ABSTRACT Previous studies indicate that estrogen positively regulates lung cancer progression. Understanding the reasons will be beneficial for treating women with lung cancer in the future.

In this study, we found that tumor formation was more significant in female EGFRL858R mice than in male mice. P53 expression levels were downregulated in the estradiol (E2)-treated lung

cancer cells, female mice with EGFRL858R-induced lung cancer mice, and premenopausal women with lung cancer. E2 increased DNA methyltransferase 1 (DNMT1) expression to enhance methylation in

the TP53 promoter, which led to the downregulation of p53. Overexpression of GFP-p53 decreased DNMT1 expression in lung cancer cells. TP53 knockout in mice with EGFRL858R-induced lung

conclusion, E2-induced DNMT1 and p53 expression were negatively regulated each other in females with lung cancer, which not only affected cancer cells but also modulated the tumor-associated

microenvironment, ultimately leading to a poor prognosis. SIMILAR CONTENT BEING VIEWED BY OTHERS FXR DEFICIENCY INDUCED FERROPTOSIS VIA MODULATION OF THE CBP-DEPENDENT P53 ACETYLATION TO

SUPPRESS BREAST CANCER GROWTH AND METASTASIS Article Open access 14 November 2024 ESTROGEN RECEPTOR BETA PROMOTES LUNG CANCER INVASION VIA INCREASING CXCR4 EXPRESSION Article Open access 21

January 2022 MACROPHAGES PROMOTE PRE-METASTATIC NICHE FORMATION OF BREAST CANCER THROUGH ARYL HYDROCARBON RECEPTOR ACTIVITY Article Open access 18 December 2024 INTRODUCTION Lung cancer,

including non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), is the leading cause of cancer-related death worldwide. Approximately 85% of all new cases are NSCLC, and

adenocarcinoma is the most common subtype of NSCLC, especially in female patients. In the past three decades, the lung cancer incidence rate has decreased approximately twofold faster in men

than in women. The clinical characteristics of men and women with lung cancer are very different. Previous studies have shown that when female patients were analyzed based on hormonal

status, premenopausal women have a worse prognosis than both men and postmenopausal women, suggesting that estrogen promotes lung cancer malignancy in premenopausal women [1,2,3]. However,

the detailed mechanism(s) remains to be elucidated. TP53 is an important tumor suppressor gene that is involved in cell proliferation and metastasis through various mechanisms, such as the

cell cycle and DNA damage repair [4, 5]. Previous studies have shown that p53 and RAS are involved in the regulation of cancer cell epithelial-mesenchymal transition (EMT) [6, 7]. p53 forms

a complex with MDM2 and Slug to promote MDM2-mediated Slug degradation [8, 9]. In addition, previous studies have indicated that p53 positively regulates miR200 to silence ZEB1, subsequently

increasing E-cadherin expression and decreasing Vimentin expression to inhibit EMT [10,11,12]. Recent studies have revealed that p53 is involved in regulating the inflammatory tumor

microenvironment and maintaining cancer stem cells (CSCs) [13]. According to previous studies, p53 may be a regulator of the NF-κB signaling pathway [14, 15]. Several lines of evidence

support the involvement of p53 in the inflammatory tumor microenvironment. First, the loss of p53 augments NF-κB transcriptional activity in response to TNF-α treatment. Second, the loss of

p53 sensitizes cells to TNF-α-induced apoptosis. Third, the loss of p53 enhances inflammatory responses by inhibiting interleukin-1 receptor antagonist (sIL-1Ra) expression. Fourth, the loss

of TP53 promotes TLR3-induced cytokine and chemokine expression. Finally, the loss of p53 affects the gene expression profile [16, 17]. In this study, we found that estrogen inhibited p53

expression and induced M2-macrophage polarization. The relationship between the loss of p53 and the tumor microenvironment in lung cancer needs to be elucidated. DNA methylation is an

important modification of the genome that modulate the gene expression and is involved in physiological and pathological conditions. Several enzymes, including DNMT1, DNMT3a, and DNMT3b,

mediate DNA methylation [18,19,20]. A recent study indicates that inhibition of DNMT1 and ERα cross-talk suppresses breast cancer by inhibiting IRF4 expression [21]. In addition, DNMT1

increases the DNA methylation of TP53 and decreases p53 expression [22, 23]. DNMT1 promotes cell proliferation by methylating the hMLH1 and hMSH2 promoters in EGFR-mutated NSCLC.

