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RASAL2, a RAS Gtpase-Activating Protein, Inhibits Stemness and Epithelial&Ndash;Mesenchymal Transition Via MAPK&Sol;SOX2

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RASAL2, a RAS Gtpase-Activating Protein, Inhibits Stemness and Epithelial&Ndash;Mesenchymal Transition Via MAPK&Sol;SOX2 Citation: Cell Death and Disease (2017) 8, e2600; doi:10.1038/cddis.2017.9 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddis RASAL2, a RAS GTPase-activating protein, inhibits stemness and epithelial–mesenchymal transition via MAPK/SOX2 pathway in bladder cancer Ke Hui1,4, Yang Gao2,4, Jun Huang1, Shan Xu1, Bin Wang1, Jin Zeng1, Jinhai Fan1, Xinyang Wang1, Yangyang Yue1, Shiqi Wu1, Jer-Tsong Hsieh3, Dalin He*,1 and Kaijie Wu*,1 Muscle-invasive or metastatic bladder cancer (BCa) is associated with a very poor prognosis, and the underlying mechanism remains poorly understood. In this study, we demonstrate RASAL2, a RAS GTPase-activating protein (RAS GAP), acts as a tumor suppressor in BCa. First, RASAL2 was downregulated in BCa specimens and inversely correlated with pathological grades and clinical stages. Furthermore, we observed that RASAL2 could inhibit BCa stemness and epithelial–mesenchymal transition (EMT) based on our gain-of-function and loss-of-function experiments. Mechanistically, we found that mitogen-activated protein kinase/ SOX2 signaling had a critical role for maintaining the stemness and mesenchymal properties of RASAL2-deficient BCa cells because inhibition of ERK activity or knockdown of SOX2 could reverse these phenotypes. Also, RASAL2 could inhibit BCa tumorigenesis and distant metastasis in vivo. Moreover, there was an inverse correlation between RASAL2 expression and the stemness/EMT status in subcutaneous xenograft and human BCa specimens. Taken together, our data indicate that RASAL2 is a tumor suppressor in BCa, and modulates cancer stemness and EMT for BCa recurrence and metastasis. Cell Death and Disease (2017) 8, e2600; doi:10.1038/cddis.2017.9; published online 9 February 2017 Bladder cancer (BCa) is one of the most common urological inactivate RAS.12,13 Several preliminary studies have reported cancers worldwide, and most of BCa are transitional cell the aberrant expression of RASAL2 in different cancer types, carcinoma.1 More than 70% cases are non-muscle-invasive including breast cancer, ovarian cancer, lung cancer and BCa (NMIBC), which can be treated with transurethral nasopharyngeal carcinoma.14–18 However, its tumor-suppres- resection. However, 450% cases will relapse and 10–30% sive or oncogenic roles in cancer development remain cases will progress into muscle-invasive BCa (MIBC), and controversial,16,18 especially, its expression and function in finally result in distant metastasis and poor prognosis of BCa are completely unknown. In this study, we will determine patients.2 So it is urgent to understand the molecular the dysregulation of RASAL2 expression in BCa specimens, mechanisms of BCa recurrence and metastasis, and explore and explore its unique roles in modulating stemness and EMT novel therapeutic targets to improve patient survival. phenotypes of BCa. Nowadays, cancer stemness has been identified in many cancer types. Cancer stem cells (CSCs) share some proper- ties with somatic stem cells, including self-renewal and multi- Results potent differentiation potential.3,4 Previous studies have revealed that cancer stemness is responsible for tumorigeni- RASAL2 is downregulated in human BCa tissues and city, therapeutic resistance, relapse and metastasis in BCa.5,6 inversely correlated with tumor grades and clinical Also, accumulating evidences suggest that epithelial– stages. To investigate the expression pattern of RASAL2 in mesenchymal transition (EMT) has an important role in the BCa tissues, we performed immunohistochemical staining enrichment of cells with CSC properties, which are believed to in our cohort including 132 cases of BCa and 10 cases of be the origin of cancer progression.7,8 Importantly, recent normal bladder epithelium specimens (Table 1). As shown in whole-genome studies have discovered the intrinsic basal and Figures 1a, b and e, we found that the expression of RASAL2 luminal MIBC subtypes associated with different chemother- protein was not only lower in BCa tissues than normal apy sensitivity or patient prognosis, in which a comprehensive epithelia, but also negatively correlated with tumor grades molecular alteration involving CSC and EMT has been well (*Po0.05, **Po0.01). Moreover, the expression of RASAL2 in characterized.9–11 All these findings provide new insights MIBC tissues was significantly lower than that in NMIBC about invasion and metastasis of BCa. tissues (Figures 1a, c and e, *Po0.05). Furthermore, we found RASAL2 is a member of RAS GTPase-activating proteins that RASAL2 expression in high-grade NMIBC tissues are (RAS-GAPs), which can catalyze GTP into GDP and lower than that in low-grade NMIBC tissues (Figures 1d and e, 1Department of Urology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China; 2Department of Urology, Shaanxi Provincial People's Hospital, Xi'an 710068, China and 3Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA *Corresponding author: Dr D He or Dr K Wu, Department of Urology, The First Affiliated Hospital of Xi'an Jiaotong University, 277, YantaWest Road, Xi'an, Shaanxi 710061, China. Tel: +86 15389299128; Fax: +86 2985323203; E-mail: [email protected] or [email protected]. 