Domenico Albino

ESE3/EHF Controls Epithelial Cell Differentiation and Its Loss Leads to Prostate Tumors with Mesenchymal and Stem-like Features

Cancer stem cells (CSC) play a significant role in tumor progression, disease recurrence, and treatment failure. Here, we show that the endogenously expressed ETS transcription factor ESE3/EHF controls prostate epithelial cell differentiation and stem-like potential. We found that loss of ESE3/EHF induced epithelial-to-mesenchymal transition (EMT), stem-like features, and tumor-initiating and metastatic properties in prostate epithelial cells, and reexpression of ESE3/EHF inhibited the stem-like properties and tumorigenic potential of prostate cancer cells. Mechanistically, ESE3/EHF repressed the expression of key EMT and CSC genes, including TWIST1, ZEB2, BMI1, and POU5F1. Analysis of human tissue microarrays showed that reduced ESE3/EHF expression is an early event in tumorigenesis, frequently occurring independently of other ETS gene alterations. Additional analyses linked loss of ESE3/EHF expression to a distinct group of prostate tumors with distinctive molecular and biologic characteristics, including increased expression of EMT and CSC genes. Low ESE3/EHF expression was also associated with increased biochemical recurrence of prostate cancer and reduced overall survival after prostatectomy. Collectively, our findings define a key role for ESE3/EHF in the development of a subset of prostate tumors and highlight the clinical importance of identifying molecularly defined tumor subgroups.

RNAi-mediated silencing of Myc transcription inhibits stem-like cell maintenance and tumorigenicity in prostate cancer

Several studies link disease progression, recurrence, and treatment failures to the cancer stem-like cell (CSC) subpopulation within the heterogeneous tumor cell population. Myc is a transcription factor having a central function in stem cell biology and in human cancers. Hence, Myc represents an attractive target to develop CSC-specific therapies. Recent findings suggest that Myc transcription can be silenced using an RNA interference (RNAi)-based strategy that targets noncoding promoter-associated RNA (paRNA) overlapping the transcription start site. In this study, we investigated the effects of silencing Myc transcription on prostate CSC in cell culture and xenograft models of human prostate cancer. Treatment with an effective promoter-targeting siRNA reduced the fraction of CSCs, leading to reduced self-renewal, tumor-initiating, and metastatic capability. Combined analysis of stem-like cells and senescence markers indicated that Myc silencing triggered a phenotypic shift and senescence in the CSC subpopulation. Notably, systemic delivery of the promoter-targeting siRNA in the xenograft model produced a striking suppression in the development of prostate tumors. Our results support a pivotal role for Myc in CSC maintenance and show that Myc targeting via RNAi-based transcriptional silencing can trigger CSC senescence and loss of their tumor-initiating capability. More generally, our findings demonstrate the efficacy of RNAi-based transcriptional strategies and the potential to target regulatory noncoding paRNAs for therapeutic applications.

ETS transcription factor ESE1/ELF3 orchestrates a positive feedback loop that constitutively activates NF-κB and drives prostate cancer progression

Chromosomal translocations leading to deregulated expression of ETS transcription factors are frequent in prostate tumors. Here, we report a novel mechanism leading to oncogenic activation of the ETS factor ESE1/ELF3 in prostate tumors. ESE1/ELF3 was overexpressed in human primary and metastatic tumors. It mediated transforming phenotypes in vitro and in vivo and induced an inflammatory transcriptome with changes in relevant oncogenic pathways. ESE1/ELF3 was induced by interleukin (IL)-1β through NF-κB and was a crucial mediator of the phenotypic and transcriptional changes induced by IL-1β in prostate cancer cells. This linkage was mediated by interaction of ESE1/ELF3 with the NF-κB subunits p65 and p50, acting by enhancing their nuclear translocation and transcriptional activity and by inducing p50 transcription. Supporting these findings, gene expression profiling revealed an enrichment of NF-κB effector functions in prostate cancer cells or tumors expressing high levels of ESE1/ELF3. We observed concordant upregulation of ESE1/ELF3 and NF-κB in human prostate tumors that was associated with adverse prognosis. Collectively, our results define an important new mechanistic link between inflammatory signaling and the progression of prostate cancer.

