Introduction
Lung cancer is a primary cause of tumor-related
mortality across the world, with >2 million cases diagnosed
worldwide, contributing to almost 1.8 million deaths annually.
Non-small cell lung cancer (NSCLC) being the most prevalent subtype
and constituting 85% of all cases (1). NSCLC is characterized by chemotherapy
resistance and high invasiveness, which contribute to its poor
prognosis (2). Despite advances in
diagnosis and treatment, patient outcomes have shown limited
improvement, rendering the overall prognosis largely unsatisfactory
(3). Senescence serves a key role
in responding to chemotherapy and suppressing tumorigenesis in
vivo and in vitro (4).
Correspondingly, the ability of tumor cells to bypass senescence is
an important contributor to tumor growth and progression both at
the early stage of carcinogenesis and during treatment failure.
Despite this, the mechanisms underpinning this escape remain
elusive due to the theoretical irreversibility of senescence
(5); identifying these specific
mechanisms is therefore key in the development of novel therapeutic
strategies against NSCLC.
Notably, the tumor-promoting effect of amphiregulin
(AREG) has been established in different cancer types, including
epithelial ovarian and pancreatic cancer (6,7). AREG
expression, transcriptionally induced by incense burning smoke or
its component auramine, has been shown to enhance NSCLC progression
and sensitize NSCLC cells to EGFR tyrosine kinase inhibitors
(8). However, the specific role of
AREG in the senescence escape of NSCLC cells remains to be further
studied.
Musculoaponeurotic fibrosarcoma oncogene homolog K
(MAFK) was selected as the key regulatory factor belong to the
basic leucine zipper family, possessing evolutionarily conserved
basic ZIP domains (9,10). MAFK is a small protein with a
molecular weight of ~18 kDa in the MAF family, located
predominantly in the nucleus (11).
In addition, MAFK demonstrates tumor-promoting activity in lung
adenocarcinoma (12). Senescent
cells modify the immediate microenvironment using a
senescence-associated (SA) secretome, a process termed SA secretory
phenotype (SASP) (13). It was
hypothesized that chemotherapy-induced cell senescence may regulate
MAFK expression in cells via SASP. Notably, SASP can secrete
inflammatory factors and chemokines, with the proinflammatory
factor IL-1β promoting the transcription of MAFF, a functionally
relevant paralog of MAFK (13,14).
Thus, chemotherapy-induced cell senescence may activate MAFK
expression through inflammatory factors. Despite this, it remains
unclear whether MAFK regulates AREG to mediate the escape of NSCLC
cells from drug-induced senescence.
In the present study, the effect of the interplay
between MAFK and AREG on escape of NSCLC cells from doxorubicin
(DOX)-induced senescence was investigated, with the aim of gaining
novel insights into NSCLC treatment.
Materials and methods
Bioinformatic analysis
Human senescence-related genes were obtained using
the Aging Atlas database (ngdc.cncb.ac.cn/aging/index), with 2,916
relevant gene targets screened with a relevance score threshold of
>5. Differentially expressed genes (DEGs) induced by
chemotherapy were identified using the R package ‘limma’ (version
4.4.1; Posit Software) using the Gene Expression Omnibus
(ncbi.nlm.nih.gov/geo/) dataset GSE6410 (15), with thresholds of log2
fold change >0.3 and P<0.05. KEGG enrichment analysis
(https://www.kegg.jp/) of intersection genes was
performed using the clusterProfiler package (version 4.4.1), with
significant pathways defined as P<0.05.
Cell culture, treatment and
transfection
NSCLC cell lines A549 (cat. no. CL-0016) and PC-9
(cat. no. CL-0668) were maintained in media (cat. nos. CM-0016 and
CM-0668; all Procell Life Science and Technology Co., Ltd.) in a
humidified atmosphere with 5% CO2 at 37°C. All cells
were authenticated through short tandem repeat analysis and tested
for Mycoplasma contamination. Untreated cells were
designated as the control group.