SUV39H1-mediated DNMT1 participates in the epigenetic regulation of Smad3 in cervical cancer [24, 25]. In this study, we found that estrogen-induced DNMT1 increased the DNA methylation of

TP53 to repress p53 expression, which positively affected DNMT1 expression, thus promoting poor prognosis of lung cancer by modulating EMT and the microenvironment. RESULTS E2-MEDIATED

INHIBITION OF P53 IS RELATED TO A POOR PROGNOSIS IN FEMALES WITH LUNG CANCER According to a previous study, estrogen may positively regulate lung cancer development [26,27,28], but the exact

mechanisms need to be clarified. In this study, we used the tet-ON system, which is a well-known conditional transgenic system, to express EGFRL858R, which leads to cancer development [29,

30]. Herein, we first showed that lung cancer development was worse in female EGFRL858R-induced mice than in male mice (Fig. 1A) and ovariectomized female mice (Fig. 1B). However, the

molecular mechanism by which E2 positively regulates lung cancer progression remains unclear. First, next-generation sequencing (NGS) of A549 and E2-A549 cells was performed to study the

gene expression profiles (Fig. 1D). There were 2354 genes and 2064 genes that were negatively and positively regulated respectively, by E2 treatment in A549 lung cancer cells (Fig. 1D). The

mRNA and protein levels of p53 were dramatically decreased in estrogen-treated A549 and PC14 cells (Fig. 1C,D). In addition, 51 p53-regulated genes, including 31 downregulated and 20

upregulated genes [31], were also regulated in E2-A549 cells, suggesting that E2-mediated inhibition of p53 regulated the expression of many p53 target genes during lung cancer progression

(Fig. 1D and Supplementary Fig. 1). Taken together, these findings suggest that estrogen can decrease p53 expression by inhibiting its transcriptional activity in the early period of lung

cancer progression. To study the role of p53 in sex-dependent lung cancer progression, TP53 was knocked out in mice with EGFRL858R-induced lung cancer and EGFRL858R x TP53+/− male and female

mice to delineate the role of p53 in sex-dependent lung cancer development induced by doxycycline for 1.5 months (Fig. 2A and Supplementary Fig. 2). The tumor burdens of doxycycline-induced

lung cancer mice were determined by measuring the increase in the lung area compared to that of normal mice. The tumor burden in female mice was worse than that in male mice (Fig. 2A, a),

but there was no significant difference in tumor size between male and female TP53-knockout mice, suggesting that the loss of p53 is the major reason for the poor prognosis in females with

lung cancer mice (Fig. 2A, b and Supplementary Fig. 2). Cancer development in several mice was induced by continuous doxycycline until death to study the effect of TP53 mutations on survival

rates. The survival rates of TP53-wt females were lower than that of TP53-wt male mice with EGFRL858R-induced lung cancer (Fig. 2B, a). However, there was no significant difference between

TP53-knockout male and female mice with EGFRL858R-induced lung cancer (Fig. 2B, b). According to previous studies, the TP53 mutation rate is higher in women with lung cancer than in men [32,

33]. We examined the TP53 genotype in lung cancer cohorts from the TCGA database to analyze the TP53 mutation rate in the early and late stages of lung cancer (Fig. 2C and Supplementary

Table 2). The survival rates of TP53-wt females were lower than that of TP53-wt males with EGFR mutations (Fig. 2C, a). Interestingly, in the early stage, there was no difference in the TP53

mutation rate between men and women with lung cancer. However, the TP53 mutation rate was higher in women with late-stage lung cancer than in men with late-stage men lung cancer (Fig. 2C,

b). Based on these findings, we believe that estrogen decreases p53 expression in the early stage of lung cancer progression in women, subsequently inducing genomic instability and leading

to TP53 mutation in the late stage, which ultimately results in a poor prognosis. In conclusion, these data indicate that E2-mediated inhibition of p53 in early-stage lung cancer may be

related to the poor prognosis of women with lung cancer compared with men with lung cancer. E2-INDUCED DNMT1 EXPRESSION INCREASES DNA METHYLATION OF P53 TO INHIBIT ITS TRANSCRIPTIONAL

ACTIVITY A previous study indicates that DNA methylation in the TP53 promoter region is increased during cancer progression, thereby silencing p53 expression [23, 34]. Our NGS gene

expression profile also showed that DNMT1 was increased in E2-treated A549 lung cancer cells (Supplementary Fig. 1). The mRNA and protein levels of DNMT1 were increased in E2-treated A549

and PC14 lung cancer cells (Fig. 3A, B). E2 treatment decreased the level of p53, and knockdown of DNMT1 increased the level of p53 in PC14 cells (Fig. 3C), suggesting that E2 increases

DNMT1 to inhibit p53 expression. Because p53 can repress DNMT1 expression in colon cancer cells [35], here we also showed that the overexpression of GFP-p53 in H1299L858R and A549 cells

significantly decreased the level of DNMT1. Furthermore, knockout of TP53 in mice with EGFRL858R-induced lung cancer increased the level of DNMT1 (Fig. 3D, right panel). The promoter of

