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ASIC1 and ASIC3 Contribute to Acidity-Induced EMT of Pancreatic Cancer Through Activating Ca2+/Rhoa Pathway

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ASIC1 and ASIC3 Contribute to Acidity-Induced EMT of Pancreatic Cancer Through Activating Ca2+/Rhoa Pathway Citation: Cell Death and Disease (2017) 8, e2806; doi:10.1038/cddis.2017.189 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddis ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca2+/RhoA pathway Shuai Zhu1,4, Hai-Yun Zhou1,2,4, Shi-Chang Deng1,3,4, Shi-Jiang Deng1, Chi He1, Xiang Li1, Jing-Yuan Chen1, Yan Jin1, Zhuang-Li Hu2, Fang Wang2, Chun-You Wang1 and Gang Zhao*,1 Extracellular acid can have important effects on cancer cells. Acid-sensing ion channels (ASICs), which emerged as key receptors for extracellular acidic pH, are differently expressed during various diseases and have been implicated in underlying pathogenesis. This study reports that ASIC1 and ASIC3 are mainly expressed on membrane of pancreatic cancer cells and upregulated in pancreatic cancer tissues. ASIC1 and ASIC3 are responsible for an acidity-induced inward current, which is required for elevation of intracellular Ca2+ concentration ([Ca2+]i). Inhibition of ASIC1 and ASIC3 with siRNA or pharmacological inhibitor significantly decreased [Ca2+]i and its downstream RhoA during acidity and, thus, suppressed acidity-induced epithelial–mesenchymal transition (EMT) of pancreatic cancer cells. Meanwhile, downregulating [Ca2+]i with calcium chelating agent BAPTA-AM or knockdown of RhoA with siRNA also significantly repressed acidity-induced EMTof pancreatic cancer cells. Significantly, although without obvious effect on proliferation, knockdown of ASIC1 and ASIC3 in pancreatic cancer cells significantly suppresses liver and lung metastasis in xenograft model. In addition, ASIC1 and ASIC3 are positively correlated with expression of mesenchymal marker vimentin, but inversely correlated with epithelial marker E-cadherin in pancreatic cancer cells. In conclusion, this study indicates that ASICs are master regulator of acidity-induced EMT. In addition, the data demonstrate a functional link between ASICs and [Ca2+]i/RhoA pathway, which contributes to the acidity-induced EMT. Cell Death and Disease (2017) 8, e2806; doi:10.1038/cddis.2017.189; published online 18 May 2017 Pancreatic cancer is one of the most aggressive cancers with metabolism shifts to glycolysis even when there is enough O2. a 5-year survival rate of about 5% and a median survival rate of This shift remarkably increases glucose metabolism, which about 6 months.1 This poor prognosis and ultimate cause of leads to a rapid production of lactic acids.8 Moreover, the death are mainly due to metastatic invasion. Strikingly, recent insufficient perfusion and chaotic vascular structure of tumor studies suggest that pancreatic cancer metastasizes very further increase the accumulation of lactic acid and H+.9 Thus, 2 early in their development. Therefore, it is critical to identify malignant tumors display an acidic extracellular pH (pHe) the underlying molecular mechanisms of invasion and when compared with normal tissue under physiological metastasis of pancreatic cancer.3 Epithelial–mesenchymal conditions.10–12 Interestingly, extracellular acidity as a favoring transition (EMT) has a major role in tumor invasion and factor of tumor progression and metastatic dissemination is metastasis. In the process of EMT, the cells lose epithelial involved in EMT. Melanoma cells, which are exposed to acidic characteristics and acquire invasive properties and stem cell- extracellular environment, display a significant EMT profile like features.4 EMT is associated with poor survival in and an increased invasiveness in vitro and in vivo.13 Acidic surgically resected pancreatic cancer.5 Signaling molecules, extracellular pH induces the expression of EMT regulator, such as Snail, which have an essential role during EMT, including ZEB1, Twist1 and Twist2, accompanied with promote metastatic process of pancreatic cancer.6 Specifi- enhancing cell motility and invasion in lung carcinoma.14 cally, our previous study reveals that both chronic pancreatitis Meaningfully, our previous work reveals that acidic micro- and pancreatic cancer demonstrated active EMT process, environment induces EMT of pancreatic cancer cells through which also indicates the pivotal role of EMT in tumorigenesis of regulating miR-652/ZEB1 pathway.15 In this study, we work to pancreatic cancer.7 However, the precise mechanism for investigate how the acidic extracellular pH, a chemical initiation of EMT in pancreatic cancer has been not signaling of metabolic microenvironment, is detected by elucidated fully. pancreatic cancer cells and further promotes