ASIC1 And ASIC3 Contribute To Acidity-induced EMT Of Pancreatic Cancer Through Activating Ca²⁺/RhoA Pathway

Subject:Our previous data demonstrated acidic microenvironment can promote metastasis of pancreatic cancer by inducing epithelial mesenchymal transition(EMT),while its mechanism is far from elucidated.Since acid-sensing ion channels(ASIC)plays pivotal function in most of the acidity-associated physiological,the present study aims to explore whether ASIC 1/3 contribute to the acidic-induced EMT of pancreatic cancer.Methods:The expression of ASIC 1/3 was detected in pancreatic cancer and adjacent noncancerous tissues by qRT-PCR,immunofluorescence and immunohistochemical staining.The expression of ASIC1/3 in pancreatic cancer cell lines(PANC-1,SW1990,BxPC-3 and AsPC-1)and normal pancreatic ductal cells(HPDE)was detected by RT-PCR,qRT-PCR and Western blot.Patch clamp was employed to test the activity of ASIC 1/3.The effects of ASIC 1/3 on acidity-induced invasion,migration and EMT of pancreatic cancer cells were evaluated both in vitro and in vivo.The concentration of intracellular calcium was showed by calcium imaging.The activity of RhoA was measured to study its role in ASIC1/3-calcium pathway.Moreover,the intracellular calcium and RhoA activity was suppressed by siRNA or specific inhibitor respectively to evaluate their effects on acidity-induced EMT in pancreatic cancer cells.Results:ASIC1 and ASIC3 are expressed on the membrane of pancreatic cancer cells,which is in charge of an acidity-induced,amiloride-inhibitable,inward current.Inhibition of ASIC1 and ASIC3 suppresses EMT of pancreatic cancer cells during acidic conditions.Acidity elevates intracellular Ca2+ concentration and RhoA activity,which is decreased by inhibition of ASIC1 and ASIC3.The acidity-induced EMT is impaired by calcium chelating agent BAPTA-AM or knockdown of RhoA.In nude mice xenograft model,knockdown of ASIC 1 and ASIC3 significantly decrease liver and lung metastasis of pancreatic cancer cells.ASIC1 and ASIC3 are overexpressed in pancreatic cancer tissues compared to paracarcinoma tissues,and associated with the expression ofEMT marker.Conclusion:ASIC1 and ASIC3 transduce the acidic pH to elevation of[Ca2+]i concentration and RhoA activity,promoting EMT of pancreatic cancer cells during acidic microenvironment.microenvironment on invasion and Our study elaborates an integrated signaling pathway to account for the effect of acidic metastasis of pancreatic cancer.