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Published in final edited form as: Exp Cell Res. 2022 Feb 8;414(1):113053. doi: 10.1016/j.yexcr.2022.113053

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line☆

Nao Muraki a,1, Mizuki Yamada a,1, Hinako Doki a,1, Riho Nakai a,1, Kazuki Komeda a,b, Daiki Goto b, Nozomi Kawabe a, Kohei Matsuoka a, Miyoko Matsushima a, Tsutomu Kawabe a, Ichidai Tanaka b, Masahiro Morise b, Jerry W Shay c, John D Minna d, Mitsuo Sato a,*
PMCID: PMC10228173  NIHMSID: NIHMS1899667  PMID: 35149086

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.

Keywords: KRAS, oncogene, telomerase, senescence

1. Introduction

KRAS is the most frequently mutated driver proto-oncogene in non-small cell lung cancer (NSCLC); however, the development of mutant KRAS-targeted drugs has been hampered primarily because of the technical difficulties in developing potent compounds to inhibit constitutively activated signaling of mutant KRAS [1]. Recently, several promising compounds specifically targeting the KRASG12C mutation accounting for 44% of KRAS mutations in NSCLCs have been developed, and show clinical activity [2-5]. Nevertheless, their efficacy is below expectations, and the development of therapeutics for other mutant KRAS types remains unsuccessful. Therefore, we still need to find additional oncogenic KRAS induced vulnerabilities to therapeutically exploit. An important approach would be to understand the mechanism (s) that normal cells invoke when initially exposed to strong oncogenic KRAS signals which leads to induction of cellular senescence. This type of senescence is called oncogene-induced senescence (OIS) [6]. OIS was first reported by Serrano et al.; they discovered that the introducing mutant RAS into normal fibroblasts induced senescence [7]. Other oncogenes, including mutant BRAFV600E, have also been demonstrated to cause OIS [8]. OIS functions as a safeguard system that prevents normal cells from oncogenic transformation [9-11,16]. Therefore, overt cancer cells with activated oncogenes that induce OIS could bypass OIS. The mechanism(s) through which KRAS-mutated lung cancer cells acquire OIS-bypassing ability during carcinogenesis is not fully understood. Importantly, such mechanism(s) potentially could also provide important “therapeutic windows.”

To clarify mechanisms of bypassing KRASV12-induced OIS during lung cancer carcinogenesis, several normal human bronchial epithelial cells strains (HBECs) immortalized without viral oncoproteins serve as powerful tools, partially because these cells have intact p53 function, a major OIS regulator [6,7,12]. We previously demonstrated that the combinatorial introduction of both hTERT, the catalytic subunit of human telomerase, and mouse Cdk4, but not either alone, reproducibly immortalized normal human bronchial epithelial cells [12]. hTERT and Cdk4 were introduced to overcome replicative senescence and p16INK4-mediated stress response, respectively. To transform one clonal cell line derivative, HBEC3 cells to a complete malignant tumorigenic state, we had to provide a combination of at least three further oncogenic changes such as exogenous mutant KRASV12, shRNA-mediated p53 knockdown, and exogenous MYC (c-myc) overexpression, which we could then demonstrate by tumor formation injection in immunodeprived mice [13,14].

A previous study reported that KRASV12 oncogene induces senescence in unimmortalized HBECs [15]. However, in our abovementioned study, we anticipated that two molecular manipulations (telomerase upregulation and abrogation of the p16/RB pathway) used for immortalizing HBEC cells allowed them to bypass mutant KRASV12-induced OIS [13]. Nevertheless, introducing KRASV12 alone induced HBEC3 cells to undergo morphological changes suggestive of senescence, including enlarged and flattened shape and vacuolation, and expression of senescence-associated β-galactosidase staining (SA-βG) [13]. Therefore, we returned to the immortalized HBEC model, which provides for a wide array of manipulation to study steps in oncogenic KRAS induced OIS to determine how these function as a barrier(s) for malignant transformation. In the current study, we have prepared a doxycycline inducible KRASV12 HBEC strain to study the mechanism and efficacy of OIS.

