Role Of Store-operated Ca²⁺ Entry In Invasion And Migration Of Glioma

Background: Gliomas are the most common primary tumors in the central nervous system(CNS), and glioblastoma multiforme(GBM) has the poorest prognosis among glioma types. Even with the current optimal therapeutic strategies, glioma cells always disperse along thin and elongated anatomic structures. For this reason, glioma cells can not be completely resected by surgical treatment. The remaining cancer cells resist to the radiotherapy and the chemotherapy, and continue to disperse in brain, which leads to recurrence and poor prognosis. The ubiquitous intracellular second messenger Ca2+ plays an important role in many fundamental physiological processes, including cell motility. Recent research indicates that Ca2+ also contributes to the malignant behaviors in tumors, such as invasion and metastasis. Store-operated Ca2+ entry(SOCE), which is initiated by the depletion of intracellular Ca2+ stores, is an important pathway in nonexcitable cells. SOCE is mediated by stromal interacting molecule-1(STIM1) and calcium release-activated calcium channel protein 1(Orai1). The present study aimed to explore the specific mechanisms of SOCE in the processes of glioma migration and invasion, which is helpful for providing potential targets for future therapeutic strategies.Methods: The expression of STIM1 and Orai1, two key components of SOCE, was examined in glioma samples and glioma cell lines by immunohistochemistry and western blot analysis. Both pharmacological intervention(SKF96365) and RNA interference against Orai1 were employed to investigate the role of SOCE in glioma cell migration and invasion in vitro. Orai1 c DNA consturct was used to ruscue the expression of Orai1. Western blot analysis and q RT-PCR were employed to confirm the efficiency of RNA interference and re-expression construct. The intracellular Ca2+ was quantified through Fluo-4/AM based Ca2+ measurement. The effect of SOCE on cell migration and invasion was explored by wound healing assay and transwell invasion assay. The morphology of glioma cells and focal adhesions were observed by immunofluorescent labeling for vinculin, a major component of focal adhesions.The expression of several epithelial-to-mesenchymal transition(EMT) related markers, such as E-cadherin, N-cadherin, and vimentin, was detected using western blot anslysis. The protein and phosphorylation expression of proline-rich tyrosine kinase 2(Pyk2), an intracellular calcium-dependent tyrosine kinase that is activated through phosphorylation, was detected by western blot analysis. Then, Pyk2 was downregulated through RNAi to investigate if SOCE functions through Pyk2 pathway. Similarly, SKF96365 and RNA interference against Orai1 were employed to investigate the role of SOCE in rat C6 glioma cell migration and invasion both in vitro and in vivo.Results: STIM1 was commonly low expressed in non-neoplastic brain tissues, low-grade glioma samples, and high-grade glioma samples. Orai1 expression was elevated in glioma tissues and several glioma cell lines compared with non-neoplastic brain tissues, and positively correlated with the WHO grading of glioma. Orai1 silencing reduced the amplitude of Ca2+ influx in U251/SNB19 glioma cells. Either inhibition of SOCE by a pharmacological inhibitor or Orai1 downregulation suppressed glioma cell migration and invasion. SKF96365 and Orai1 downregulation induced large focal adhesions and slowdown of focal adhesion turnover. SKF96365 and Orai1 downregulation elevated the expression of E-cadherin and reduced the expression of N-cadherin and vimentin. SKF96365 and Orai1 downregulation reduced the phosphorylation of Pyk2. Re-expression of Orai1 can rescue all of the changes described above resulting from Orai1 downregulation. Pyk2 silencing inhibits cell invasion, induces large focal adhesions, and inhibits EMT-like in glioma cells again. SKF96365 and Orai1 downregulation suppressed C6 glioma cell migration and invasion in vitro. Orai1 downregulation suppressed C6 glioma cell invasion and cluster around the perivascular niche.Conclusions:SOCE enhances glioma cell migration and invasion via regulation of focal adhesion turnover and induction of EMT-like. In addition, Pyk2 is an important immediate effector of SOCE that mediates downstream mechanisms. Thus, SOCE is likely to become a novel therapeutic approach in treating malignant glioma. Orai1may become a molecular target for glioma diagnosis and treatment.