To induce senescence, A549 and PC-9 cells were
exposed to 100 nM DOX (cat. no. HY-15142A; MedChemExpress) for 4
days at 37°C (16). For generating
persistent A549 or PC-9 cell (PAC/PPC), the senescent cells were
washed with PBS (cat. no. C0221A; Beyotime Biotechnology) and
stimulated with fresh media containing 10% FBS (cat. no. C0252;
Beyotime Biotechnology) for 10 days at 37°C.
pGPU6-cloned short hairpin RNA against AREG
(pGPU6-shAREG; cat. no. C02001; 5′-CCTCTTTCCAGTGGATCATAA-3′),
pEX-2-cloned plasmids overexpressing MAFK (pEX-2-MAFK; cat. no.
C05002) and their respective negative controls (NCs) pGPU6-cloned
shNC (cat. no. C02001; 5′-CCTAAGGTTAAGTCGCCCTCG-3′) and
pEX-2-cloned NC (cat. no. C05002) were procured from Shanghai
GenePharma Co., Ltd. A total of 0.5 µg of each plasmid was used for
transfection.
A total of 2 days after treatment, A549 and PC-9
cells (~80% confluence during the emergence phase of persistent
cells) were transfected (24 h at 37°C) with shAREG, shNC, plasmids
overexpressing MAFK or the empty vector NC using
Lipofectamine™ 3000 Transfection Reagent (cat. no.
L3000015; Thermo Fisher Scientific, Inc.). For western blotting,
cells were cultured in DMEM (cat. no. C0891; Beyotime
Biotechnology) containing 10% FBS for 24 h following transfection,
and then harvested for lysis 48 h after transfection.
SA-β-galactosidase (SA-β-gal)
assay
Using a SA-β-gal assay kit (cat. no. 9860; Cell
Signaling Technology, Inc.), SA-β-gal activity was measured to
assess A549 and PC-9 cell senescence. Briefly, cells were fixed
with 4% paraformaldehyde (cat. no. C104190; Shanghai Aladdin
Biochemical Technology Co., Ltd.) at room temperature for 15 min
and treated with SA-β-gal reaction solution overnight at 37°C. A
TE2000 fluorescence microscope (Nikon Corporation) was utilized to
detect the positively stained cells in 20 randomly selected fields
of view at ×200 magnification. The activity of SA-β-gal was
determined by the proportion of the positively stained cells
(4).
Flow cytometry assay
The proportion of senescent cells (designated as
persistent A549/PC-9 senescent cells; PAS/PPS) and proliferating
cells (designated as persistent A549/PC-9 dividing cells; PAD/PPD)
within the emerging cell population (defined as PACs/PPCs in the
preceding methods) was detected through a flow cytometry assay.
Trypsinized PAS and PPS cells were washed with PBS, permeabilized
using cold 70% ethanol (cat. no. E130059; Shanghai Aladdin
Biochemical Technology Co., Ltd.) at 4°C for 30 min and washed
twice in Teknova 1X PBS with 0.1% Tween 20 and 1% BSA (cat. no.
11020021; Thermo Fisher Scientific, Inc.). A total of
2×105 cells was incubated with FITC mouse anti-human
Ki67 (cat. no. 556026) or FITC mouse IgG1 (cat. no. 550616; both BD
Pharmingen™; BD Biosciences) as control isotype for 30
min at room temperature. The forward-scatter (FSC)/side-scatter
(SSC) values and the corresponding Ki67 plots were analyzed using
the Attune™ NxT flow cytometer (cat. no. A24860; Thermo
Fisher Scientific, Inc.) and the results were calculated using
FlowJo software (version 10.8.1; FlowJo; BD Biosciences) (4). Based on Ki67 expression levels, cells
with any detectable Ki67 positivity (Ki67+) were defined
as the proliferating population (PAD/PPD), while Ki67-negative
(Ki67−) cells were collected as the senescent population
(PAS/PPS), respectively, using a BD FACSAria III cell sorter (BD
Biosciences). The sorted cells were collected for subsequent
analyses.
