BACKGROUND & AIMS:
Background:Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target. Overexpression of EGFR is frequently observed in hepatocellular carcinoma (HCC) and EGFR activation has been proven to be a potential determinant of primary resistance of HCC cells to sorafenib. In our previous study, we found 13 missense mutations in EGFR exon 19-23 from hepatocellular carcinoma (HCC) tissues, but the functions of these mutations have not been determined. This study aims to determine the kinase activity and sensitivity to erlotinib, a 1st-generation EGFR-tyrosine kinase inhibitor (TKI), of seven HCC-derived mutants (K757E, N808S, R831C, V897A, P937L, T940A, and M947T).
Results:Using transduction of pBabe-puro retroviral vector with or without EGFR, we constructed and determined the function of EGFRs in NIH-3T3 cells stably harboring each of the seven mutants, as well as the erlotinib-sensitive L858R-mutant, the erlotinib-resistant T790M-mutant, and EGFR wild type (WT). Our results indicate that the seven mutants are functioning, EGF-dependent, EGFRs. Cells harboring six of the seven mutants could generate some level of EGFR phosphorylation in the absence of EGF, indicating some constitutive kinase activity, but all of the seven mutants remain primarily EGF-dependent. Our results demonstrate that erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs: complete apoptosis and autophagy (cleavage of both caspase-3 and PARP, and marked LC3-II increment) in L858R-mutant; partial apoptosis and autophagy (only cleavage of caspase-3, and moderate LC3-II increment) in WT and HCC-derived mutants; and no apoptosis and minimal autophagy (no cleavage of caspase-3 and PARP, and minimal LC3-II increment) in T790M-mutant. The seven HCC-derived mutants are erlotinib-resistant, as treatment with erlotinib up to high concentration could only induce partial inhibition of EGFR phosphorylation, partial or no inhibition of AKT and ERK phosphorylation, and partial apoptosis and autophagy.
Conclusion:The seven HCC-derived EGFR mutants in this study are functioning, EGF-dependent, and erlotinib-resistant. Erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs. The degree of inhibition of EGFR phosphorylation by erlotinib is the determining factor for the degree of apoptosis and autophagy amongst cells harboring EGFR mutants. This study paves the way for further investigation into the sensitivity of these HCC-derived mutants to the 3rd-generation irreversible EGFR-TKI, osimertinib.
背景与目标:
背景:表皮生长因子受体(EGFR)已成为重要的治疗靶点。在肝细胞癌(HCC)中经常观察到EGFR的过表达,并且已证明EGFR激活是HCC细胞对索拉非尼原发耐药性的潜在决定因素。在我们先前的研究中,我们在肝细胞癌(HCC)组织的EGFR外显子19-23中发现了13个错义突变,但这些突变的功能尚未确定。这项研究旨在确定七个HCC衍生突变体(K757E,N808S,R831C,V897A,P937L,T940A和M947T)的激酶活性和对第一代EGFR酪氨酸激酶抑制剂(TKI)的厄洛替尼的敏感性。
结果:通过转导含或不含EGFR的pBabe-puro逆转录病毒载体,我们构建并确定了EGFR在稳定携带7个突变体以及厄洛替尼敏感的L858R突变体,厄洛替尼-的NIH-3T3细胞中的功能。抗性的T790M突变体和EGFR野生型(WT)。我们的结果表明,这七个突变体是功能性的,依赖EGF的EGFR。携带7个突变体中的6个的细胞在没有EGF的情况下可以产生一定水平的EGFR磷酸化,表明某些组成性激酶活性,但所有7个突变体仍然主要依赖于EGF。我们的结果表明,厄洛替尼在具有不同EGFR的细胞之间诱导不同程度的凋亡和自噬:在L858R突变体中完全凋亡和自噬(裂解caspase-3和PARP,并标记LC3-II增量); WT和HCC衍生突变体中的部分细胞凋亡和自噬(仅裂解caspase-3,且LC3-II中等增加);并且在T790M突变体中没有凋亡和最小限度的自噬(不切割caspase-3和PARP,最小的LC3-II增量)。七个HCC衍生突变体均具有埃洛替尼抗性,因为高浓度的埃洛替尼治疗只能诱导EGFR磷酸化的部分抑制,AKT和ERK磷酸化的部分抑制或无抑制,部分细胞凋亡和自噬。
结论:本研究中的七个HCC衍生的EGFR突变体具有功能性,EGF依赖性和埃洛替尼耐药性。厄洛替尼在具有不同EGFR的细胞之间诱导不同程度的凋亡和自噬。厄洛替尼对EGFR磷酸化的抑制程度是决定携带EGFR突变体的细胞凋亡和自噬程度的决定因素。这项研究为进一步研究这些HCC衍生突变体对第三代不可逆EGFR-TKI奥西替尼的敏感性铺平了道路。