Jayant K. Rane

MicroRNA Expression Profile of Primary Prostate Cancer Stem Cells as a Source of Biomarkers and Therapeutic Targets

UNLABELLED MicroRNA (miRNA) expression profiles were generated from prostate epithelial subpopulations enriched from patient-derived benign prostatic hyperplasia (n=5), Gleason 7 treatment-naive prostate cancer (PCa) (n=5), and castration-resistant PCa (CRPC) (n=3). Microarray expression was validated in an independent patient cohort (n=10). Principal component analysis showed that miRNA expression is clustered by epithelial cell phenotype, regardless of pathologic status. We also discovered concordance between the miRNA expression profiles of unfractionated epithelial cells from CRPCs, human embryonic stem cells (SCs), and prostate epithelial SCs (both benign and malignant). MiR-548c-3p was chosen as a candidate miRNA from this group to explore its usefulness as a CRPC biomarker and/or therapeutic target. Overexpression of miR-548c-3p was confirmed in SCs (fivefold, p<0.05) and in unfractionated CRPCs (1.8-fold, p<0.05). Enforced overexpression of miR-548c-3p in differentiated cells induced stemlike properties (p<0.01) and radioresistance (p<0.01). Reanalyses of published studies further revealed that miR-548c-3p is significantly overexpressed in CRPC (p<0.05) and is associated with poor recurrence-free survival (p<0.05), suggesting that miR-548c-3p is a functional biomarker for PCa aggressiveness. Our results validate the prognostic and therapeutic relevance of miRNAs for PCa management while demonstrating that resolving cell-type and differentiation-specific differences is essential to obtain clinically relevant miRNA expression profiles. PATIENT SUMMARY We report microRNA (miRNA) expression profiles of epithelial cell fractions from the human prostate, including stem cells. miR-548c-3p was revealed as a functional biomarker for prostate cancer progression. The evaluation of miR-548c-3p in a larger patient cohort should yield information on its clinical usefulness.

Advanced prostate cancer—a case for adjuvant differentiation therapy

The development of novel therapies such as abiraterone acetate and sipuleucel-T has improved the outlook for patients with advanced-stage and castration-resistant prostate cancer. However, the beneficial effects of these drugs are only measured in months. Moreover, the National Institute for Health and Clinical Excellence in the UK had ruled that the use of abiraterone acetate was not cost-effective before cost revision by the manufacturers. The FDA statement asserting that the use of 5α-reductase inhibitors for prostate cancer chemoprevention could increase the risk of developing high-grade prostate cancer also indirectly questions the value of direct androgen response manipulation for long-term benefit. These reports illustrate the need for a fresh and comprehensive analysis of advanced prostate cancer pathology to promote the next generation of effective adjuvant therapies. One such avenue is that of differentiation therapy, which seeks to promote the differentiation of cancer stem cells into a phenotype more sensitive to anticancer therapy than their parents. Using differentiation therapy with current antiandrogen therapies should augment our armoury of treatment for the management of advanced prostate cancer.

Telomerase Activity and Telomere Length in Human Benign Prostatic Hyperplasia Stem-like Cells and Their Progeny Implies the Existence of Distinct Basal and Luminal Cell Lineages

UNLABELLED Benign prostatic hyperplasia (BPH) treatments have changed little over many years and do not directly address the underlying cause. Because BPH is characterised by uncontrolled cell growth, the chromosomal telomeres should be eroded in the reported absence or low levels of telomerase activity, but this is not observed. We investigated the telomere biology of cell subpopulations from BPH patients undergoing transurethral resection of prostate (TURP). Measurement of TERC, TERT, and telomerase activity revealed that only the epithelial stem-like and progenitor fractions expressed high levels of telomerase activity (p<0.01) and individual enzyme components (p<0.01). Telomerase activity and TERT expression were not detected in stromal cells. Telomere length measurements reflected this activity, although the average telomere length of (telomerase-negative) luminal cells was equivalent to that of telomerase-expressing stem/progenitor cells. Immunohistochemical analysis of patient-derived BPH arrays identified distinct areas of luminal hyperproliferation, basal hyperproliferation, and basal-luminal hyperproliferation, suggesting that basal and luminal cells can proliferate independently of each other. We propose a separate lineage for the luminal and basal cell components in BPH. PATIENT SUMMARY We unexpectedly found an enzyme called telomerase in the cells that maintain benign prostatic hyperplasia (BPH), suggesting that telomerase inhibitors could be used to alleviate BPH symptoms.