Reverse transcription-quantitative PCR
(RT-qPCR)
Total RNA was extracted from A549 and PC-9 cells
using TRI reagent® (cat. no. 93289; Sigma-Aldrich; Merck
KGaA), followed by quantification using a DR6000 UV
spectrophotometer (HACH China). Complementary DNA was synthesized
using SuperScript™ IV reverse transcriptase (cat. no.
18090010; Thermo Fisher Scientific, Inc.) at 50°C for 10 min.
RT-qPCR analysis was performed with TB Green® Premix Ex
Taq™ II (cat. no. RR820A; Takara Bio, Inc.) on the CFX
Connect™ Real-Time PCR Detection System (cat. no.
1855201; Bio-Rad Laboratories, Inc.). The RT-qPCR conditions were
as follows: Denaturation at 95°C for 5 min, followed by 40 cycles
of 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. The
relative expression of MAFK and AREG was calculated using the
2−ΔΔcq method and normalized to that of GAPDH (17). Primers were as follows: MAFK
forward, 5′-GAGAGTGGCTCACACGTCG-3′ and reverse,
5′-CTGCTCACCGTCAAATGATGG-3′; AREG forward,
5′-GTGGTGCTGTCGCTCTTGATA-3′ and reverse,
5′-CCCCAGAAAATGGTTCACGCT-3′; IL-6 forward,
5′-CGAGCCCACCGGGAACGAAA-3′ and reverse,
5′-GCTTCGTCAGCAGGCTGGCAT-3′; IL-8 forward,
5′-TTTTGCCAAGGAGTGCTAAAGA-3′ and reverse,
5′-AACCCTCTGCACCCAGTTTTC-3′ and GAPDH forward,
5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse,
5′-GGCTGTTGTCATACTTCTCATGG-3′.
Chromatin-immunoprecipitation (ChIP)
assay
A549 and PC-9 cells were cross-linked with
formaldehyde (cat. no. F111941; Shanghai Aladdin Biochemical
Technology Co., Ltd.) for 10 min at room temperature and the
reaction was terminated by 5 min incubation with 0.125 M glycine
(cat. no. 1686386; Sigma-Aldrich; Merck KGaA). Cells were washed
three times in cold PBS, scraped and washed three times again.
Pellets were resuspended in 1 ml lysis buffer (cat. no. RABLYSIS1;
Sigma-Aldrich; Merck KGaA). Protease and phosphatase inhibitors
(cat. no. PPC1010; Sigma-Aldrich; Merck KGaA) were added to all
buffers. Samples were incubated for 15 min at 4°C and mixed by
vortex for 30 sec every 2 min. Following centrifugation at 7,200 ×
g for 10 min at 4°C, the supernatant was removed. Pellets were
resuspended in 500 µl SDS lysis buffer (cat. no. 20-163;
Sigma-Aldrich; Merck KGaA) to obtain DNA fragments. The supernatant
was centrifuged at 7,200 × g for 10 min at 4°C and diluted using IP
buffer (cat. no. 20-153; Sigma-Aldrich; Merck KGaA). Each 500 µl
extract was incubated with 5 µl dithiothreitol (0.1 M;
Sigma-Aldrich; Merck KGaA) and 15 µl Protein A/G magnetic beads
(cat. no. 88802; Thermo Fisher Scientific, Inc.) pre-conjugated
with 2 µg specific antibodies overnight at 4°C. After washing in
Tris-EDTA buffer (cat. no. 93283; Sigma-Aldrich; Merck KGaA), 200
µl fresh elution buffer (1% SDS and 0.1 M sodium bicarbonate) was
used to elute samples. Following reversal of the DNA-protein
cross-links, DNA was purified using a DNA Purification kit (cat.
no. D0033; Beyotime Biotechnology) and subjected to PCR
amplification as aforementioned targeting the AREG promoter region.
