Division of Host Defense Sciences, Dept. of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Japan.
Division of Host Defense Sciences, Dept. of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Japan; Dept. of Respiratory Medicine, Nagoya University Graduate School of Medicine, Japan.
Exp Cell Res. 2022 May 1;414(1):113053. doi: 10.1016/j.yexcr.2022.113053. Epub 2022 Feb 8.
Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called "oncogene-induced senescence (OIS)", prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRAS induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRAS-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called "HBEC3KT"), immortalized with hTERT ("T") and Cdk4 ("K"), was used in this study. HBEC3 that expressed mutant KRAS in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRAS expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRAS caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRAS expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated β-galactosidase staining (SA-βG) method. Furthermore, mutant KRAS enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRAS reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRAS-induced senescence. This suggests that OIS does not efficiently suppress KRAS-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.
突变型 KRAS 是肺腺癌中最常见的（约 30%）驱动癌基因，它使正常上皮细胞发生衰老。这种现象称为“癌基因诱导的衰老（OIS）”，可防止突变型 KRAS 诱导的恶性转化。我们之前报道过，突变型 KRAS 在一组通过 hTERT 和 Cdk4 永生化的正常人类支气管上皮细胞系中诱导 OIS。了解这一重要癌症预防机制的机制和效果是一个关键的知识空白。因此，本研究调查了突变型 KRAS 在上调端粒酶的同时，通过 p16/RB 通路失活在正常支气管上皮细胞中诱导的 OIS。本研究使用了正常（非转化和非致瘤）人支气管上皮细胞系 HBEC3（也称为“HBEC3KT”），该细胞系通过 hTERT（“T”）和 Cdk4（“K”）永生化。建立了一种以四环素调控方式表达突变型 KRAS 的 HBEC3（命名为 HBEC3-RIN2）。突变型 KRAS 的受控诱导表达导致 HBEC3-RIN2 细胞发生部分上皮-间充质转化，同时上调 ZEB1 和 SNAIL 的表达。突变型 KRAS 使大多数 HBEC3-RIN2 发生形态变化；提示衰老，这与增强的自噬通量有关。在表达突变型 KRAS 后，只有一小部分 HBEC3-RIN2 细胞通过衰老相关β-半乳糖苷酶染色（SA-βG）方法检测到衰老。此外，突变型 KRAS 通过增加磷酸化 AKT 和 ERK 的表达来增强细胞生长，通过比色增殖测定法、液体和软琼脂集落形成测定法进行评估，但不影响细胞分裂或细胞周期状态。有趣的是，突变型 KRAS 通过延长半衰期减少 p53 蛋白表达，但增加 p21 蛋白表达。这些结果表明，hTERT/Cdk4 永生化的正常支气管上皮细胞系对突变型 KRAS 诱导的衰老具有一定的抗性。这表明在同时发生端粒酶上调和 p16/Rb 通路失活的情况下，OIS 不能有效地抑制 KRAS 诱导的转化。
