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BTG2 Loss and Mir-21 Upregulation Contribute to Prostate Cell Transformation by Inducing Luminal Markers Expression and Epithelial–Mesenchymal Transition

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BTG2 Loss and Mir-21 Upregulation Contribute to Prostate Cell Transformation by Inducing Luminal Markers Expression and Epithelial–Mesenchymal Transition Oncogene (2013) 32, 1843–1853 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE BTG2 loss and miR-21 upregulation contribute to prostate cell transformation by inducing luminal markers expression and epithelial–mesenchymal transition V Coppola1,6, M Musumeci1,6, M Patrizii1, A Cannistraci1, A Addario1, M Maugeri-Sacca` 2, M Biffoni1, F Francescangeli1, M Cordenonsi3, S Piccolo3, L Memeo4, A Pagliuca1, G Muto5, A Zeuner1, R De Maria2,6 and D Bonci1,6 Prostate cancer is one of the leading causes of cancer-related death in men. Despite significant advances in prostate cancer diagnosis and management, the molecular events involved in the transformation of normal prostate cells into cancer cells have not been fully understood. It is generally accepted that prostate cancer derives from the basal compartment while expressing luminal markers. We investigated whether downregulation of the basal protein B-cell translocation gene 2 (BTG2) is implicated in prostate cancer transformation and progression. Here we show that BTG2 loss can shift normal prostate basal cells towards luminal markers expression, a phenotype also accompanied by the appearance of epithelial–mesenchymal transition (EMT) traits. We also show that the overexpression of microRNA (miR)-21 suppresses BTG2 levels and promotes the acquisition of luminal markers and EMT in prostate cells. Furthermore, by using an innovative lentiviral vector able to compete with endogenous mRNA through the overexpression of the 30-untranslated region of BTG2, we demonstrate that in prostate tumor cells, the levels of luminal and EMT markers can be reduced by derepression of BTG2 from microRNA-mediated control. Finally, we show that the loss of BTG2 expression confers to non-tumorigenic prostate cells ability to grow in an orthotopic murine model, thus demonstrating the central role of BTG2 downregulaton in prostate cancer biology. Oncogene (2013) 32, 1843–1853; doi:10.1038/onc.2012.194; published online 21 May 2012 Keywords: prostate cancer; BTG2; microRNA; basal and luminal phenotype; EMT INTRODUCTION their epithelial characteristics, whereas acquiring a mesenchymal- Although much progress has been made in prostate cancer like phenotype associated with the acquisition of migratory and diagnosis and management over the last few decades, prostate invasive traits.12 The basal cell compartment is also a major site of tumors still represent one of the leading causes of cancer-related expression of the B-cell translocation gene 2 (BTG2).13 BTG2 is a death in men.1 The malignant transformation can occur through pan-cell cycle modulator and a major effector of p53-induced progressive acquisition of genetic and epigenetic lesions, coupled proliferation arrest that exerts its biological activity by inhibiting with increasing microenvironmental stimuli. However, early the expression of cyclin D1 and the phosphorylation of Rb.14–16 In molecular events underlying the oncogenic process in the addition, knockdown of BTG2 revealed its cooperation with the prostate remain largely unknown. Prostate acini are composed RAS oncogene in inducing cell transformation.17 The tumor- of a luminal compartment of differentiated cells surrounded by suppressive role of BTG2 is supported by its downregulation in an undifferentiated layer of basal cells. A transit-amplifying several tumors, including clear cell renal carcinoma,18 breast population composed of intermediate progenitors retaining cancer19 and prostate adenocarcinoma.20 both basal and luminal markers mirrors a continuum in the It is well established that post-transcriptional modifications differentiation program.2 Basal cells have been traditionally modulate gene expression. MicroRNAs (miRs) are small, non- considered the cells of origin of prostate cancer.3 However, how coding, single-stranded RNAs that suppress mRNA translation of cancer cells may both express luminal cell markers4,5 and have specific target genes.21 A compelling body of evidence indicates functional properties of basal cells, such as unlimited self-renewal that miRs control nearly all basic cell functions22 and that their capacity6 and activation of survival mechanisms, continues to deregulation is deeply implicated in tumor development and remain controversial.7 Unlike prostate cancer cells, the basal layer progression.23–25 miR-21 upregulation occurs in many cancers of normal prostate acini expresses considerable levels of DNp63a, including prostate tumors.26–28 It has also been correlated with an isoform of p63 involved in stemness maintenance and tumor aggressiveness, androgen-dependent and independent differentiation control during prostate organogenesis.8,9 In prostate cancer growth, and metastatic spread.29,30 Here we addition, DNp63 loss has been associated with enhanced demonstrate a novel role of miR-21 in driving prostate cells motility and epithelial–mesenchymal transition (EMT),10,11 a toward acquiring a malignant phenotype via the downregulation trans-differentiation process through which cancer cells lose of BTG2. 1Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita`, Rome, Italy; 2Regina Elena Cancer Institute, Rome, Italy; 3Department of Histology, Microbiology and Medical Biotechnologies, University of Padua School of Medicine, Padua, Italy; 4Department of Experimental Oncology, Mediterranean Institute of Oncology, Catania, Italy and 5Department of Urology, S. Giovanni Bosco Hospital, Turin, Italy. Correspondence: Professor R De Maria, Regina Elena Cancer Institute, Rome, Italy or Dr D Bonci, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita`, Rome, Italy. E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. Received 30 June 2011; revised and accepted 5 April 2012; published online 21 May 2012 BTG2 loss and miR-21 upregulation V Coppola et al 1844 RESULTS as a transcriptional regulator, we investigated whether p63 BTG2 is downregulated in prostate tumors and cancer cell lines reduction might result in similar features and potentially mediate We evaluated, by immunohistochemistry, the expression of BTG2 the effects of BTG2 downregulation. We therefore performed RNA in five specimens of prostate tumors and five normal prostate interference of p63 in RWPE-1 cells, observing a strong upregula- tissues. The analysis confirmed that BTG2 expression is confined to tion of luminal markers CK8-18 and AR (Supplementary Figure the basal cell layer of normal tissues (60–70% of cells), with a S2B). Therefore, BTG2 and p63 interference both result in the acquisition of a luminal phenotype. As BTG2 has been reported to prevalent staining of both nuclei and cytoplasm, and a minority of 17,35 either nuclear or cytoplasmatic localization. BTG2 staining was suppress RAS activity, we investigated whether the reduction negative in luminal cells of both normal prostate acini and tumor of p63 in BTG2-interfered cells could be because of the activation tissues (Figure 1a). However, in some cases, BTG2 was detected in of the ERK signaling. The concurrent interference of KRAS was indeed able to rescue p63 levels (Supplementary Figure S2C). p63 luminal cells of acini dispersed in tumor areas, as highlighted by 10 co-staining of BTG2 and cytokeratins 8 and 18 (CK8-18; Figure 1b). loss has also been associated with EMT, the process through To further corroborate the downregulation of BTG2 in prostate which epithelial cells acquire a more elongated, motile phenotype tumors, we evaluated its expression in primary cells isolated from associated with the expression of mesenchymal markers like five prostate tumors. Flow cytometry analysis confirmed BTG2 vimentin and fibronectin, and the loss of the epithelial adhesion downregulation in cancer cells as compared with normal primary molecule E-Cadherin. We verified this mechanism in prostate cells cells (Supplementary Figure S1A). We validated the specificity of by evaluating the levels of vimentin upon interference and our BTG2 staining by BTG2 immunoprecipitation and detection overexpression of p63. In RWPE-1 cells, RNA interference against with a non-commercial anti-BTG2 antibody31 (Supplementary all p63 and DNp63 isoforms resulted in upregulation of vimentin Figure S1B). To investigate the consequences of BTG2 down- (Supplementary Figure S2D). Conversely, in RWPE-2 cells, which regulation in prostate cancer, we evaluated BTG2 levels in our express higher levels of vimentin, the overexpression of DNp63 experimental model, comparing its expression in three established reduced this mesenchymal marker, whereas a TAp63-carrying prostate cell lines representing different phases of prostate plasmid did not (Supplementary Figure S2D). This suggests that tumorigenesis: (i) immortalized non-tumorigenic RWPE-1 cells, (ii) only DNp63 isoforms can control the expression of vimentin. We early tumoral RWPE-2 cells and (iii) metastasis-derived PC3 cells. therefore evaluated whether BTG2 interference might also result Cytofluorimetric analysis revealed a progressive reduction of BTG2 in EMT. Flow cytometry and immunofluorescence analyses expression in this series of cell lines (Figure 1c). Notably, in RWPE-1 showed mesenchymal markers induction in BTG2-interfered cells cells, BTG2 protein is strongly expressed in the nuclei of small (Figure 2d and Supplementary Figure S2E). Similarly, E-Cadherin round cells, while residing in the cytoplasm of larger cells staining underlined a partial disorganization of epithelial
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