Antibodies were as follows: AREG (cat. no. sc-74501; 1:200; Santa
Cruz Biotechnology, Inc.), MAFK (cat. no. ab229766; 1:200; Abcam)
and control IgG (cat. no. 3900; 1:200; Cell Signaling Technology,
Inc.). The regions were amplified using the primer for AREG
promoter region from −1,263 to −1,249 relative to the transcription
start site (forward, 5′-TTTGGACTACGCACTGTGAT-3′; and reverse,
5′-CAGTGCATCTGTCCTAATTCTGG-3′).
Dual-luciferase reporter assay
A549 and PC-9 cells were co-transfected with
MAFK-overexpressing plasmids/empty vectors (pEX-2 vector;
GenePharma Co., Ltd.) and AREG wild-type (WT) or mutant (MUT) by
Lipofectamine 3000 Transfection Reagent (cat. no. L3000015; Thermo
Fisher Scientific, Inc.) at 37°C. After 48 h of transfection,
Firefly and Renilla luciferase activities within cells were
determined using the Dual-Luciferase® Reporter Assay
System (cat. no. E1910; Promega Corporation) according to the
manufacturer's instructions. Data were measured using a Veritas
Microplate Luminometer (Turner BioSystems; Promega
Corporation).
Western blotting
Expression of MAFK, AREG and senescence-related
protein p21 wild-type p53-activated fragment 1 (p21waf1) in A549
and PC-9 cells was measured through western blotting. RIPA lysis
buffer (cat. no. 20-188; Sigma-Aldrich; Merck KGaA) was used for
cell lysis. Proteins were quantified using a BCA Protein Assay kit
(cat. no. 7780S; Cell Signaling Technology, Inc.). Equal amounts of
protein were separated by 12% SDS-PAGE and transferred to PVDF
membranes (cat. no. 88518; Thermo Fisher Scientific, Inc.).
Following 2 h incubation in 5% non-fat milk at room temperature,
the membranes were incubated overnight at 4°C with the following
primary antibodies: MAFK (cat. no. ab50322; 17 kDa; 1:4,000;
Abcam), AREG (cat. no. ab89119; 28 kDa; 1:1,000; Abcam), p21waf1
(cat. no. 2947; 21 kDa; 1:1,000; Cell Signaling Technology, Inc.)
and heat-shock cognate protein 70 (loading control; cat. no.
ab51052; 71 kDa; 1:1,000; Abcam). Membranes were washed three times
with TBS with 0.1% Tween-20 (Beijing Solarbio Science &
Technology Co., Ltd.) and incubated for 1 h with the horseradish
peroxidase-conjugated secondary goat anti-mouse (cat. no. A3562)
and anti-rabbit IgG (cat. no. AP132; both 1:3,000; both
Sigma-Aldrich; Merck KGaA) at room temperature. Visualization was
performed using Pierce™ ECL Plus Western Blotting
Substrate (cat. no. 32132X3; Thermo Fisher Scientific, Inc.). The
blots were detected using FluorChem M Chemiluminescence Imaging
Analysis System (Protein Simple; Bio-Techne) and semi-quantified
using ImageJ software (version 1.53; National Institutes of
Health).
Colony formation assay
A cell colony is a cluster of cells formed by the
growth of a single ancestor cell in culture; in the present colony
formation assay, a colony was defined as containing ≥60 cells. A549
and PC-9 cells subjected to DOX treatment, AREG knockdown or MAFK
overexpression were seeded into a 6-well plate (cat. no. 140675;
Thermo Fisher Scientific, Inc.) with 1×103 cells/well
and maintained in culture medium at 37°C. After 9 days, the
colonies were exposed to 4% formaldehyde for 10 min at room
temperature, then stained with 0.1% crystal violet (cat no. G1062;
Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at
room temperature. Colony images were acquired using a light
microscope (IX73, Olympus, Japan) and captured with a digital
camera (Canon EOS 857D; Canon, Inc.) was employed to capture the
colonies. Colony numbers were manually counted in three random
fields per well.