the transduction Recent studies have noted that the acidity is an intrinsic of EMT as biological signaling. hallmark of tumor microenvironment. Owing to the ‘Warburg Acid-sensing ion channels (ASICs), an H+-gated subgroup effect’, which is an intrinsic characteristic in malignant cells, of the epithelial Na+ channel/degenerin (ENaC/DEG) 1Department of Pancreatic Surgery, Pancreatic Disease Institute, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China and 3Department of Gastrointestinal Surgery, Union Hospital West Campus, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China *Corresponding author: G Zhao, Department of Pancreatic Surgery, Pancreatic Disease Institute, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Avenue 1277, Wuhan 430022, China. Tel: +86 27 85351621; Fax: +86 27 85351669; E-mail: [email protected] 4These authors contributed equally to this work. Received 11.12.16; revised 02.3.17; accepted 14.3.17; Edited by R Johnstone ASIC1 and ASIC3 in pancreatic cancer S Zhu et al 2 superfamily, are triggered by increased extracellular In consistence with the results in pancreatic cancer cell acidification.16,17 To date, six ASIC subunits have been lines, the immunofluorescence assay showed that ASIC1 and identified: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC3 in pancreatic cancer specimens were increased ASIC4.18 The pH values required for half-maximal activation of compared with paired adjacent noncancerous tissues 19 ASICs (pH0.5) are varied from 4.9 to 6.8. ASICs are involved (Figure 1e). Therefore, these results indicate that ASIC1 and in most of the acidity-associated physiological and pathologi- ASIC3 are functionally expressed in pancreatic cancer cells. cal functions in nervous system disease.20,21 It has been reported that high-grade glioma cells express voltage-inde- pendent, amiloride-inhibitable inward Na+ current, which is Inhibition of ASIC1 and ASIC3 suppresses acidity- attributed to mixed ASIC1 and ASIC2, but not exists in normal promoted invasion and migration of pancreatic cancer astrocyte or low-grade glioma. Moreover, inhibition of this cells. In order to evaluate the role of ASIC1 and ASIC3 in conductance decreases the growth and migration of glioma acidity-promoted invasion and migration, the expression of cells.22 Other research has shown that ASIC1 is highly ASIC1 and ASIC3 was downregulated with siRNA. The si-2 expressed in D54-MG human glioblastoma multiform cells of ASIC1 siRNA and si-2 of ASIC3 siRNA had the most than that of primary human astrocytes. Knockdown of ASIC1 distinct restraining effect on the expression of mRNA and or using pharmacological inhibitor effectively abolishes the protein, respectively (Supplementary Figures S2A and B). whole-cell patch-clamp current and inhibits tumor migration in After knockdown of ASIC1 and ASIC3 separately or glioma cells.23 Given that ASIC1 and ASIC3 are the most simultaneously (Supplementary Figure S2C), the acidity- 19 promoted invasion and migration of pancreatic cancer cells susceptible to acidic pHe and function as acidic pH sensors, we investigate whether ASIC1 and ASIC3 are expressed in were inhibited (Figures 2a and b). However, simultaneous pancreatic cancer and contribute to acidity-induced EMT of inhibition of ASIC1 and ASIC3 did not have synergistic effect. pancreatic cancer. Meanwhile, knockdown of ASIC1 and ASIC3 with siRNA did not display remarkable effect on viability of PANC-1 and BxPC-3 cells (Figure 2c). Thus, the results of Transwell assay Results and wound-healing test were attributed to the decreased activity of invasion and migration but not proliferation ASIC1 and ASIC3 are functionally expressed on inhibition. Furthermore, amiloride, a nonspecific inhibitor to pancreatic cancer cells. It has been well demonstrated ASICs, distinctly attenuated the acidity-promoted invasion that ASICs are involved in numerous physiological and and migration of PANC-1 and BxPC-3 cells (Figures 2d pathological process in nervous system, whereas there is and e). Similarly, the specific inhibitor for ASIC1, PcTx1 rare research about the expression and function of ASICs in demonstrated inhibitory effects to acidity-promoted invasion non-nervous system. Given that ASIC1 and ASIC3 are the and migration of pancreatic cancer cells (Supplementary most sensitive to acidic pHe and function as acidic pH Figures S3A and B). These results suggest that ASIC1 and sensors, we first detected the expression of ASIC1 and ASIC3 have an important role in acidity-promoted invasion ASIC3 in pancreatic cancer cells. Reverse transcription-PCR and migration. (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) displayed that the mRNA of ASIC1 and ASIC3 in pancreatic cancer cell lines (PANC-1, SW1990,
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