2. Materials and methods

2.1. Cell lines and tissue culture

Three lung cancer cell lines and an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line, HBEC3, were purchased from ATCC (Manassas, USA) or were derived from the Hamon Center Collection (University of Texas Southwestern Medical Center, Dallas, TX, USA) [12,17]. The lung cancer cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Wako, Japan) supplemented with 10% fetal bovine serum (FBS). The HBEC3 cells were cultured in keratinocyte serum-free medium (Life Technologies, USA) supplemented with 50 ng/ml bovine pituitary extract and 5 ng/ml epidermal growth factor.

2.2. RNA extraction and quantitative real-time reverse transcription PCR (qRT-PCR)

Cells were harvested for extracting RNA two weeks after initiating mutant KRASV12 induction. Total RNAwas isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany) and was reverse-transcribed with a Superscript IV First-Strand Synthesis System using a random primer system (Thermo Fisher Scientific, USA). Quantitative real-time PCR analysis of CDH1, VIMENTIN, TWIST, ZEB1, SLUG, SNAIL, p53, p21 and GAPDH used as an internal control, was conducted using SYBR Green Master Mix (Thermo Fisher Scientific). The primer sequences are listed in Supplementary Table S1.

2.3. Vector construction and viral transduction

A mutant KRASV12 amplicon was amplified from a pLenti-KRASV12 vector [18]. The HBEC3 cell line-expressing mutant KRASV12 in a doxycycline-regulated manner was established using the ViraPower HiPerform T-Rex Gateway Vector kit (Thermo Fisher Scientific) (designated as HBEC3-RIN2). Viral transduction of HBEC3 cells was performed using ViraPower (Thermo Fisher Scientific) according to the manufacturer’s protocol. Expression of mutant KRASV12 was induced by adding doxycycline (Dox) (Sigma-Aldrich) to the culture medium at final concentrations of 0, 1, 10, and 100 ng/ml.

2.4. Autophagy analysis

Autophagy was analyzed using Autophagy Watch (MBL, Japan) and CYTO-ID (Enzo Life Sciences, USA).

2.5. Senescence analysis

Three weeks after initiating mutant KRASV12 induction, senescence was analyzed by using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, USA). Averaged percentages of senescent cells were calculated by counting all staining-positive and negative cells (approximately 5000 cells) in eight microscopic fields at a magnification of × 400.

2.6. Cell growth assays

A colorimetric proliferation assay was conducted using the WST-1 assay kit (Roche, Switzerland) according to the manufacturer’s protocol. Liquid and soft agar colony formation assays were conducted as described previously [14]. To determine the effects of mutant KRASV12 expression on proliferation, 2000 cells were plated in 12-well plates in triplicates and viable or dead cells were counted after trypan blue staining 12 d after transduction.

2.7. Cell cycle analysis

Cells were harvested for cell cycle analysis three weeks after initiating mutant KRASV12 induction. The cells were, fixed, treated with RNase A, and stained with propidium iodide using the BD Cycletest Plus Reagent Kit (BD Bioscience, USA) according to manufacturer’s instructions, and analyzed by flow cytometry for DNA synthesis and cell cycle status using a FACSCalibur flow cytometer (BECKMAN COULTER, USA). Results were analyzed using the Flow Jo program (BD Bioscience).

2.8. Western blotting

Western blotting was performed as described previously using whole cell lysates [14]. Cells were harvested to prepare cell lysates two weeks after initiating of mutant KRASV12 induction. Primary antibodies used were mouse anti-E-cadherin (Cat#610181, BD Biosciences), mouse anti-Vimentin Cat#550513, BD Biosciences), mouse anti-p53 (Cat#sc-126, Santa Cruz, USA), mouse anti-p21 Waf/Cip/CDKN1A (Cat#sc-6246, Santa Cruz), mouse anti-KRAS (Cat#sc-30, Santa Cruz), rabbit anti-AKT (pan) (Cat#4691, Cell Signaling Technology), rabbit anti-phospho-AKT (ser 473) (Cat#4060, Cell Signaling Technology), rabbit anti-p44/42MAPK(Erk1/2) (Cat#9102, Cell Signaling Technology), rabbit anti-phospho-p44/42MAPK(Erk1/2) (Cat#4370, Cell Signaling Technology), mouse anti-Actin (Cat#A2228, Sigma-Aldrich), mouse anti-Vinculin (Cat#SC-73614, Santa Cruz), rabbit anti-α-tubulin (Cat# PM054-7, MBL Life Science, USA), and rabbit anti-phospho-p53 (Ser 15) (Cat#9284, Cell Signaling Technology) antibodies. Actin, Vinculin, or α-tubulin protein levels were used as a control for the adequacy of equal protein loading. Anti-rabbit or anti-mouse (GE Healthcare, Buckinghamshire, England) antibodies were used at 1:2000 dilution as secondary antibodies.