Statistical analysis
Statistical analyses were performed using GraphPad
Prism (version 8.0; Dotmatics). All data are presented as the mean
± SD of three independent experiments. Two-group comparisons were
performed using the independent unpaired sample t-test. Multi-group
comparisons were performed using one-way ANOVA, followed by Tukey's
post hoc test. Normality (Shapiro-Wilk test) and homogeneity of
variance (Levene's test) were also assessed. P<0.05 was
considered to indicate a statistically significant difference.
Results
DOX induces senescence and suppresses
proliferation of NSCLC cells, while significantly enhancing the
proliferative capacity in persistent cells
A total of 138 senescence-related DEGs were
identified by intersecting genes from Aging Atlas and the GSE6410
dataset (Fig. 1). Kyoto
Encyclopedia of Genes and Genomes enrichment analysis revealed that
these genes were significantly enriched in key signaling pathways
including ‘cell cycle’ and ‘MAPK signaling pathway’ (Fig. 2A). Among the DEGs, AREG was markedly
involved in four pathways (Fig. 2B and
C) and exhibited the most significant expression difference.
Based on its role in a number of senescence-related pathways and
pronounced differential expression, AREG was selected as the target
gene for experimental validation. To explore how DOX influenced
NSCLC cell senescence, A549 and PC-9 cells were exposed to 100 nM
DOX for 4 days. The expression of senescence-related protein
p21waf1 in the cells was identified and western blotting results
exhibited DOX upregulated p21waf1 expression in A549 and PC-9 cells
(Fig. 3A-C). The proportion of
SA-β-gal-positive cells and expression of IL-6 and IL-8 in both
A549 and PC-9 cells increased after DOX treatment (Fig. 3D-I). These experimental data
revealed DOX induced NSCLC cell senescence. Persistent cells were
generated from A549 and PC-9 cells. DOX suppressed A549 and PC-9
cell proliferation but enhanced persistent cell proliferation
(Fig. 3J and K). Therefore, DOX
induced senescence and suppressed proliferation of NSCLC cells,
while enhancing the proliferative capacity in persistent cells.
 | Figure 3.DOX induces senescence and suppresses
proliferation of NSCLC cells, while enhancing proliferation of
persistent cells. (A) Western blot analysis of senescence-related
protein p21waf1 levels in (B) A549 and (C) PC-9 cells following DOX
treatment for 4 days. The effect of DOX treatment for 4 days in (D)
A549 and (E) PC-9 cell senescence was detected by SA
β-galactosidase staining. Scale bar, 50 µm. Magnification, ×200.
Expression of the SASP-related factors IL-6 and IL-8 was analyzed
by RT-qPCR: (F) IL-6 in A549 cells, (G) IL-8 in A549 cells, (H)
IL-6 in PC-9 cells and (I) IL-8 in PC-9 cells. The proliferation of
DOX-treated (J) A549 and (K) PC-9 cells and PACs/PPCs was assessed
by colony formation assay. **P<0.01 and ***P<0.001 vs.
control. DOX, doxorubicin; NSCLC, non-small cell lung cancer;
HSC70, heat-shock cognate protein 70; PAC, persistent A549 cell;
PPC, persistent PC-9 cell; p21waf1, p21 wild-type p53-activated
fragment 1; SA, senescence-associated. |
Senescent cells constitute the
majority of persistent cells and DOX suppresses MAFK and AREG
expression in NSCLC cells and enhances expression in persistent
cells
Proportions of senescent (PAS/PPS cells) and
proliferating cells (PAD/PPD cells) in emerging cells (PACs/PPCs)
were detected by Ki67 staining (Fig.
