CLCA4 Inhibits Bladder Cancer Cell Proliferation, Migration, and Invasion by Suppressing the PI3K/AKT Pathway

www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 54), pp: 93001-93013 Research Paper CLCA4 inhibits bladder cancer cell proliferation, migration, and invasion by suppressing the PI3K/AKT pathway Teng Hou1,*, Lijie Zhou1,*, Longwang Wang1,2, Gallina Kazobinka1, Xiaoping Zhang1 and Zhaohui Chen1 1Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2Department of Urology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330008, China *These authors have contributed equally to this work Correspondence to: Zhaohui Chen, email: [email protected] Keywords: CLCA4, bladder cancer, PI3K/Akt, proliferation, metastasis Received: July 21, 2017 Accepted: August 17, 2017 Published: October 09, 2017 Copyright: Hou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Calcium activated chloride channel A4 (CLCA4), a tumor suppressor, was shown to contribute to the progression of several human cancers, while its role in bladder carcinoma remains unclear. In this study, we showed CLCA4 expression was down- regulated in bladder carcinoma tissues and cells compared to adjacent non-tumor tissues and normal urothelial cells. Low CLCA4 expression was correlated with larger tumor size, advanced tumor stage, and poor prognosis in bladder carcinoma patients. Overexpression of CLCA4 profoundly attenuated the proliferation, growth, migratory and invasive capabilities of bladder cancer cells, whereas CLCA4 knockdown had the opposite effect. Mechanistically, CLCA4 is involved in PI3K/AKT signaling and its downstream molecules can promote bladder cancer cell proliferation. Additionally, CLCA4 could mediate the migration and invasion of bladder cancer cells by regulating epithelial-mesenchymal transition and PI3K/Akt activation. This study suggests that CLCA4 may represent a promising prognostic biomarker for bladder cancer and provides a possible mechanism for bladder cancer growth and invasion. INTRODUCTION development and identify effective therapeutic strategies to improve patient survival. Bladder cancer represents the most common Calcium-activated chloride channel (CLCA) cancer of the urinary tract. Overall, the annual incidence regulators are a family of proteins that are characterized rate is 80.5/100,000 in China, with an estimated by a symmetrical multiple cysteine motif in the amino- mortality rate of 32.9/100,000 [1]. Although the clinical terminal tail [3]. The human CLCA locus is located on treatment outcomes for bladder cancer have improved chromosome 1p31-1p22 [4]. As outwardly rectified in recent decades, the survival time of patients with chloride channel modulators, CLCA proteins are activated invasive stage tumors or metastatic diseases remains by calcium to play a role in chloride conductance in very short. In particular, the median survival time of epithelial cells [5]. In mammals the CLCA protein family these patients is about 14 months, while the long-term members are distinguished by the von Willebrand factor disease-free survival rate is only 15% [2]. Although type A domain, the structure of metalloprotease, and tumor progression and migration are the most ruinous the capability to self-cleave [6]. Studies have shown aspects of bladder cancer and directly impact the clinical that CLCA members were involved in a diverse range survival of patients, the underlying mechanisms of these of biological processes including cell differentiation, processes remain elusive. Therefore, it is necessary to adhesion, apoptosis, and the development of inflammatory investigate the underlying mechanisms of bladder cancer processes in the airway [7]. In addition, some reports have www.impactjournals.com/oncotarget 93001 Oncotarget revealed involvement of CLCA1, CLCA2, and CLCA4 expression pattern in 196 bladder cancer specimens in tumor progression. CLCA1 may contribute to the by real-time PCR. The correlation analysis of CLCA4 increased spontaneous differentiation and reduced cell expression revealed a significant association between proliferation of colorectal cancer [8], and is thus associated CLCA4 expression and tumor size (P = 0.001) and tumor with favorable prognosis of colorectal cancer patients stage (T classification) (P = 0.034) (Table 1). However, [9]. As a p53-inducible senescence regulator, CLCA2 there was no significant correlation between CLCA4 negatively mediates tumor cell invasion [10], while loss expression and other clinicopathologic factors, such as of CLCA2 facilitates epithelial-to-mesenchymal transition age, sex, or tumor grade. Kaplan–Meier survival analysis and implicates higher chance of cancer metastasis [11]. revealed