2.9. Cycloheximide chase assay

Two weeks after initiating KRASV12 expression induction, cells treated with 5 ng/ml of cycloheximide (Sigma-Aldrich) were harvested for Western blotting of p21 at time points of 0, 0.5, 1, and 2 h. The intensities of p21 bands in Western blotting normalized to β-actin were semi-quantitated by using the image-J software. Fitting of linear regression was done to calculate half-life times.

2.10. Statistics

IBM (International Business Machine) Statistical Package for the Social Sciences v.28(IBM, USA) was used for all statistical analyses in this study. Significant differences (P < 0.05) between control (Dox:0 ng/ml) and mutant KRASV12-induced cells (Dox:1, 10, and 100 ng/ml) cells were analyzed by Dunnett’s test.

3. Results

3.1. Mutant KRASV12 expression induces partial epithelial-to-mesenchymal transition and morphological changes associated with enhanced autophagic flux in HBEC3-RIN2

We obtained tightly regulated protein expression of mutant KRASV12 in HBEC3-RIN2 (Fig. 1A). Maximum levels of mutant KRASV12 levels in HBEC3-RIN2 were comparable to those observed in patient derived NSCLC lines with mutant KRAS (Fig. 1A) [19]. Previous studies have demonstrated that the RAS signaling induces epithelial-to-mesenchymal transition (EMT) [20,21], and others have also shown that EMT allows normal epithelial cells to bypass OIS [22,23]. This prompted us to evaluate whether mutant KRASV12 induced EMT in our model system. Upon KRASV12 expression, HBEC3-RIN2 cells underwent partial EMT, as demonstrated by increased levels of Vimentin (mesenchymal marker) but exhibited unchanged E-cadherin (epithelial marker) levels (Fig. 1A). To identify the transcription factors that were predominantly involved in this partial EMT, we conducted qRT–PCR for four major master EMT-inducing transcription factors. We found that turning on KRASV12 expression was associated with ~5–8 fold upregulated levels of ZEB1 and SNAIL, suggesting that they played roles in the partial EMT phenotype (Fig. 1B).

Fig. 1. Mutant KRASV12 induces partial epithelial-to-mesenchymal transition and morphological changes associated with enhanced autophagic flux in HBEC3-RIN2.

Fig. 1.

Fig. 1.

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A Mutant KRASV12 protein is induced by adding doxycycline (Dox) in a dose-dependently. Upon KRASV12 expression. HBEC3-RIN2 cells undergo partial epithelial to mesenchymal transition (EMT), as demonstrated by increased Vimentin (mesenchymal marker) levels and unchanged E-cadherin (epithelial marker) levels. Cells were harvested for cell lysates two weeks after the initiating mutant KRASV12 induction.

B qRT-PCR of four major master EMT-inducing transcription factors reveals that ZEB1 and SNAIL are upregulated (upper). The ratio of Vimentin to E-cadherin expression as an index that represents the degree of mesenchymal phenotype is shown in the lower graph. Cells were harvested for extracting RNA two weeks after initiating mutant KRASV12 induction.

C Upon mutant KRASV12 expression, HBEC3-RIN2 cells undergo morphological changes of becoming larger, more rounded and more vacuolized. Images were taken 1 week after initiating mutant KRASV12 induction.

D CYTO-ID, an autophagosome-specific staining, reveals that the vacuoles induced by mutant KRASV12 expression are autophagosomes. Images were taken 4 d after initiating mutant KRASV12 induction.

