role as clinical biomarkers. In this study, AR-V7 expression was assessed in an isogenic model of enzalutamide resistance. The model consisted of age matched control cells and two sub-line clones displaying varied resistance to enzalutamide. circRNA profiling was performed on the panel using a high throughout microarray assay. Bioinformatic analysis identified a number of differentially expressed circRNAs and predicted five miRNA binding sites for each circRNA. miRNAs were stratified based on known associations with prostate cancer, and targets were validated using qPCR. Overall, circRNAs were more often down regulated in resistant cell lines compared with control (588 vs . 278). Of particular interest was hsa_circ_0004870, which was down-regulated in enzalutamide resistant cells (p ≤ 0.05, vs . sensitive cells), decreased in cells that highly express AR (p ≤ 0.01, vs . AR negative), and decreased in malignant cells (p ≤ 0.01, vs . benign). The associated parental gene was identified as RBM39 , a member of the U2AF65 family of proteins. Both genes were down-regulated in resistant cells (p < 0.05, vs . sensitive cells). This is one of the first studies to profile and demonstrate discrete circRNA expression patterns in an enzalutamide resistant cell line model of prostate cancer. Our data suggests that hsa_circ_0004870, through RBM39 , may play a critical role in the development of enzalutamide resistance in CRPC.

IntroductionPreliminary evidence supports the beneficial role of physical activity on prostate cancer outcomes. This phase III randomised controlled trial (RCT) is designed to determine if supervised high-intensity aerobic and resistance exercise increases overall survival (OS) in patients with metastatic castrate-resistant prostate cancer (mCRPC).Methods and analysisParticipants (n=866) must have histologically documented metastatic prostate cancer with evidence of progressive disease on androgen deprivation therapy (defined as mCRPC). Patients can be treatment-naïve for mCRPC or on first-line androgen receptor-targeted therapy for mCRPC (ie, abiraterone or enzalutamide) without evidence of progression at enrolment, and with no prior chemotherapy for mCRPC. Patients will receive psychosocial support and will be randomly assigned (1:1) to either supervised exercise (high-intensity aerobic and resistance training) or self-directed exercise (provision of guidelines), stratified by treatment status and site. Exercise prescriptions will be tailored to each participant’s fitness and morbidities. The primary endpoint is OS. Secondary endpoints include time to disease progression, occurrence of a skeletal-related event or progression of pain, and degree of pain, opiate use, physical and emotional quality of life, and changes in metabolic biomarkers. An assessment of whether immune function, inflammation, dysregulation of insulin and energy metabolism, and androgen biomarkers are associated with OS will be performed, and whether they mediate the primary association between exercise and OS will also be investigated. This study will also establish a biobank for future biomarker discovery or validation.Ethics and disseminationValidation of exercise as medicine and its mechanisms of action will create evidence to change clinical practice. Accordingly, outcomes of this RCT will be published in international, peer-reviewed journals, and presented at national and international conferences. Ethics approval was first obtained at Edith Cowan University (ID: 13236 NEWTON), with a further 10 investigator sites since receiving ethics approval, prior to activation.Trial registration numberNCT02730338.

SUMMARY 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, 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.

Early growth response-1 ( EGR1) is a transcription factor correlated with prostate cancer (PC) progression in a variety of contexts. For example, EGR1 levels increase in response to suppressed androgen receptor signaling or loss of the tumor suppressor, PTEN. EGR1 has been shown to regulate genes influencing proliferation, apoptosis, immune cell activation, and matrix degradation, among others. Despite this, the impact of EGR1 on PC metastatic colonization is unclear. We demonstrate using a PC model (DU145/RasB1) of bone and brain metastasis that EGR1 expression regulates angiogenic and osteoclastogenic properties of metastases. We have shown previously that FN14 (TNFRSF12A) and downstream NF-κB signaling is required for metastasis in this model. Here we demonstrate that FN14 ligation also leads to NF-κB-independent, MEK-dependent EGR1 expression. EGR1 -depletion in DU145/RasB1 cells reduced both the number and size of metastases but did not affect primary tumor growth. Decreased EGR1 expression led to reduced blood vessel density in brain and bone metastases as well as decreased osteolytic bone lesion area and reduced numbers of osteoclasts at the bone-tumor interface. TWEAK (TNFSF12) induced several EGR1 -dependent angiogenic and osteoclastogenic factors (e.g. PDGFA, TGFB1, SPP1, IL6, IL8, and TGFA, among others). Consistent with this, in clinical samples of PC, the level of several genes encoding angiogenic/osteoclastogenic pathway effectors correlated with EGR1 levels. Thus, we show here that EGR1 has a direct effect on prostate cancer metastases. EGR1 regulates angiogenic and osteoclastogenic factors, informing the underlying signaling networks that impact autonomous and microenvironmental mechanisms of cancer metastases.