Activation of the Lin28/let-7 Axis by Loss of ESE3/EHF Promotes a Tumorigenic and Stem-like Phenotype in Prostate Cancer

Although cancer stem-like cells (CSC) are thought to be the most tumorigenic, metastatic, and therapy-resistant cell subpopulation within human tumors, current therapies target bulk tumor cells while tending to spare CSC. In seeking to understand mechanisms needed to acquire and maintain a CSC phenotype in prostate cancer, we investigated connections between the ETS transcription factor ESE3/EHF, the Lin28/let-7 microRNA axis, and the CSC subpopulation in this malignancy. In normal cells, we found that ESE3/EHF bound and repressed promoters for the Lin28A and Lin28B genes while activating transcription and maturation of the let-7 microRNAs. In cancer cells, reduced expression of ESE3/EHF upregulated Lin28A and Lin28B and downregulated the let-7 microRNAs. Notably, we found that deregulation of the Lin28/let-7 axis with reduced production of let-7 microRNAs was critical for cell transformation and expansion of prostate CSC. Moreover, targeting Lin28A/Lin28B in cell lines and tumor xenografts mimicked the effects of ESE3/EHF and restrained tumor-initiating and self-renewal properties of prostate CSC both in vitro and in vivo These results establish that tight control by ESE3/EHF over the Lin28/let-7 axis is a critical barrier to malignant transformation, and they also suggest new strategies to antagonize CSC in human prostate cancer for therapeutic purposes. Cancer Res; 76(12); 3629-43. ©2016 AACR.

EC-70124, a Novel Glycosylated Indolocarbazole Multikinase Inhibitor, Reverts Tumorigenic and Stem Cell Properties in Prostate Cancer by Inhibiting STAT3 and NF-κB

Cancer stem cells (CSC) contribute to disease progression and treatment failure in prostate cancer because of their intrinsic resistance to current therapies. The transcription factors NF-κB and STAT3 are frequently activated in advanced prostate cancer and sustain expansion of prostate CSCs. EC-70124 is a novel chimeric indolocarbazole compound generated by metabolic engineering of the biosynthetic pathways of glycosylated indolocarbazoles, such as staurosporine and rebeccamycin. In vitro kinome analyses revealed that EC-70124 acted as a multikinase inhibitor with potent activity against IKKβ and JAK2. In this study, we show that EC-70124 blocked concomitantly NF-κB and STAT3 in prostate cancer cells and particularly prostate CSCs, which exhibited overactivation of these transcription factors. Phosphorylation of IkB and STAT3 (Tyr705), the immediate targets of IKKβ and JAK2, respectively, was rapidly inhibited in vitro by EC-70124 at concentrations that were well below plasma levels in mice. Furthermore, the drug blocked activation of NF-κB and STAT3 reporters and suppressed transcription of their target genes. Treatment with EC-70124 impaired proliferation and colony formation in vitro and delayed development of prostate tumor xenografts. Notably, EC-70124 had profound effects on the prostate CSC subpopulation both in vitro and in vivo. Thus, EC-70124 is a potent inhibitor of the NF-κB and STAT3 signaling pathways and blocked tumor growth and maintenance of prostate CSCs. EC-70124 may provide the basis for developing new therapeutic strategies that combine agents directed to the CSC component and the bulk tumor cell population for treatment of advanced prostate cancer. Mol Cancer Ther; 15(5); 806–18. ©2016 AACR.

MicroRNA-424 impairs ubiquitination to activate STAT3 and promote prostate tumor progression

Mutations and deletions in components of ubiquitin ligase complexes that lead to alterations in protein turnover are important mechanisms in driving tumorigenesis. Here we describe an alternative mechanism involving upregulation of the microRNA miR-424 that leads to impaired ubiquitination and degradation of oncogenic transcription factors in prostate cancers. We found that miR-424 targets the E3 ubiquitin ligase COP1 and identified STAT3 as a key substrate of COP1 in promoting tumorigenic and cancer stem-like properties in prostate epithelial cells. Altered protein turnover due to impaired COP1 function led to accumulation and enhanced basal and cytokine-induced activity of STAT3. We further determined that loss of the ETS factor ESE3/EHF is the initial event that triggers the deregulation of the miR-424/COP1/STAT3 axis. COP1 silencing and STAT3 activation were effectively reverted by blocking of miR-424, suggesting a possible strategy to attack this key node of tumorigenesis in ESE3/EHF-deficient tumors. These results establish miR-424 as an oncogenic effector linked to noncanonical activation of STAT3 and as a potential therapeutic target.