E Western blotting of LC-3 reveals that mutant KRASV12 enhances autophagic flux. Cells were harvested 4 d after initiating mutant KRASV12 induction.

With the increased mutant KRASV12 levels, HBEC3-RIN2 cells underwent morphological changes, becoming larger, more rounded, and more vacuolized, suggesting senescence (Fig. 1C). CYTO-ID, an autophagosome -specific staining [24,25], revealed that these vacuoles were autophagosomes [24,25], suggesting that mutant KRASV12-induced autophagy (Fig. 1D). To confirm this finding, we performed Western blotting of LC-3 (an autophagy marker) and found that the mutant KRASV12 expression was associated with enhanced autophagy (Fig. 1E).

Senescence-associated heterochromatin foci is a phenomenon, first described by Narita et al. they found that human diploid fibroblasts undergoing H-rasV12-induced senescence displayed large nucleoli with punctate DNA foci visualized by DAPI staining that can be easily distinguished from control cells that contain small nucleoli and a more uniform DAPI staining pattern [26]. However, we did not observe large nucleoli or punctate DNA foci in HBEC2-RIN2 cells expressing mutant KRASV12 (Fig. 1D), suggesting that HBEC3-RIN2 cells are resistant to mutant KRASV12-induced senescence.

3.2. Mutant KRASV12-induced senescence occurs in only in a small subset of the population without impairing overall proliferation resulting in enhanced clonal survivability in HBEC3-RIN2

SA-βG staining showed that in response to KRASV12 expression, there was an increase in the number of positive cells, but the percentages were relatively low (<2%) (Fig. 2A). The number of SA-βG positive cells tended to positively correlate with KRASV12 expression levels, but was not statistically significant. By contrast, hydrogen peroxide treatment generated ~55% SA-βG positive cells (Fig. 2A). Mutant KRASV12 expression enhanced clonal growth in both anchorage-dependent and -independent growth (soft agar colony formation) (Fig. 2B and C). Mutant KRASV12 increased the expression of phosphorylated AKT and ERK proteins in both the presence and absence of EGF (Fig. 2D). This suggested that the mutant KRASV12-induced clonal survivability is associated with, and is probably partially attributable to RAF/MEK/ERK and PI3K/AKT/mTOR pathway activation. Cell viability evaluated by colorimetric analysis revealed that KRASV12 enhanced the viability (Fig. 2E), but not cell numbers at 12 d after KRASV12 expression (Fig. 2F). We and other note that the cell viability evaluated by colorimetric assays does not necessarily accurately reflect the number of cells because colorimetric assays generally measure the activity of enzymes in mitochondria [27]. Cell cycle analysis demonstrated that KRASV12 expression did not affect the cell cycle population (Fig. 2G).

Fig. 2. Mutant KRASV12 induces senescence only in a small population without impairing proliferation but enhances clonal growth ability in HBEC3-RIN2.

Fig. 2.

Fig. 2.

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A Three weeks after initiating mutant KRASV12 induction, SA-βG staining reveales that KRAS expression increases the number of positive cells, although at low percentages (< 2%). The numbers of positive cells tend to positively correlate with KRASV12 expression levels but there is no statistical significance. Cells treated with hydrogen peroxide were used as positive controls. Averaged percentages of senescent cells from eight microscopic fields at a magnification of ×400 are shown as mean± standard deviation (SD).

B., C Mutant KRASV12 expression enhances clonal growth in both anchorage dependent (B) and independent (C) growth. Results are shown as mean ± SD. ** indicates P < 0.01 (Dunnett’s test).

D Mutant KRASV12 increases the expression of phosphorylated AKT and ERK proteins in both the presence and absence of EGF.

E Cell viability evaluated by colorimetric analysis shows KRASV12 enhances viability. Results are shown as mean ± SD. *, and ** indicate P < 0.05, and P < 0.01 (Dunnett’s test), respectively.

F Cell number counted at 12 d after transduction shows that KRASV12 expression does not affect the numbers of cells. Results are shown as mean ± SD.

