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). 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. 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.

中文翻译：

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HCC 衍生的 EGFR 突变体具有功能性、EGF 依赖性和厄洛替尼耐药性

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 活化是 HCC 细胞对索拉非尼的原发性耐药的潜在决定因素。在我们之前的研究中，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。本研究旨在确定七种 HCC 衍生突变体（K757E、N808S、R831C、V897A、P937L、T940A 和 M947T）的激酶活性和对厄洛替尼（一种第一代 EGFR 酪氨酸激酶抑制剂 (TKI)）的敏感性。使用带有或不带有 EGFR 的 pBabe-puro 逆转录病毒载体的转导，我们构建并确定了 EGFR 在 NIH-3T3 细胞中的功能，这些细胞稳定地携带了七种突变体，以及厄洛替尼敏感的 L858R 突变体、厄洛替尼耐药的 T790M 突变体和 EGFR 野生型 (WT)。我们的结果表明，这七种突变体具有功能性、EGF 依赖性、EGFR。含有七种突变体中的六种的细胞可以在没有 EGF 的情况下产生某种程度的 EGFR 磷酸化，这表明存在一些组成型激酶活性，但所有七种突变体仍然主要依赖于 EGF。我们的研究结果表明，厄洛替尼在含有不同 EGFR 的细胞中诱导不同程度的细胞凋亡和自噬：L858R 突变体中的完全细胞凋亡和自噬（caspase-3 和 PARP 均被切割，并且 LC3-II 显着增加）；部分凋亡和自噬（仅切割 caspase-3，和中度 LC3-II 增加）在 WT 和 HCC 衍生的突变体中；在 T790M 突变体中没有细胞凋亡和最小的自噬（没有 caspase-3 和 PARP 的切割，以及最小的 LC3-II 增量）。七种 HCC 衍生的突变体对厄洛替尼具有耐药性，因为使用高达高浓度的厄洛替尼治疗只能诱导部分抑制 EGFR 磷酸化、部分或不抑制 AKT 和 ERK 磷酸化以及部分细胞凋亡和自噬。本研究中的七种 HCC 衍生的 EGFR 突变体具有功能性、EGF 依赖性和厄洛替尼耐药性。厄洛替尼在具有不同 EGFR 的细胞中诱导不同程度的细胞凋亡和自噬。厄洛替尼对 EGFR 磷酸化的抑制程度是携带 EGFR 突变体的细胞凋亡和自噬程度的决定因素。