MTOR Enhancement Of Store-operated Calcium Entry Restricts TSC Tumor Development

The receptor tyrosine kinase (RTK)-phosphatidylinositol 3-kinase (PI)-AKT-mammalian target of rapamycin (mTOR) signaling cascade plays a central role in regulating cell growth, proliferation, survival and differentiation. Components of this pathway include many proto-oncogenes, such as RTKs, PIK, AKT and mTOR, and tumor suppressors, such as PTEN, TSC 1 and TSC2. Due to recurrent gain-of-function mutations of proto-oncogenes and loss-of-function mutations of tumor suppressors, RTK-PI3K-AKT-mTOR signaling cascade is one of the most frequently activated signaling pathways in human cancers. Tuberous Sclerosis Complex (TSC), caused by inactivation of tumor suppressor complex TSC1/TSC2 and subsequent hyperactive mTOR and decreased AKT1 activity, is a benign tumor syndrome afflicting multiple organs. However, the precise mechanisms downstream of the mTOR signaling that regulate tumor development remain largely unclear.Calcium ion (Ca+), as an important second messenger, can regulate a wide variety of cellular processes, such as cell growth, differentiation, proliferation, and death. Disturbance of cellular Ca2+homeostasis may cause various diseases, including cancer. Several studies showed that TRPC (transient receptor potential cation channel) proteins were very important for prostate cancer and liver cancer development, SCPA2 (secretory pathway Ca2+-ATPase) and Orail (the pore-subunit protein of store-operated calcium channel, SOC) could promote breast tumor growth, the endoplasmic reticulum Ca2+ sensor STIM1 (stromal interaction molecule 1) and Orail were essential for breast tumor invasion and metastasis. In this study, we have founded that mTOR enhances store-operated calcium entry (SOCE), and then restrictes TSC tumor development.Using calcium imaging method, we first measured the Ca2+istribution in both normal mouse embryonic fibroblasts (MEFs) (Tsc2+/+MEFs) and hyperactive mTOR MEFs(sc2-1MEFs). Between normal MEFs and Tsc2 null MEFS, no difference was found for the cytosolic Ca2+concentrations ([Ca2+]c) at resting state and the ER Ca2-concentrations ([Ca2+]ER.However, following ER Ca2+depletion, the Ca2+entry through SOC was greatly enhanced in Tsc2 null MEFs comparing to wt MEFs, and this augmentation was blocked by mTOR inhibitor, rapamycin, indicating that mTOR positively regulates SOCE. Then we identified that the elevation of SOCE in Tsc2-' MEFs was due to up-regulation of STIM1 and Orail/TRPCl. Moreover, inhibition of SOCE either by knocking down STIM1 or exogenous expression of dominant-negative Orail promoted TSC tumor development. Considering the decreased AKT1 activity due to negative feedback regulation of hyperactive mTOR in TSC tumor, we checked the effect of SOCE on AKT1 activity. AKT1 activity was significantly recovered after suppression of SOCE in Tsc2 null cells. Further more, inhibition of SOCE promoted angiogenesis of TSC tumors derived from Tsc2 null cells in nude mice.We conclude that mTOR enhancement of SOCE restricts TSC tumor development, which may contribute to the benign nature of tumors observed in TSC patients. Moreover, the removal of SOCE-mediated mTOR suppression of AKT by rapamycin may explain the limited efficacy of rapamycin in the treatment of TSC patients.