Investigation Of Risk Factors Associated With Insulinoma Tumorgenesis And Role Of MTOR/PRAS40/P70S6K Signal Pathway In Its Carcinogenesis

BackgroundInsulinomas are the most common functional islet cell tumor, accounting for 70% to 80% of all pancreatic neuroendocrine neoplasms, however, insulinomas are relatively rare with an incidence of approximately four cases per million per year. There are few studies particularly aimed at investigating the possible risk factors for the pathogenesis of insulinoma and its etiology was poorly understood because of its rarity. More and more studies had confirmed that activation of mTOR played a significant role in tumor formation in many human cancers and PRAS40 was a key regulator of mTORC1, however, there has no study that focus on the mTOR/PRAS40/P70S6K signal pathway in insulinomas carcinogenesis.Objective(1) Investigate possible risk factors associated with the incidence of insulinoma in Chinese population;(2) Compare activation and positive rate of mTOR and its downstream P70S6K between insulinoma tissue and normal pancreatic tissue;(3) Evaulate influence of mTOR inhibitor and dural PI3K/mTOR inhibitor on the mTOR signal pathway activity and influence on the profileration, insulin secretion and apoptosis of an insulinoma cell line-INS-1;(4) Observe the influence of PRAS40 knockdown on the mTOR and P70S6K phosphorylation level and INS-1 cell profileration.Methods(1) We conducted a retrospective hospital-based case-control study in Peking Union Medical College Hospital (PUMCH) in China. Patients who were diagnosed with insulinomas between January 2000 and January 2010 were enrolled into the case group.The controls subjects were patients who underwent operations not because of benign or malignant tumors at the department of general surgery of PUMCH during the same time frame. Medical records of patients and controls were systematically reviewed, data including demographic information, possible risk factors (height, weight, smoking, alcohol use, family history of PETs or other cancers and their personal medical history) were collected for analysis. Univariate and multivariate analysis were applied to determine the independent risk factors for occurrence of insulinoma.(2) Immunohistochemical staining and western blotting were applied to evaluate the expression of p-mTOR and its downstream p-P70S6K in the insulinoma and normal pancreatic tissue, mRNA level of these two gene were also examined by the RT-PCR technique;(3) An insulinoma cell line (INS-1) was treated with inhibitors of mTOR (rapamycin) or dual PI3K/mTOR inhibitor (NVP-BEZ235) in vitro, RT-PCR and western boltting were applied to evaluate their influence on the expression of mTOR and P70S6K. MTT assay was used and cell cycle and apoptosis were analyzed by flow cytometry, insulin secretion was evaluated by GSIS;(4) PRAS40-siRNA was transferred into INS-1 cell to knockdown expression of PRAS40, Taqman real-time PCR was used to evaluate silencing effect, western boltting was applied to assess phosphorylation level of mTOR and P70S6K and MTT assay was used to judge profileration level.Results(1) Rural residency(OR=4.950, P＜0.0001), family history of pancreatic endocrine tumor (OR=16.754, P=0.007), family history of other cancers (OR=2.360, P=0.037) were risk factors for the carcinogenesis of insulinoma, wheather smoking and alcohol consumption were risk factors of insulinoma should be further studied;(2) Positive expression of p-mTOR and p-P70S6K were higher in insulinoma tumor specimens than the normal pancreatic islet, which were both confirmed by the immunohistochemical staining and western blotting methods, mRNA level of mTOR and P70S6K were also higher in insulinoma tumor tissues than normal pancreatic tissue;(3) Both rapamycin and NVP-BEZ235 had significant influence on the expression of mTOR and P70S6K, The mRNA and phosphorylation protein level of these two genes were significantly decreased after incubated 48 hours with different concentrations of rapamycin or NVP-BEZ235, which resulted in inhibiting the INS-1 cell proliferation, insulin secretion and inducing apoptosis. We also found that high concentrations of rapamycin can induce elevation of p-mTOR contrarily, however, NVP-BEZ235 had better influence on inhibiting the cell proliferation and inducing apoptosis;(4) mRNA level of PRAS40 was successfully decreased by transferring PRAS40-siRNA into INS-1 cell, level of p-mTOR and p-P70S6K were elevated, which promoting the proliferation of INS-1 cell.Conclusions(1) Rural residency, family history of pancreatic endocrine tumor and family history of other cancers are risk factors for the carcinogenesis of insulinoma;(2) mTOR/P70S6K signaling pathway is actived in insulinoma, this pathway may be involved in the carcinogenesis of insulinoma;(3) NVP-BEZ235 can inhibit activity of mTOR/P70S6K pathway effectively, which provide guidance for forthcoming malignant insulinoma treatment;(4) PRAS40 is a negative regulator of mTOR in insulinoma cell line, the mechanism requires further studies, this gene may be a new target for the treatment of insulinoma or other tumors.