4A-C). The dividing PAD/PPD subpopulation within emerging cells
can be identified by low FSC and SSC profiles and high Ki67
staining intensity (18,19). Flow cytometry suggested that PAD/PPD
subpopulations exhibited a relatively high proliferative activity,
yet PAS/PPS subpopulations demonstrated low proliferation (Fig. 4A-C). The percentage of
SA-β-gal-positive PAS/PPS cells was also higher compared with that
of PAD/PPD cells (Fig. 4D and E),
suggesting that the surviving cells were primarily senescent cells
but contained a minority of proliferating cells. The RT-qPCR
results showed that MAFK and AREG expression levels were
significantly higher in PAD/PPD compared with in PAS/PPS
subpopulations in A549 and PC-9 cells, implying that MAFK and AREG
upregulation was associated with proliferating cells (Fig. 4F-I). DOX downregulated the levels of
MAFK and AREG in A549 and PC-9 cells, but the levels were
significantly enhanced in PACs/PPCs (Fig. 4J-M). The transfection efficiency of
shAREG in emerging A549 and PC-9 cells was demonstrated by
downregulated AREG (Fig. 4N and O).
Consequently, senescent cells constituted the majority of
persistent cells and DOX suppressed MAFK and AREG expression in
NSCLC cells, but expression was enhanced in persistent cells.
 | Figure 4.Senescent cells constitute the
majority of persistent cells and DOX suppresses MAFK and AREG
expression in NSCLC cells but enhances expression in persistent
cells. (A-C) Emerging cells (PACs/PPCs) were analyzed by flow
cytometry following Ki67 staining. Cells were gated based on low
(green) or high (red) FSC/SSC values and Ki67 expression was
analyzed for each population. The PAC/PPC population was
heterogeneous and composed of ~60–70% senescent cells named PAS/PPS
cells and 30–40% of proliferating cells named PAD/PPD cells. (D and
E) Emerging cells were sorted by flow cytometry according to low
(PAD/PPD) or high (PAS/PPS) FSC/SSC values and SA-β-gal activity
was analyzed. Scale bar, 50 µm. Magnification, ×200. (F-I) MAFK and
AREG expression in PACs/PPCs was examined by RT-qPCR. (J-M) MAFK
and AREG expression in DOX-treated A549 and PC-9 cells and
PACs/PPCs was examined by RT-qPCR and GAPDH was used as an internal
standard. (N and O) Transfection efficiency of shAREG in emerging
A549 and PC-9 cells was examined using western blotting.
**P<0.01 and ***P<0.001 vs. control; ##P<0.01
and ###P<0.001 vs. shNC; ^^^P<0.001 vs.
PAD; &&&P<0.001 vs. PPD. DOX,
doxorubicin; MAFK, musculoaponeurotic fibrosarcoma oncogene homolog
K; AREG, amphiregulin; NSCLC, non-small cell lung cancer, PAC/PPC,
persistent A549/PC-9 cells; FSC/SSC, forward-scatter/side-scatter;
PAS/PPS, persistent A549/PC-9 senescent; PAD/PPD, persistent
A549/PC-9 dividing; SA-β-gal, senescence-associated
β-galactosidase; RT-qPCR, reverse transcription-quantitative PCR;
HSC70, heat-shock cognate protein 70; sh, short hairpin; NC,
negative control. |
MAFK overexpression reverses the
inhibitory effect of AREG knockdown on proliferation of emerging
NSCLC cells
To evaluate how AREG affected the proliferation of
emerging NSCLC cells, A549 and PC-9 cells during emergence were
transfected with shAREG or shNC. AREG knockdown suppressed cell
proliferation (Fig. 5A and B). Flow
cytometry and SA-β-gal assay showed AREG knockdown markedly
increased the proportion of senescent PACs/PPCs while decreasing
the proportion of proliferating cells (Fig. 5C-H).