G Cell cycle analysis shows that KRASV12 expression does not affect the cell cycle population. Cells were harvested and fixed for cell cycle analysis three weeks after initiating mutant KRASV12 induction.

These results indicate that the acute expression of mutant KRASV12 in HBEC3-RIN2 induced senescence only in a small population without impairing proliferation but enhanced the clonal growth ability in both anchorage-dependent and -independent conditions.

3.3. Mutant KRASV12 decreases p53 expression but increases p21 protein expression by prolonging its half-life in HBEC3-RIN2

Two pivotal pathways, the p16/RB and the p21/p53 pathways, regulate cellular senescence induced by mutant KRASV12 [6,7] To obtain molecular insights into why mutant KRASV12 in HBEC3-RIN2 cells did not cause significant growth suppression through OIS, we investigated the status of the p21/p53 pathway in KRASV12-induced HBEC3-RIN2 cells because the p16/RB pathway has been abrogated by Cdk4 overexpression by our immortalization process [12,14,28]. We hypothesize that the p21/p53 pathway could be abrogated by mutant KRASV12, resulting in an OIS-resistant phenotype. We observed that mutant KRASV12 modestly suppressed p53 protein expression but increased p21 protein expression at the highest levels of KRASV12 activation (Fig. 3A). While p53 protein expression was only modestly altered, p53 mRNA expression was suppressed 5-fold by mutant KRASV12, suggesting that suppression of p53 protein expression by mutant KRASV12 occurred primarily at a transcriptional level (Fig. 3B). A previous study reported that mutant KRAS but not mutant HRAS or NRAS reduced p53 expression in cancer cell lines [29]. Furthermore, a recent study demonstrated that a reduction in p53 protein expression induced by mutant KRAS, occurs through impaired stability of p53 protein due to reduced phosphorylation at Ser-15 [30]. The study conducted KRAS knockdown experiments using the A549 lung cancer cell line harboring wildtype p53 and mutant KRAS as a model system. However, in our HBEC3-RIN2 system, p53 phosphorylated at Ser-15 was not detected in mutant KRASV12-on or -off cells (Supplementary Figure. S1). Unexpectedly, p21 mRNA expression was unaffected by mutant KRASV12 despite increased p21 protein expression (Fig. 3B). This result suggested that mutant KRASV12 expression prolongs the half-life of p21 protein. To examine this possibility, we conducted a cycloheximide chase assay and found that mutant KRASV12 prolonged 2-fold the half-life of p21 protein expression (Fig. 3C). These data showed that in response to mutant the KRASV12 expression, HBEC3-RIN2 cells increased protein expression of p21 by prolonging its half-life. However, this p21 induction by mutant KRAS did not apparently induce senescence significantly as only a few HBEC3-RIN2 cells underwent OIS in response to mutant KRASV12 expression (Fig. 2A).

Fig. 3. Mutant KRASV12 decreases p53 expression but increases p21 protein expression by prolonging its half-life in HBEC3-RIN2.

Fig. 3.

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A Mutant KRASV12 suppresses p53 protein expression but increases p21 protein expression.

B A qRT-PCR analysis reveals that mutant KRASV12 expression results in downregulated p53 and unchanged p21 mRNA expression. qPCR was performed in triplicate. Results are shown as mean ± SD. ** indicates P < 0.01 (Dunnett’s test).

C Cycloheximide chase assay reveals that mutant KRASV12 prolongs the half-life of p21.

4. Discussion

Our results demonstrate that activated expression of mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence simultaneously with partial changes of EMT, but only ~2% of the cells undergo full OIS with expression of SA-βG. Because of this, we did not see growth or viability suppression and actually found a several fold increase in anchorage-dependent and -independent colony formation. Furthermore, we found that the mutant KRASV12 expression reduced the p53 protein expression in HBEC3-RIN2 cells at the transcriptional level but, unexpectedly, increased p21 protein by prolonging its half-life. These results can be interpreted to indicate that normal human bronchial epithelial cells immortalized by hTERT and Cdk4 are at least partially resistant to mutant KRASV12-induced OIS. These findings have a critical ramification of developing therapeutics of OIS induction for human lung cancer [6], because hTERT upregulation and the p16/CDK4/RB pathway abrogation occur almost universally in human lung cancer pathogenesis [31]. Thus, building on the information in this report including the induction of partial EMT, upregulation of p21 by half life alteration, and downregulation of p53 mRNA expression we need to explore ways to overcome this OIS resistance.

