The Mechanism Of RhebY35N Mutation Driven Tumor Growth

Objective: In recent years,with development of gene sequencing technology,more and more species genome was reported,the cancer genome sequencing will also become a kind of accurate tumor treatment strategy.Generally,gene mutation reduces tumor by activating or becoming an oncogene.Rheb is a Ras family GTPase,which binds to and activates mammalian target of rapamycin complex 1(mTORC1)when GTP loaded.Recently,cancer genome sequencing efforts have identified over 30 Rheb mutations in different human cancers.RhebTyr35 is an apparent hotspot for mutations.Another study also demonstrated that Rheb-Y35 N activated m TORC1 in a 293 T cell transient overexpression assay.The function of this mutation is unclear.In this paper,it explores the potential function of RhebY35 N mutation in cell proliferation and signal pathways and finds a therapeutic strategy for cancer patients with this mutation.Methods: The stably transfected 293 T cell line with overexpression of RhebY35 N mutant is constructed by transfecting RhebY35 N plasmid and the signaling pathways regulated by Rheb Y35 N are analyzed by immunoblotting and immunoprecipitation using specific drugs of those pathways.The effect of RhebY35 N on tumor growth and proliferation and the therapeutic effect of the drug are examined by constructing nude mouse model of RhebY35 N overexpressing NIH3T3 cell line.Results: The study demonstrates that RhebY35 N activates not only mTOR pathway but MAPK pathway persistently.Moreover,the analyses show that Rheb could directly bind to AMPK? to regulate its activity and RhebY35 N mutant competitively binds AMPK? to inhibit RhebWT mediated AMPK activation.RhebY35 N impaires AMPK? activation in response to AICAR or nutrient depletion.Finally in vivo combination treatments combining mTORC1 and MAPK inhibitors were very effective in preventing RhebY35 N NIH3T3 cell growth as xenografts.In summary,results demonstrate that Rheb Y35 N is an oncogenic mutation and activates both mTORC1 and MAPK pathways.These findings suggest that cancers with RhebY35 N mutations may be uniquely sensitive to combination treatment with rapamycin and an ERK inhibitor.