 | Figure 5.AREG knockdown suppresses
proliferation and promotes senescence of NSCLC cells. The
proliferation of emerging (A) A549 and (B) PC-9 cells following
AREG knockdown was evaluated by colony formation assay. Emerging
cells (PACs/PPCs) were analyzed by flow cytometry following Ki67
staining: (C) Representative Ki67 staining plots in A549 cells, (D)
representative Ki67 staining plots in PC-9 cells, (E)
quantification of Ki67-positive A549 PAC subpopulations (PAD and
PAS), and (F) quantification of Ki67-positive PC-9 PPC
subpopulations (PPD and PPS). Cells were gated into two populations
based on low (green) or high (red) FSC/SSC values and Ki67
expression was analyzed for each population. Emerging (G) A549 and
(H) PC-9 cells were sorted by flow cytometry according to low
(PAD/PPD) or high (PAS/PPS) FSC/SSC values and
senescence-associated β-galactosidase activity was analyzed. Scale
bar, 50 µm. Magnification, ×200. ###P<0.001 vs. shNC;
^^^P<0.001 vs. PAD;
&&&P<0.001 vs. PPD. MAFK,
musculoaponeurotic fibrosarcoma oncogene homolog K; AREG,
amphiregulin; NSCLC, non-small cell lung cancer; PAC/PPC,
persistent A549 or PC-9 cells; FSC/SSC,
forward-scatter/side-scatter; PAS/PPS, persistent A549 or PC-9
senescent; PAD/PPD, persistent A549 or PC-9 dividing; sh, short
hairpin; NC, negative control. |
ChIP showed that MAFK directly bound to the promoter
region of AREG (Fig. 6A and B). The
transfection efficiency of plasmids overexpressing MAFK in A549 and
PC-9 cells was corroborated by upregulated MAFK in western blotting
(Fig. 6C-E). A dual-luciferase
reporter assay was used to evaluate the targeted regulation between
MAFK and AREG. Luciferase activity was significantly increased by
WT AREG and MAFK, but only slightly increased by MUT AREG and MAFK,
implying that MAFK promoted luciferase activity in
AREG-WT-transfected A549 and PC-9 cells (Fig. 6F and G). MAFK overexpression
enhanced the proliferation of emerging A549 and PC-9 cells and
reversed the anti-proliferative effect of AREG knockdown (Fig. 6H and I), indicating that the
pro-proliferative effect of MAFK was not solely mediated by AREG
but may also involve additional target genes or pathways.
Collectively, MAFK overexpression reversed the inhibitory effect of
AREG knockdown on proliferation of emerging NSCLC cells.
Discussion
Senescence is a tumor-suppressive mechanism that
causes permanent proliferation arrest responding to
chemotherapy-induced genotoxic stress or an carcinogenic insult
(4). DOX triggers senescence in
varying cell types, including breast cancer cells, a number of
which can emerge after chemotherapy and reproliferate (20). Escape from cell senescence induction
is an effective mechanism of chemoresistance (21). It has been established that DOX
induces senescence and suppresses the proliferation of NSCLC cells,
while promoting proliferation in persistent cells, suggesting that
NSCLC cells capable of escaping senescence possess enhanced
survival and proliferation abilities (22). Given that the surviving cells were
primarily composed of senescent cells, the mechanism of NSCLC cells
evading DOX-induced senescence was investigated in the present
study.
AREG was identified as a human senescence-related
gene affected by chemotherapeutic drugs using bioinformatic tools.
AREG has been demonstrated to serve a regulatory role in multiple
tumors, including HER2-positive breast, colorectal and endometrial
cancer (23–25). The role of AREG in cancer cells
treated with DOX has been reported. For example, AREG knockdown
potentiates DOX-induced autophagy and apoptosis by regulating
endoplasmic reticulum stress in glioblastoma cells (26). AREG enhances migration and
resistance to DOX in chondrosarcoma cells via the MAPK pathway
(27). Furthermore, Elangovan et
al (28) suggested fos-like
antigen-1 promotes Kras-induced lung cancer through AREG and cell
survival-associated gene regulation. Hsu et al (29) demonstrated that lung
tumor-associated dendritic cell-derived AREG enhances cancer
development and progression. Transcriptional coactivator with
PDZ-binding motif sensitizes EGFR-WT NSCLC to gefitinib by
activating AREG transcription (30). AREG-triggered EGFR activation
confers crizotinib resistance of echinoderm microtubule-associated
protein-like 4-anaplastic lymphoma kinase-positive lung cancer
in vivo, and this resistance can be reversed by EGFR
inhibitors (31).
