Importantly Vascular Disrupting Agent distinct somatic
pses like the EGFR. Importantly, distinct somatic point mutations in RAS genes turn these proto oncogenes into oncogenes by affecting their intrinsic GTPase activity thereby forcing constitutive activation of RAS proteins and stimulating downstream signaling pathways such as mitogen activated protein kinases and phosphoinositide 3 kinase. Recent studies investigated the frequency of RAS gene point mutations in distinct solid tumors and demonstrated the KRAS gene to be more frequently mutated than NRAS or HRAS mainly in tumors of the pancreas, the lung, or the colon. Furthermore, clinical trials studying therapeutic efficacy of EGFR directed antibodies like cetuximab or panitumumab showed that patients experiencing KRASmutated colorectal tumors do not respond to these agents.
This led to label restrictions for EGFR Abs in CRC patients bearing wildtype KRAS tumors. EGFR Abs are able to elicit distinct effector mechanisms for tumor cell destruction: Fab mediated effects comprise inhibition of ligandbinding or tumor cell growth, apoptosis induction, as well as EGFR down modulation, whereas Fc mediated effector mechanisms are triggered through the Fc region by binding either complement component C1q to induce complement dependent cytotoxicity or Fc receptors on effector cells to trigger Ab dependent cell mediated cytotoxicity or phagocytosis. Both Fab and Fc mediated effector mechanisms have been suggested to be important for therapeutic outcome of EGFR Abs with recent studies demonstrating a strong impact of Fc mediated effector mechanisms on the efficacy of EGFR Abs.
Furthermore, distinct genetic polymorphisms that determine the binding affinity and ADCC efficacy for FcγRIIa and FcγRIIIa have been described and were linked to clinical outcome of therapeutic EGFR Abs in metastatic CRC . Considering these findings, it might be hypothesized that Fc mediated mechanisms of EGFR Abs play important roles in tumor cell destruction. However, whether oncogenic mutations in the KRAS gene directly affect ADCC or CDC activity, as described for Fab mediated effector mechanisms, has to our knowledge not been previously investigated. In this study, we observed that oncogenic KRASG12V signaling is accompanied by down regulation of EGFR transcript and protein levels in a C/EBP dependent manner.
Decreased EGFR cell surface expression was accompanied by diminished ADCC as well as CDC and might in part explain the lack of efficacy of EGFR Abs in the therapy for KRAS mutated tumors. Materials and Methods Blood Donors Experiments reported here were approved by the Ethics Committee of the Christian Albrechts University, Kiel, Germany, in accordance with the Declaration of Helsinki. Blood donors were randomly selected from healthy volunteers, who gave written informed consent before analyses. Cell Lines and Antibodies Human epidermoid carcinoma cell line A431 was kept in RPMI 1640 medium, whereas A549, HCT 116, and A1207 cell lines were cultivated in Dulbecco modified Eagle medium. Cell culture media were supplemented with 10% heat inactivated fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin. EGFR directed antibodies used in this study: C225, 2F8, and HuMab 003. VH and VL regions of the humanized EGFR directed IgG1 antibody H425 were cloned into an expr .