Synergistic Targeting Of Insulin Receptor And KIT In Imatinib-Resistant Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumor(GIST)is the most common mesenchymal and solid tumor of the gastrointestinal tract(GI).The majority of GISTs(>80%)occurrence is related to oncogene kit mutation.Imatinib(IM),a small-molecule tyrosine kinase inhibitor(TKI),specially inhibits tyrosine kinase receptor(RTK)KIT,and has become an effective drug for treatment of inoperable and malignant GIST.However,GIST patients develop resistance to imatinib eventually.Therefore,there is an urgent need to find novel therapeutic strategies to address IM-resistance.Phospho-RTK assay was performed in imatinib-resistant and-sensitive GIST cell lines and patient tumor tissue,and the result showed that insulin receptor(IR)is strongly activated only in imatinib-resistant cell lines(GIST430 and GIST48)but not in imatinib-sensitive GIST cell lines(GIST882 and GIST-T1).Immunoblotting and immunoprecipitation confirmed that phospho-IR was strongly expressed in imatinib-resistant GIST lines and primary tumor sample,but not in imatinib-sensitive GIST cell lines.In order to investigate the functional roles of activated IR in GISTs,linsitinib(LST),a IR/IGF1R specific small-molecule inhibitor,was used to study the effects of IR and KIT signaling in imatinib-resistant(GIST430 and GSIT48)and-sensitive(GIST882)GIST cell lines.Immunoblotting showed that activation of AKT and mTOR substrate S6 was inhibited in dose-dependent manner after treatment with LST for 6 hours in GIST430 and GIST48,but not in GIST882.In addition,inhibition of IR little affected phospho-KIT expression in three GIST cell lines.Additive effects were obtained through a coordinated attack on IR and KIT by LST and IM,respectively,as demonstrated by immunoblots,cell viability,colony formation,apoptosis,wound healing,and cell cycle analyses,showing that combination inhibition of IR and KIT,in imatinib-resistant GIST cell lines inactived greater PI3K/AKT/mTOR signaling intermediate AKT and S6,and induced greater cell apoptosis,anti-proliferative and anti-migration effects,and cell cycle arrest,compared to either intervention alone,but not in imatinib-sensitive GIST cell line.These novel findings underscore that IR activation play the crucial oncogenic roles to imatinib-resistant GIST cell proliferation and survival.These compelling pro-apoptotic and anti-proliferative responses indicate that combination inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs.Furthermore,we investigated the mechanisms of IR activation in GISTs by transcriptome sequencing,qRT-PCR,ir sequencing,co-IP,and immunoblotting after KIT inhibition by using sunitinib.The results showed that IR phosphorylation was independent of KIT activation,no IR mutation,IR overexpression,and interaction of IR and KIT were found.Ligand IGF2 overexpression but not IGF1 or insulin expression may contribute IR activation in imatinib-resistant GISTs.