In the present study, DOX downregulated AREG in
NSCLC cells, while AREG was upregulated in NSCLC cells escaping
senescence, indicating that AREG upregulation may contribute to
senescence escape in NSCLC. In addition, AREG knockdown suppressed
proliferation of emerging NSCLC cells. DOX induces DNA damage to
promote cancer cell senescence and AREG repairs DNA damage
(32–34). Thus, upregulated AREG may repair
DOX-induced DNA damage to promote the senescence escape of NSCLC
cells.
MAFK, a transcription factor targeting AREG in lung
tissue, was selected as the present target gene based on a
literature search and comparative analysis. MAFK implicated in
multiple disease processes, including thrombocytopenia, neuronal
disorder, diabetes and carcinogenesis (35). A previous study demonstrated a
tumor-promoting role of MAFK. For example, MAFK induces
epithelial-mesenchymal transition and malignant progression of
triple-negative breast cancer cells by targeting the gene encoding
the transmembrane glycoprotein non-metastatic melanoma protein B
(36). Wnt1-induced MAFK expression
promotes osteosarcoma cell proliferation (11). Transcription factor nuclear factor
E2-related factor 2/MAFK signaling regulates rat placental
glutathione S-transferase gene during hepatocarcinogenesis
(37). Furthermore, previous
research has revealed the promoting effect of MAFK on lung
adenocarcinoma (12). The present
study found DOX suppressed MAFK expression in NSCLC cells, while
MAFK was upregulated in NSCLC cells escaping senescence. However,
the mechanisms by which the interaction between MAFK and AREG
affects senescence escape of DOX-treated NSCLC cells remains
unknown. The present data demonstrated that MAFK overexpression
reversed the inhibitory effect of AREG knockdown on proliferation
of emerging NSCLC cells, suggesting that MAFK promoted AREG
expression to facilitate the senescence escape of NSCLC cells.
However, the hypothesis that MAFK overexpression partially
compensated AREG loss indicates a more complex, multi-faceted role,
potentially involving the regulation of additional effectors beyond
AREG. To uncover these alternative mechanisms, future studies
should identify the full spectrum of MAFK target genes (through
chromatin IP-sequencing for example) and assess their individual
and combined contribution to proliferation in the absence of AREG
to clarify whether MAFK activates a parallel proliferation factor
pathway, directly regulates cell cycle components or employs other
strategies to sustain proliferation independently of AREG.
Senescent cells are characterized by SASP, which
secretes inflammatory factors and chemokines, and IL-1β can enhance
the transcription of a paralog of MAFK, namely MAFF (13,14).
Therefore, DOX may promote NSCLC cell senescence and the senescent
cells subsequently secrete inflammatory factors and chemokines via
the SASP program to activate MAFK in some cells, which results in
senescence escape. This escape process may involve the facilitation
of DNA damage repair, as the successful bypass of senescence
typically requires the resolution of the initial damage signal
(38). However, direct evidence
supporting the role of DNA damage repair in this context remains
lacking. Future studies should use direct assays such as γ-H2AX
foci quantification and comet assays to test this hypothesis.
Overall, the present study demonstrated that DOX
suppresses MAFK and AREG expression in NSCLC cells, while their
levels are upregulated in NSCLC cells escaping from senescence.
Furthermore, MAFK induced senescence escape of DOX-treated NSCLC
cells by promoting AREG. To the best of our knowledge, the present
study is the first to demonstrate the role of MAFK in the
senescence escape of NSCLC cells.
Acknowledgements
Not applicable.
Funding
Funding: No funding was received.
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
NF and GL conceived and designed the study. JS, QW
and LF collected the data. NF, JS, QW and LF analyzed and
interpreted the data. GL and LF wrote and revised the manuscript.
All authors have read and approved the final manuscript. NF and GL
confirm the authenticity of all the raw data.
Ethics approval and consent to
participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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