Mutant KRASV12-induced partial EMT, as shown by increased Vimentin and unchanged E-cadherin protein expression in HBEC3-RIN2, associated with the upregulation of ZEB1and SNAIL but not TWIST or SLUG. Several studies have reported that KRAS activation induces EMT in human cancers, including ovarian and pancreatic cancers [20,21]. Similar to our results, one of these studies found that ZEB1and SNAIL plays a crucial role in mutant KRAS-induced EMT in pancreatic cancer [20]. In addition, we and others have demonstrated a critical role of ZEB1 in regulating EMT in human lung cancers [32-34]. However, no studies have examined which master EMT-inducing transcription factors play a crucial role in mutant KRAS-induced EMT in normal lung epithelial cells. Therefore, our investigations suggest the potential critical roles of ZEB1 and SNAIL in this type of EMT. Moreover, as EMT allows OIS bypassing [22,23], we hypothesize that the partial EMT-induced by mutant KRASV12 in HBEC3-RIN2 cells contributes, at least in part, to the OIS-resistant phenotype.

Furthermore, we found that mutant KRASV12 increased autophagic vacuole formation in HBEC3-RIN2 cells. Our previous study showed that mutant KRASV12 induced vacuole formation in HBEC3 cells [13]. In this study, through CYTO-ID staining, we demonstrate that the vacuoles are autophagic vacuoles. A previous study found that mutant KRASV12 induces autophagic vacuole formation in normal breast epithelial cells [35]. Additionally, the study further demonstrated that the enhanced autophagy contributed to mutant KRASV12-induced malignant transformation by performing ATG5 and ATG7 (two important autophagic-specific genes) silencing. To investigate whether enhanced autophagy in HBEC3-RIN2 cells also contributes to malignant transformation, we examined the effects of the knocking down ATG4B, ATG5, and ATG7 on the growth of HBEC3-RIN2 cells, but did not observe any significant difference between mutant KRASV12-on and -off cells (data not shown). Thus, further studies are needed to investigate the roles of autophagy in mutant KRASV12-induced malignant transformation in normal lung epithelial cells.

Cycloheximide chase assay for p21 protein indicated that the half-life of p21 was prolonged ~2-fold by mutant KRASV12 expression. Furthermore, a previous study reported that AKT activation prolongs the half-life of p21 by phosphorylating its two sites (Thr [145] and Ser [146]) in the carboxyl terminus [36]. Since we observed AKT activation in HBEC3-RIN2 cells expressing mutant KRASV12 (Fig. 2D), we speculate that the prolonged half-life of p21 was attributable to AKT activation, induced by mutant KRASV12.

In conclusion, the combinatorial introduction of hTERT and Cdk4 appears to confer partial resistance to mutant KRASV12-induced OIS on normal human bronchial epithelial cells. However, further analysis will be required to elucidate the underlying molecular mechanisms of this OIS-resistant phenotype since such knowledge will contribute to the development of OIS-inducing therapeutics for lung cancer.

Supplementary Material

Muraki Exp Cell Res Suppl Files

Acknowledgements

This work was supported, in part, by Grant-in-Aid for Exploratory Research 19K22617 and Grant-in-Aid for Scientific Research (B) 21H02924 for M. Sato, and Lung Cancer SPORE (P50 CA070907), and CPRIT RP160652 for J. Minna, J. Shay.

Footnotes

Declaration of competing interest

On behalf of all authors above, I certify that all the authors except John D. Minna declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. John D. Minna receives royalties from the NIH, USA and UT Southwestern for distribution of human cell lines.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.yexcr.2022.113053.

☆

JDM receives royalties from the NIH, USA and UT Southwestern for distribution of human cell lines. The other authors do not have any conflicts of interest to declare.

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