KRAS 유전자가 돌연변이에 의해 활성화된 세포 주에서 MEK저해제의 선택적 활성 및 기전에 관한 연구

Abstract

EGFR tyrosine kinase inhibitors (TKIs) have shown promising efficacy in the treatment of tumors with EGFR mutations and amplifications. However, TKIs have also proven ineffective against the majority of tumors with EGFR wild-type (WT) alleles. Therefore, novel strategies for the treatment of cancer patients with tumors harboring EGFR WT alleles have yet to be thoroughly delineated. Among EGFR WT tumors, KRAS is frequently mutated in the tumors, resulting in activation of the RAF/MEK/ERK signaling pathway. However, targeting this signaling axis has been a challenging issue for the treatment of KRAS mutant (MT) cancers. Here, I analyzed the activities of a MEK inhibitor, selumetinib (AZD6244), investigated the mechanisms of action, and provided the basis for a rational combination strategy against human EGFR WT/KRAS MT cancers. First, using a panel of human EGFR WT gastric cancer cell lines, I demonstrated that gastric cancer cells harboring the KRAS mutation were selectively sensitive to MEK inhibition as compared with those cells harboring both KRAS and PI3K mutations and KRAS WT alleles. However, all cell lines were found to be resistant to EGFR inhibition. The results from Western blots and phospho-protein arrays showed that in MEK inhibitor resistant cell lines, AKT was activated via the EGFR/HER3/PI3K pathway following selumetinib (AZD6244) treatment. Blockade of this feedback mechanism via the targeting of MEK and EGFR resulted in detectable synergistic effects in some cell lines both in vitro and in vivo. Second, to further investigate EGFR/AKT activation by MEK inhibition, I performed cDNA microarray in 4 KRAS MT cancer cells showing variable responses to AZD6244. Here, I found that the expression of mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR, was most strongly increased in LOVO and SNU668 cells with low IC50(<0.1umol/L) of AZD6244, while reduced in HCT116 and AGS cells with high IC50(>0.8umol/L). As compared with changes of MIG6 expression in LOVO and SNU668 cells, AZD6244-resistant HCT116 and AGS cells showed down-regulation of MIG6 expression and concomitant up-regulation of EGFR and AKT phosphorylation. Reconstitution of MIG6 expression enhanced sensitivity to AZD6244 in resistant cells, while knockdown of MIG6 expression increased resistance to AZD6244 in sensitive cells. Finally, I observed that combination of MEK and EGFR inhibitor blocked both EGFR and AKT activation, and synergistically inhibited cell proliferation in HCT116 cells. Third, given that cancer cells have multiple genetic alterations combinatorial therapeutic strategy is demanded for effective cancer therapy. To address this, I first characterized MEK dependence in four NSCLC cells. Two cells (H358, A549) carried KRAS mutation only, and the other two (H23, H157) harbored co-mutation of KRAS/PTEN. H358 cells with KRAS mutation only were sensitive to MEK inhibition. However, the other KRAS MT A549 cells and KRAS/PTEN co-MT H23 and H157 cells were resistant to MEK inhibition. Compared to KRAS MT/PTEN WT cells, STAT3 was significantly activated following MEK inhibition in KRAS/PTEN co-MT cells. Combined STAT3 inhibition by a JAK2 inhibitor or gene knockdown with MEK inhibition blocked STAT3 activation, synergistically suppressed cell growth, and induced apoptosis in co-mutant cells. In conclusion, I provide the basis for a rational combination strategy against human EGFR WT gastric cancers, predicated on the understanding of cross-talk between the MEK and EGFR pathways. Furthermore, I found that mitogen-inducible gene 6 (MIG6) was critical roles between MEK and EGFR pathways in EGFR WT/KRAS MT cancer cells. Finally, I showed that EGFR WT/KRAS MT cancer cells were frequently co-mutated with phosphatase and tensin homolog (PTEN), resulting in resistance to MEK inhibition. Hence, in order to achieve the maximum effect of MEK inhibitors in EGFR WT/KRAS MT cancer, combinatorial approach was needed due to compensatory signaling activation by additional genetic alteration and/or endogenous negative inhibitor.