Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell
Nao Muraki1, Mizuki Yamada1, Hinako Doki1
1Division of Host Defense Sciences, Dept. of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Japan.
Abstract:
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 KRASV12 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 KRASV12-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 KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated β-galactosidase staining (SA-βG) method. Furthermore, mutant KRASV12 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 KRASV12 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 KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.
Insights
Mutant KRAS oncogene induces senescence, a cancer prevention mechanism. However, in immortalized bronchial cells with upregulated telomerase and inactivated p16/RB pathway, KRAS-induced senescence is less effective, promoting transformation.
Area of Science:
- Oncology
- Cell Biology
- Molecular Biology
Background:
- Mutant KRAS is a frequent driver oncogene in lung adenocarcinoma (~30%).
- Oncogene-induced senescence (OIS) is a tumor-suppressive mechanism preventing malignant transformation.
- Previous studies showed mutant KRASV12 induces OIS in a subset of immortalized human bronchial epithelial cells.
Purpose of the Study:
- Investigate the mechanism and efficacy of OIS in normal bronchial cells with upregulated telomerase and p16/RB pathway inactivation.
- Determine if OIS effectively suppresses KRASV12-induced transformation under these specific cellular conditions.
Main Methods:
- Utilized a doxycycline-inducible system in an immortalized human bronchial epithelial cell line (HBEC3KT) expressing mutant KRASV12 (HBEC3-RIN2).
- Assessed OIS using senescence-associated β-galactosidase staining (SA-βG).
- Evaluated cell proliferation, colony formation, epithelial-to-mesenchymal transition (EMT), autophagic flux, and key protein expressions (ZEB1, SNAIL, p53, p21, AKT, ERK).
Main Results:
- Mutant KRASV12 induced partial EMT, enhanced autophagic flux, and increased cell growth and colony formation.
- Only a small subset of HBEC3-RIN2 cells underwent senescence upon KRASV12 induction.
- Mutant KRASV12 reduced p53 but increased p21 protein expression by prolonging its half-life, without affecting cell division or cell cycle.
Conclusions:
- An hTERT/Cdk4-immortalized normal bronchial epithelial cell line exhibits partial resistance to mutant KRASV12-induced senescence.
- OIS is not fully efficient in suppressing KRASV12-induced transformation when telomerase is upregulated and the p16/RB pathway is inactivated.
- These findings highlight the complex interplay between OIS, cellular immortalization, and oncogene-driven transformation in lung cancer.
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