Inhibition of the glucocorticoid receptor results in an enhanced miR-99a/100-mediated radiation response in stem-like cells from human prostate cancers

Radiation therapy is a major primary treatment option for both localized early stage prostate cancer, and for advanced, regionally un-resectable, cancer. However, around 30% of patients still experience biochemical recurrence after radiation therapy within 10 years. Thus, identification of better biomarkers and new targets are urgently required to improve current therapeutic strategies. The miR-99 family has been shown to play an important role in the regulation of the DNA damage response, via targeting of the SWI/SNF chromatin remodeling factors, SMARCA5 and SMARCD1 in cell line models. In the present study, we have demonstrated that low expression of miR-99a and miR-100 is present in cell populations which are relatively radiation insensitive, for example in prostate cancer stem cells and in castration-resistant prostate cancer. Additionally, treatment of cells with the synthetic glucocorticoid, Dexamethasone resulted in decreased miR-99a and 100 expression, suggesting a new mechanism of miR-99a and 100 regulation in androgen-independent prostate cells. Strikingly, treatment of prostate cells with the glucocorticoid receptor inhibitor, Mifepristone was found to sensitize prostate cells to radiation by increasing the levels of miR-99a and miR-100. These results qualify the miR99 family as markers of radiation sensitivity and as potential therapeutic targets to improve efficiency of radiotherapy.

Conserved Two-Step Regulatory Mechanism of Human Epithelial Differentiation

Summary Human epithelia are organized in a hierarchical structure, where stem cells generate terminally differentiated cells via intermediate progenitors. This two-step differentiation process is conserved in all tissues, but it is not known whether a common gene set contributes to its regulation. Here, we show that retinoic acid (RA) regulates early human prostate epithelial differentiation by activating a tightly coexpressed set of 80 genes (e.g., TMPRSS2). Response kinetics suggested that some of these genes could be direct RA targets, whereas others are probably responding indirectly to RA stimulation. Comparative bioinformatic analyses of published tissue-specific microarrays and a large-scale transcriptomic data set revealed that these 80 genes are not only RA responsive but also significantly coexpressed in many human cell systems. The same gene set preferentially responds to androgens during terminal prostate epithelial differentiation, implying a cell-type-dependent interplay between RA and tissue-specific transcription factor-mediated signaling in regulating the two steps of epithelial differentiation.

Construction of therapeutically relevant human prostate epithelial fate map by utilising miRNA and mRNA microarray expression data

Background:Objective identification of key miRNAs from transcriptomic data is difficult owing to the inherent inconsistencies within miRNA target-prediction algorithms and the promiscuous nature of miRNA-mRNA target relationship.Methods:An integrated database of miRNAs and their ‘relevant’ mRNA targets was generated from validated miRNA and mRNA microarray data sets generated from patient-derived prostate epithelial normal and cancer stem-like cells (SCs) and committed basal (CB) cells. The effect of miR-542-5p inhibition was studied to provide proof-of-principle for database utility.Results:Integration of miRNA-mRNA databases showed that signalling pathways and processes can be regulated by a single or relatively few miRNAs, for example, DNA repair/Notch pathway by miR-542-5p, P=0.008. Inhibition of miR-542-5p in CB cells (thereby achieving miR-542-5p expression levels similar to SCs) promoted efficient DNA repair and activated expression of Notch reporters, HES1 and Survivin, without inducing dedifferentiation into SCs.Conclusions:Our novel framework impartially identifies therapeutically relevant miRNA candidates from transcriptomic data sets.

Abstract 5198: Elucidation of dynamic prostate epithelial hierarchy: Insights into transcriptional and epigenetic regulatory mechanisms