The ETS factor ESE3/EHF represses IL-6 preventing STAT3 activation and expansion of the prostate cancer stem-like compartment

Metastatic prostate cancer represents a yet unsolved clinical problem due to the high frequency of relapse and treatment resistance. Understanding the pathways that lead to prostate cancer progression is an important task to prevent this deadly disease. The ETS transcription factor ESE3/EHF has an important role in differentiation of human prostate epithelial cells. Loss of ESE3/EHF in prostate epithelial cells determines transformation, epithelial-to-mesenchymal transition (EMT) and acquisition of stem-like properties. In this study we identify IL-6 as a direct target of ESE3/EHF that is activated in prostate epithelial cells upon loss of ESE3/EHF. ESE3/EHF and IL-6 were significantly inversely correlated in prostate tumors. Chromatin immunoprecipitation confirmed binding of ESE3/EHF to a novel ETS binding site in the IL-6 gene promoter. Inhibition of IL-6 reverted transformation and stem-like phenotype in tumorigenic ESE3/EHF knockdown prostate epithelial cell models. Conversely, IL-6 stimulation induced malignant phenotypes, stem-like behavior and STAT3 activation. Increased level of IL-6 was observed in prostatospheres compared with adherent bulk cancer cells and this was associated with stronger activation of STAT3. Human prostate tumors with IL-6 elevation and loss of ESE3/EHF were associated with STAT3 activation and displayed upregulation of genes related to cell adhesion, cancer stem-like and metastatic spread. Pharmacological inhibition of IL-6/STAT3 activation by a JAK inhibitor restrained cancer stem cell growth in vitro and inhibited self-renewal in vivo. This study identifies a novel connection between the transcription factor ESE3/EHF and the IL-6/JAK/STAT3 pathway and suggests that targeting this axis might be preferentially beneficial in tumors with loss of ESE3/EHF.

A promoter-proximal transcript targeted by genetic polymorphism controls E-cadherin silencing in human cancers

Long noncoding RNAs are emerging players in the epigenetic machinery with key roles in development and diseases. Here we uncover a complex network comprising a promoter-associated noncoding RNA (paRNA), microRNA and epigenetic regulators that controls transcription of the tumour suppressor E-cadherin in epithelial cancers. E-cadherin silencing relies on the formation of a complex between the paRNA and microRNA-guided Argonaute 1 that, together, recruit SUV39H1 and induce repressive chromatin modifications in the gene promoter. A single nucleotide polymorphism (rs16260) linked to increased cancer risk alters the secondary structure of the paRNA, with the risk allele facilitating the assembly of the microRNA-guided Argonaute 1 complex and gene silencing. Collectively, these data demonstrate the role of a paRNA in E-cadherin regulation and the impact of a noncoding genetic variant on its function. Deregulation of paRNA-based epigenetic networks may contribute to cancer and other diseases making them promising targets for drug discovery.

Abstract 2625: Targeting prostate cancer stem cells (CSCs) with the novel BET bromodomain (BRD) protein inhibitor OTX015