4These authors contributed equally to this work. Received 04.7.16; revised 15.12.16; accepted 22.12.16; Edited by M Agostini RASAL2 inhibits BCa stemness and EMT K Hui et al 2 Table 1 Distribution by tumor characteristics for our patients with bladder *Po0.05). Also, we analyzed RASAL2 expression in another cancer cohort of BCa tissues profiled by microarray from NCBI GEO. Mean age at diagnosis (years) 68.6 Consistently, we further observed that RASAL2 mRNA was Gender (n) downregulated in human BCa tissues, and there was a Male 108 negative correlation between RASAL2 and BCa grades and Female 24 stages (Supplementary Figure 1A, *Po0.05, ***Po0.001), Stage (n) indicating that RASAL2 loss may be associated with BCa Ta–166progression. T2–466 Grade (n) PUNLMP 11 RASAL2 modulates the stemness properties of BCa Low grade 40 cells. Cancer stemness is one of the hotspot mechanisms High grade 81 leading to BCa development.5 Therefore, we applied both Total (n) 132 gain-of-function and loss-of-function strategies to further Figure 1 Expression of RASAL2 in BCa tissues. (a) Representative pictures of IHC staining of RASAL2 in normal urothelium, papillary urothelial neoplasm of low malignant potential (PUNLMP), low grade/NMIBC and high grade/MIBC. The scale bar represents 100 μm. (b and c) Quantification and percentage analysis of RASAL2 staining in normal urothelium and BCa tissues with different grades (b) and stages (c) were shown. Normal urothelium (NU, n = 10), PUNLMP (LMP, n = 11), low-grade BCa (LG, n = 40), high- grade BCa (HG, n = 81), NMIBC (n = 66) and MIBC (n = 66). (d) Quantification analysis of RASAL2 staining in NMIBC tissues with different grades. (e) Heatmap for IHC of RASAL2 staining in BCa tissues with different grades and stages. *Po0.05, **Po0.01 Cell Death and Disease RASAL2 inhibits BCa stemness and EMT K Hui et al 3 Figure 2 RASAL2 suppresses stemness in BCa cells. (a) Quantitative real-time RT-PCR and western blotting analysis of RASAL2 levels in human BCa cell lines. (b) Quantitative real-time RT-PCRand western blotting analysis of RASAL2 levels in 5637 cells transfected with RASAL2 shRNAs and scramble shRNA, or T24 cells infected with RASAL2 lentivirus and scramble control. (c) Tumorsphere assay of 5637 cells transfected with RASAL2 shRNAs and scramble shRNA, or T24 cells infected with RASAL2 lentivirus and scramble control. The tumorsphere number was counted and plotted, and percentage of tumorspheres with diameters o50 μm, 50–100 μmor4100 μmwas calculated and plotted. The scale bar represents 100 μm. (d) Colony formation assay of 5637 cells transfected with RASAL2 shRNAs and scramble shRNA, or T24 cells infected with RASAL2 lentivirus and scramble control. The colonies number was counted and plotted. (e) Western blotting analysis of RASAL2, CD44 and SOX2 in 5637 cells transfected with RASAL2 shRNAs and scramble shRNA, or T24 cells infected with RASAL2 lentivirus and scramble control. GAPDH was used as internal loading control. *Po0.05, **Po0.01 explore the roles of RASAL2 in BCa stemness. As screening RASAL2 modulates EMT in BCa cells. Current studies in Figure 2a by quantitative RT-PCR and western blotting have reported that cancer stemness and EMT always appear assays, RASAL2 is highly expressed in SVHUC-1, an coincidently, and EMTendows cells with a more CSC-like and immortalized normal bladder epithelial cell line, whereas a mesenchymal phenotype.19 Indeed, 5637 cells revealed a relatively lower expression was detected in all BCa cell lines. cobblestone-like morphology; however, RASAL2-deficient Fortunately, we successfully established the stable 5637 5637 cells displayed an elongated fibroblastoid appearance, sublines with endogenous RASAL2 knockdown and T24 as well as increased cell migration and invasion in vitro sublines with ectopic RASAL2 overexpression (Figure 2b). (Figures 3a, **Po0.01, ***Po0.001). Also, western blotting Indeed, we noticed that knockdown of RASAL2 expression in results showed that epithelial marker (i.e., E-cadherin) 5637 cell lines enhanced the ability of tumorsphere and decreased but mesenchymal markers (i.e., vimentin, ZEB1) colony formation, meanwhile overexpression of RASAL2 in increased in RASAL2 knockdown 5637 cells compared T24 cells could decrease this ability (Figures 2c and d, with control (Figure 3b). In contrast, RASAL2 overexpression *Po0.05, **Po0.01). Consistently, western blotting data in T24 cells reversed EMT, and inhibited cell migration clearly showed that levels of potential stemness markers o o SOX2 and CD44 protein increased in 5637 cells after and invasion (Figures 3c and d, **P 0.01, **P 0.001). RASAL2 knockdown, however, ectopic RASAL2 expression Similar results were shown in 253J and 253J-BV cells could downregulate SOX2 and CD44 expression in T24 cells (Supplementary Figure 2). These data suggest that RASAL2 (Figure 2e). These data indicate that RASAL2 loss could could suppress BCa migration and invasion through potentiate CSC phenotype of BCa cells.
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