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The aim of present investigation is to elucidate the complex stem cell dynamics within prostate cancer, which can be utilized to design novel diagnostic and therapeutic strategies for the management of prostate cancer. In order to determine precise transcriptional and microRNA regulatory mechanisms modulating stem cell self-renewal and differentiation, unique cellular assays have been developed in our lab that utilize homogeneous fractionated cell populations enriched from primary patient prostate cultures. Using a prospective bioinformatic analysis of gene expression data from Birnie et. al., 2008, we have identified LCN2, CEACAM6, and S100p as candidate genes for regulation of prostate stem cell differentiation. Their over-expression in differentiated cells, as compared to stem cells, was validated in respective cells enriched from cultures obtained from BPH, cancer and castration resistant prostate cancer samples and from primary human prostate cancer xenografts. Interestingly, the analysis of 25,000 published human Affymetrix microarray chips revealed that LCN2, CEACAM6, and S100p have a more similar expression pattern than that of any other genes in the entire human genome, suggesting that they may have common function and are co-regulated. Indeed, the promoter analysis showed that the promoters (1kb from TSS) of all these genes have common binding sites for 40 transcription factors with very high affinity and P < 0.001. Most of these transcription factors have a well-documented role in cell differentiation (RA, AR, and NANOG) and prostate carcinogenesis (NF-kB). Significant up-regulation in the expression of these genes in prostate cell lines after treatment with all-trans retinoic acid and androgen analogue R1881 further suggested the role of AR and RA in prostate differentiation. Along with transcriptional regulation, Agilent v3 miRNA microarray data revealed obviously distinct miRNA expression profiles in stem and differentiated prostate epithelial cells, confirming crucial role of miRNA in main taining epithelial hierarchies, in prostate. We anticipate that evaluation of integrative transcriptional (LCN2-CEACAM6-S100p)-microRNA regulatory network, with further functional studies, will comprehensively establish a detailed knowledge base for potential regulatory mechanisms involved in prostate stem cell and prostate cancer stem cell differentiation. These insights will be valuable to formulate efficient ‘differentiation therapy’ for the management of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5198. doi:1538-7445.AM2012-5198

A detailed analysis of gene expression in human basal, luminal, and stromal cell populations from benign prostatic hyperplasia tissues and comparisons with cultured basal cells

The understanding of common prostatic disorders has been restricted by both cellular heterogeneity and the scarcity of established cell lines, although organoid technology, based on primary cultures, promises much for the future. In particular, little is known about the aetiology of benign prostatic hyperplasia (BPH), for which culture of single cell types might not accurately not reflect the stromal and epithelial overgrowths observed in tissues [1]. To address the applicability of primary cell culture models of prostate disease, we compared cell type–specific mRNA expression patterns in BPH tissues and primary basal cells cultured from the same transurethral biopsies. We resolved cellular heterogeneity by direct tissue fractionation into luminal cells (Lin /CD31 /EpCAM/CD24), basal cells (Lin /CD31 / EpCAM/CD44), and stromal cells (Lin /CD31 /EpCAM ) obtained from three patient samples before analysis using GeneChip 2.0 human transcriptomic arrays (Affymetrix, Santa Clara, CA, USA) [1,2]. Unsupervised clustering and principal component analysis (Fig. 1A) of the microarray data revealed that subpopulations clustered together in a cell type–specific manner. However, even short-term culture in a defined medium induced significant transcriptomic changes in basal cells. This was confirmed by quantitative real-time polymerase chain reaction analysis for representative genes (Fig. 1B). We next sought to put the principal differences in gene expression between cell populations into a disease context using gene set enrichment analysis. When nonmalignant BPH basal cells were grown in a defined, serum-free culture medium previously shown to amplify skin epithelial stem cells, differential overexpression of gene sets associated with (1) stemness, (2) aggressive breast and prostate cancers, and (3) STAT5 signalling were observed. These results support the findings from our own and other laboratories that