In human neoplasias small subpopulations of cancer cells with stem cell-like properties are thought to play a role in progression, metastasis, disease recurrence and treatment failure. Effective targeting of CSCs may thus provide the basis for a paradigm shift in cancer therapy. Epigenetic and transcriptional regulators such as BRD proteins (BRD2/3/4) may contribute to the CSC phenotype and as such represent druggable targets for CSC-directed therapies. No data are currently available on the anti-CSC activity of BRD protein inhibitors. OTX015 is an orally bioavailable small molecule BRD protein inhibitor. It demonstrates broad anticancer activity in vitro and is currently under clinical evaluation in hematologic malignancies and solid tumor patients, including castrate-resistant prostate cancer. We investigated the effects of OTX015 on the phenotypes of bulk tumor cells and CSCs in a panel of human prostate cancer cell lines, including the androgen receptor positive (LNCaP, VCaP and 22RV1) and negative (DU145 and PC3) prostate cell lines. Similar levels of BRD proteins were present in the cell lines independent of androgen receptor (AR) status and the presence of ETS gene fusions. OTX015 strongly inhibited proliferation in all cell lines tested (IC50, 200-800 nM) by MTT assays after 72h-exposure. Soft agar clonogenic capability of prostate cancer cells was also inhibited by OTX015 (IC50, 20-100 nM). OTX015 also potently inhibited the CSC component in prostato-sphere forming assays (IC50, 1-100 nM) after 10 days of treatment. Of note, the CSC-directed activity of OTX015 was independent of the AR and ETS translocation state. Effective inhibition of CSCs by OTX015 was consistently associated with downregulation of critical CSC genes, including c-Myc and Nanog mRNA. In vivo experiments in 5 human tumor xenografts models (LNCaP, VCaP, 22RV1, DU145 and PC3) in nude mice are ongoing to evaluate antitumor and anti-CSC activity of OTX015. To our knowledge, this is the first evidence implicating BRD proteins in the expansion and maintenance of prostate CSCs independently of the specific biologic and genetic features of the bulk tumor cell population. These results suggest that the BRD protein inhibitor OTX015 could be effective for eradicating the CSC component in prostate cancer, providing the basis for novel treatment approaches. Citation Format: Gianluca Civenni, Silvia Pedrani, Sara Allegrini, Antonina Bruccoleri, Domenico Albino, Sandra Pinton, Ramon Garcia-Escudero, L9Houcine Ouafik, Esteban Cvitkovic, Giuseppina M. Carbone, Carlo V. Catapano. Targeting prostate cancer stem cells (CSCs) with the novel BET bromodomain (BRD) protein inhibitor OTX015. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2625. doi:10.1158/1538-7445.AM2015-2625

Abstract LB-201: ERG lysine methylation promotes prostate cancer progression in ERG transgenic mice

The TMPRSS2-ERG gene fusion occurs frequently in prostate cancers and leads to over-expression of the ETS transcription factor ERG. We have recently described a novel mechanism cooperating with ERG fusion and enhancing ERG oncogenic activity. We found that the protein methyltransferase Enhancer of zest homolog 2 (EZH2) interacts with ERG and catalyzes methylation of a specific lysine residue in the ERG DNA binding domain. Lysine methylation of ERG alters intra-domain dynamics leading to increased chromatin binding and transcriptional activity. These events result in the formation of ERG/EZH2 co-activator complexes on selected gene promoters and enhancers and in broad transcriptional reprogramming in prostate epithelial cells. In this study we examined whether ERG methylation and ERG/EZH2 crosstalk were associated with ERG-driven tumor progression in genetically engineered mouse models represented by mice with prostate-specific expression of ERG (Pb-Cre4; Rosa26ERG/ERG) and mice with combined prostate-specific expression of ERG and deletion of PTEN (Pb-Cre4; Ptenflox/flox; Rosa26ERG/ERG). Only the combined ERG/PTEN mice exhibit progressive disease and develop invasive adenocarcinomas, whereas ERG mice fail to do so. We detected ERG methylation exclusively in ERG/PTEN mice. Enhanced methylation was linked to increased expression and AKT-induced phosphorylation of EZH2 at Serine 21 (pS21). Consistently, we observed higher promoter occupancy by ERG/EZH2 complexes and increased expression of selected ERG/EZH2 co-regulated genes in ERG/PTEN mice. Thus, enhanced ERG methylation and EZH2 activation occur in mice with combined ERG gain and PTEN loss and are concomitant with the emergence of an invasive phenotype. Systemic treatment with pharmacological inhibitors of EZH2, such as GSK343 blocked ERG methylation and expression of ERG/EZH2 co-regulated genes in ERG/PTEN mice. Moreover, GSK343 significantly reduced prostate volume, Ki67 immuno-staining and areas of invasive adenocarcinomas compared to control mice. Relevantly, we found preferential upregulation of ERG/EZH2 co-regulated genes in human prostate cancers exhibiting combined ERG over-expression and PTEN loss. These data establish the association of ERG methylation with enhanced ERG oncogenic activity and provide mechanistic insights into the synergy between ERG gain and PTEN loss in human tumors. Furthermore, these results establish the efficacy of EZH2 inhibitors in antagonizing ERG oncogenic activity in the ERG/PTEN model providing a strong rationale for developing new therapeutic strategies for the management of ERG fusion positive prostate cancers. Citation Format: Marita Zoma, Dheeraj Shinde, Domenico Albino, Simone Mosole, Jacopo Sgrignani, Andrea Cavalli, Carlo V. Catapano, Giuseppina M. Carbone. ERG lysine methylation promotes prostate cancer progression in ERG transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-201.