The Role Of KRAS Mutations In Nuclear Factor-κB Activation And Their Effect On Chemotherapy Response And Prognosis In Colorectal Cancer

Aim: The aim of this study was to explore the role of KRAS mutations in constitutiveNF-κB activation in colorectal cancer and examined chemotherapy response,prognosis and clinical-pathological feature of colorectal cancer patients with bothKRAS mutation and NF-κB activation.Materials and Methods: Part I: NF-κB activation was analyzed byimmunohistochemistry in167primary colorectal cancer specimens in which theKRAS mutation status was confirmed. Clinical and pathologic data were extractedfrom the medical records and reviewed. We investigated the relationship betweenNF-κB activation and KRAS status, and examined chemotherapy response andclinical-pathological parameters and correlated overall survival of colorectal cancerpatients with both KRAS mutation and NF-κB activation. Part II:①SW620, HCT8,HCT116, LoVo, DLD-1colon cancer cell lines (mutated KRAS) and HT29, CaCO-2,HCA-7colon cancer cell lines (wild type KRAS) were selected in the study. The levelsof nuclear and cytoplasmic NF-kappaB-p65were detected by western blot. Coloncancer cells were treated with NF-κB inhibitor JSH-23in the different concentrationfor48h. To knock-down KRAS expression, colon cancer cell lines harboring KRASmutations were transfected by KRAS siRNA. Cells proliferation were tested by MTSassay.②We established SW620cells with stable knockdown of KRAS by KRASshRNA transfection and puromycin seclection and treat3μM or10μM ERK1/2inhibitor U0126on the SW620cells for30minites. The levels of RAS,NF-kappaB-p65, p-ERK1/2, p-IκBα and other key signal moleculors were detected bywestern blot.Results: Part I: Of167tumors screened,63(37.7%) had NF-κB activation,59(35.3%) had KRAS mutations, and30(18.0%) had both NF-κB activation and KRASmutations. The frequency of NF-κB activation in tumors with KRAS mutations was significantly higher than in tumors with wild type KRAS;50.8%vs.30.6%, P=0.012.A significantly greater percentage of patients having G12D and G12V mutations withNF-κB activation was found than patients with the G13D mutation;67.9%and55.6%vs.26.3%, P=0.029. There was no significant difference in sex, age, cigarettesmoking, tumor location, pathology and TNM stage between patients with both KRASmutations and NF-κB activation and others. Patients with both KRAS mutations andNF-κB activation had a lower objective response rate to firstline chemotherapycompared to patients with other tumors,23.8%vs.49.4%(P=0.035). Compared topatients with both KRAS mutations and NF-κB activation, overall survival wassignificantly higher in patients in other groups; median overall survival (OS)28.4months (95%CI:21.0-35.8) vs.46.3months (95%CI:39.4-53.2), Hazard Ratio (HR)0.259(95%CI:0.125-0.538), P=0.005. Part II:①Constitutive NF-κB activationwere observed in DLD-1, LOVO, and SW620cells, JSH-23decreased theproliferation of SW620, DLD-1and LoVo cells. In cell lines harboring KRASmutations, cell proliferation were significantly inhibited in SW620, DLD-1and LoVocells by KRAS siRNA transfection, whereas the suppression of KRAS had no effect onthe proliferation of HCT116or HCT8cells.②Compaired to SW620cells, markedlydecreased RAS, nuclear NF-κB-P65, p-ERK1/2and p-IκBα was observed inSW620/KRAS shRNA cells. U0126blocked ERK1/2phosphorylation and evidentlyinhibited the phosphorylation of IκBα and NF-κB activition in SW620cells.Conclusions: In this study, NF-κB activation was associated with KRAS mutation incolorectal cancer tissues. Both KRAS mutation and NF-κB activition indicate hightolerence to chemotherapy and poor prognosis for colorectal cancer patients.Constitutive activation of NF-κB was found in colon cancer cells with KRAS mutationand KRAS dependence. NF-κB activation was decreased in KRAS knockdown SW620cells probably through the RAS-ERK-IκBα pathway. Tumors with KRAS mutationsand NF-κB activation may represent a unique subtype of colorectal cancer.