Abstract 3053: Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA There is increasing evidence that the phenotype of heterogeneous prostate tumors can be determined to some extent by reversible (non-mutagenic/epigenetic) genetic mechanisms. In the complex microenvironment of a developing cancer, this is an excellent survival strategy. The target cell (and molecular targets) for primary therapeutic intervention in prostate cancer is frequently determined by hormone responsiveness. We have shown that, in cells fractionated from human cancers (relative to cell lines or multiply passaged xenografts), the human prostate cancer-inducing cell (TIC) is a primitive, more basal-like cell with stem cell characteristics and limited hormone sensitivity. The TICs, and indeed normal tissue stem cells, have proven remarkably resistant to both radiotherapy and chemotherapy, as a result of their unique stem cell-like chromatin configuration, which can be ‘unlocked’ using common epigenetic control disruptors (e.g. HDAC inhibition), resulting in differentiation and the enhancement of therapeutic efficacy. This result prompted us to explore the nature of epigenetic control in multiple cellular fractions of both basal and luminal cells purified from established prostate cell lines, primary tumor cultures, primary xenografts of prostate cancer and small directed biopsies of freshly biopsied prostate tumor tissues. The data indicate a remarkable difference between multiple ‘standard’ cell line models of prostate cancer and cell types from near-patient models. Chromatin status in terms of histone modification, genomic methylation patterns, microRNA expression are all cell-type specific, in contrast to the variable data reported in extracts of unfractionated human tumor biopsies, reflecting tissue heterogeneity. The TIC fraction has retained (or acquired) the bivalent chromatin of embryonic stem cells in a fully undifferentiated state. Dominant epigenetic signals are associated with cellular differentiation and maintenance of the proliferating luminal phenotype, which can mask underlying changes responsible for tumor induction, stem cell maintenance and metastatic capability. Therapeutic targeting of the former simply shrinks tumors without affecting metastatic induction and patient survival. Unlinked clusters of coregulated genes define the differentiation process, and contain novel molecular targets such as stem cell maintenance genes, which could render all cells in a cancer susceptible to current therapies as part of a combination treatment strategy. The lower concentrations of stem cell modifying agents (e.g. retinoic acid and HDAC inhibitors), when used as phenotype modifiers rather than toxins, in combination with more established (cytotoxic) therapies, are more likely to be well tolerated in patients, compared to the toxic clinical doses of current ‘differentiating’ agents. Citation Format: Norman J. Maitland, Davide Pellacani, Jayant K. Rane, Fiona M. Frame, Anne T. Collins. Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2014-3053

Abstract 1726: Androgenic regulation of the anti-apoptotic Bcl-2 family member Mcl-1 in prostate cancer cells.

Anti-apoptotic proteins of the Bcl-2 family are known to confer resistance against a variety of therapeutic agents. Among them is myeloid leukemia cell differentiation protein (Mcl-1), a highly regulated protein at both transcriptional and post-translational levels by a number of signaling pathways. Overexpression of Mcl-1 has been reported in biopsies of prostate cancer patients with high Gleason grades. Currently, novel anti-cancer approaches that aim to inhibit Mcl-1 are being developed. In this study we have analyzed the influence of androgens and androgen receptor (AR) signaling on Mcl-1 protein expression. Western Blot experiments with androgen-sensitive prostate cancer cell lines LNCaP and VCaP showed a consistent upregulation of Mcl-1 protein expression levels under androgen-depleted conditions, i.e. in steroid-free medium. Addition of 0.1-10 nM of the synthetic androgen R1881 decreased Mcl-1 expression in a concentration-dependent manner in LNCaP and VCaP cells. This effect could also be observed in the castration-resistant LNCaP-abl cell line, but not in AR-negative PC3 cells. Further analysis showed that regulation of Mcl-1 was dependent on the presence of functional AR since it could not be observed in cells co-treated with R1881 and antiandrogens bicalutamide or hydroxyflutamide or after AR downregulation by siRNA. Quantitative PCR on mRNA levels revealed that Mcl-1 regulation by androgens is at a transcriptional level, while downregulation of the three known Mcl-1 E3 ligases APC/C(CDC20), SCF(FBW7), and HUWE1 (MULE) by siRNA did not influence androgenic regulation of Mcl-1 at the post-translational level. To evaluate a possible role of Mcl-1 as a resistance mechanism in cancer stem cells, its expression in basal cells isolated from patient9s biopsies was assessed. The highest expression of Mcl-1 mRNA was observed in tumors isolated from castration therapy resistant patients, followed by therapy naive samples and was the lowest in prostatic cell lines. Interestingly, after isolation of stem cells (SC), transit amplifying (TA) and committed basal (CB) cells, the highest expression of Mcl-1 mRNA could be detected in SC. Our findings indicate that androgenic signaling leads to decreased Mcl-1 expression levels in prostate cancer cell lines, which could be mediated by E2F1 transcription factor known to inhibit Mcl-1 transcription. Vice versa, absent AR signaling leads to upregulated Mcl-1 expression levels. The finding of high Mcl-1 mRNA expression in prostate cancer stem cells that do not express AR supports the hypothesis that Mcl-1 could be involved in resistance mechanisms against first- and second-line therapies. Therefore, anti-Mcl-1 therapies might be considered to improve the efficacy of androgen ablation and could furthermore target prostate cancer stem cells. Citation Format: Frederic R. Santer, Holger H. Erb, Birgit Luef, Florian Handle, Su Jung Oh, Susanne Lobenwein, Christian Ploner, Jayant Rane, Anne T. Collins, Norman J. Maitland, Zoran Culig. Androgenic regulation of the anti-apoptotic Bcl-2 family member Mcl-1 in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2013-1726