that patients with high CLCA4 expression CLCA4 is a member of the CLCA family, and had more favorable overall and recurrent-free survival the primary structure of CLCA4 is similar to CLCA1 (P<0.001 and P=0.032, respectively) than those with and 2 [12]. RT-PCR analysis revealed that high levels low CLCA4 expression (Figure 1E). Multivariable Cox of CLCA4 mRNA is expressed in human brain, testis, regression analyses revealed that low CLCA4 expression small intestine, colon and lung tissues [7]. Recently, was a risk factor for the overall and recurrent-free CLCA4 was found to be aberrantly expressed in breast survival of bladder cancer patients (P=0.001 and P=0.019, cancer, and its ectopic expression inhibited tumour cell respectively) (Table 2). growth as well as the epithelial to mesenchymal transition [13]. However, the impact of CLCA in bladder cancer CLCA4 suppresses cell viability and growth in development still needs to be veriﬁed. In this study, we bladder cancer examined the role and mechanism of CLCA4 in human bladder cancer. We demonstrated that CLCA4, whose In order to determine whether CLCA4 inhibited low expression associates with tumor aggressiveness and the growth capacity of bladder cancer cells, we further unfavorable clinical survival, promoted bladder cancer cell explored the biological significance of CLCA4 using proliferation and metastasis via the PI3K/AKT pathway. Gene Set Enrichment Analysis (GSEA) based on mRNA Together with other published data, our results showed expression data from the TCGA. The results indicated that CLCA4 might be exploited as a target for potential that low levels of CLCA4 was closely correlated with clinic treatments of patients with bladder cancer. proliferation-associated gene signature (Figure 2A). We then evaluated the role of CLCA4 on the bladder RESULTS cancer cell proliferation in established stable CLCA4- overexpressing and CLCA4-knocking down EJ and T24 CLCA4 expression is down-regulated in bladder cells (Figure 2B). CLCA4 overexpression suppressed cancer the colony forming ability of both cell lines (Figure 2C- 2D). Additionally, CLCA4 expression was negatively To investigate whether CLCA4 expression correlated with Ki67 expression from the real-time PCR is correlated with bladder cancer development and data (P = 0.002) (Figure 2E). The CCK-8 cell viability progression, we first analyzed CLCA4 expression in assays showed that overexpression of CLCA4 significantly bladder cancer cell lines and tissues. Decreased CLCA4 reduced cell viability in both cell lines, while knockdown mRNA expression was detected in 80.0% (48/60) of the of CLCA4 had the opposite effect (Figure 2F). Moreover, patients (Figure 1A). Additionally, western blot analysis GSEA results indicated that low CLCA4 expression was showed that CLCA4 expression was markedly lower in associated with cell cycle-associated gene signatures, bladder cancer than that in adjacent non-tumor tissues suggesting that CLCA4 is involved in the cell cycle (Figure 1B). In addition, both western blot (WB) and real- regulation (Figure 3A). Flow cytometry assay revealed time PCR analyses showed that CLCA4 was profoundly that overexpressing CLCA4 significantly reduced, but down-regulated in all 7 bladder cancer cell lines compared silencing CLCA4 increased the percentage of S phase with NBUCs and normal urothelial cells (Figure 1C). cells (Figure 3B). EdU incorporation assay suggested Moreover, we analyzed mRNA microarray data from that the percentages of cells with incorporated EdU was GSE13507 database, and surprisingly found the mRNA dramatically repressed in CLCA4-overexpressing cells expression of CLCA4 was drastically decreased in the and elevated in CLCA4-silencing cells (Figure 3C). bladder cancer tissues (P<0.001, Figure 1D). These results implied that CLCA4 may inhibit the cell proliferation in bladder cancer. Correlation of CLCA4 expression with clinicopathological characteristics and prognosis CLCA4 inhibits cell migration and invasion in of bladder cancer patients bladder cancer In order to explore the clinical significance of We next examined the role of CLCA4 in the CLCA4 in bladder cancer, we analyzed the CLCA4 migration and invasion of bladder cancer cells. The results www.impactjournals.com/oncotarget 93002 Oncotarget showed that CLCA4-overexpressing cells exhibited a anchorage-independent growth assay. As shown in significantly decreased migratory capacity, whereas Figure 5A, CLCA4 overexpressed cells formed a CLCA4-knockdown cells had an increased migratory smaller number and smaller colonies compared with capacity in wound healing assays. Transwell assays control

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