DNMT1 was analyzed by software, and several estrogen receptor (ER)-binding sites were found in the promoter of DNMT1. To study the effect of E2 on the transcriptional activity of DNMT1,

luciferase activity driven by the promoter of DNMT1 was studied (Fig. 3E). The data indicated that E2 treatment significantly increased luciferase activity in A549 cells (2.0-Kbp promoter)

and the loss of one estrogen receptor (ER) binding site (1.7-Kbp promoter) decreased luciferase activity, indicating that estrogen directly binds with ER to induce the transcriptional

activity of DNMT1 (Fig. 3E). In addition, the level of p53 in male lung cancer mice was higher than that in female lung cancer mice (Fig. 3F, a). To study the role of sex in p53 levels, the

ovary was removed from female lung cancer mice by ovariectomy (Fig. 3F). The levels of p53 in male mice were higher than those in female mice. When the ovaries were removed, the levels of

p53 in ovariectomized mice were greater than that in ovary-intact female lung cancer mice, indicating that estrogen is involved in the downregulation of p53 expression (Fig. 3F). Because

DNMT1 is a DNA methyltransferase, the effect of estrogen treatment on the methylation of the TP53 promoter was studied (Fig. 3G). The data indicated that the methylation of the TP53 promoter

region was increased by E2 treatment in A549 cells (Fig. 3G). To identify the detailed methylation site(s) within the TP53 promoter, the promoter activity of TP53 was studied by a

luciferase activity assay (Fig. 3H). The data indicated that the luciferase activity of the TP53 promoter was significantly decreased by E2 treatment, but this effect was abolished by

removing the methylation motifs of the TP53 promoter, especially mutating the −1045 and −949 sites, indicating that E2-induced DNMT1 enhancement of DNA methylation within the TP53 promoter

is important for decreasing p53 expression (Fig. 3H). In summary, E2 treatment of lung cancer cells increases DNMT1 expression to enhance the DNA methylation of TP53, subsequently repressing

p53 expression. E2-MEDIATED INHIBITION OF P53 INCREASES THE DIFFERENTIATION OF M2 MACROPHAGES Because previous studies have indicated a higher TP53 mutation rate in women with lung cancer

than in men with lung cancer, this might be the major reason for the poor prognosis in women. Many p53-regulated EMT- and cancer malignancy-related genes were changed by E2 treatment

(Supplementary Fig. 1) [36], but the effect of p53 on the tumor-associated microenvironment (TME) in lung cancer needs to be addressed. To study the effect of p53 on TME during lung cancer

progression, TP53 was knocked out in the EGFRL858R-, and EGFRL858R × TP53−/−-induced lung cancer mouse models (Fig. 4). Tumor size was increased in TP53-knockout mice (Fig. 1 and

Supplementary Fig. 2). Based on these results, we hypothesized that p53 regulates not only cancer cells but also other cells in the TME, such as macrophages. The conditioned medium of

H1299L858R cells with or without GFP-p53 expression was collected and then cultured with THP-1 monocytes to study the polarization of macrophage M1/M2 macrophages (Fig. 4). The data

indicated that THP-1 cells treated with conditioned medium from H1299L858R lung cancer cells with GFP-p53 overexpression decreased the levels of CD206 and IL6 but increased the level of

IL10, suggesting that p53 in lung cancer cells can inhibit the polarization of M2 macrophages, which can promote cancer metastasis (Fig. 4A). We used the M2-macrophage marker YM1 to study

the distribution of M2 macrophages around lung tumors in EGFRL858R and EGFRL858R × TP53+/− mice (Fig. 4B). Interestingly, the tumor infiltration of M2 macrophages (TIMs) was markedly

increased in TP53-knockout mice, suggesting that the loss of p53 facilitated macrophage infiltration in the lung cancer region, which might be beneficial for cancer progression (Fig. 4B).

The effect of p53-mediated secreted factors on the migration of macrophages and cancer cells was studied (Fig. 5A–C). Conditioned medium from H1299L858R cells overexpressing GFP-p53

inhibited the migration of macrophages (Fig. 5A) and cancer cells, H1299L858R (Fig. 5B) and A549 cells (Fig. 5C), suggesting that p53-mediated secreted factors from lung cancer cells are

involved in lung cancer progression by regulating cancer cells and the tumor-associated microenvironment. To study the composition of the conditioned medium, protein arrays were used to

examine the conditioned medium of H1299 and H1299L858R cells with or without GFP-p53 expression (Fig. 5D). Because we used mice with EGFRL858R-induced lung cancer to study the effect of p53

on the TME, in the protein array, we examined the differences in H1299L858R but not in H1299 lung cancer cells (Fig. 5D). Only the proteins FGF2, GDF15, and CCL5 were regulated by p53 in

H1299L858R cells but not in H1299 cells. GFP-p53 overexpression increased the levels of FGF2 and GDF15 but decreased CCL5 in H1299L858R cells (Fig. 5D). In addition, several proteins,

including VEGF, MMP9, and PTX3, were decreased in GFP-p53-expressing H1299 and H1299L858R cells, suggesting that these proteins are regulated by p53 in an EGFR mutation-independent manner

(Fig. 5D and Supplementary Fig. 3). Furthermore, we validated the expression of CCL5, GDF15, and FGF2 in GFP-p53-expressing H1299L858R cells (Fig. 5E, a) and EGFRL858R x TP53+/− mice (Fig.

5E, b). In H1299L858R cells, GFP-p53 inhibited the mRNA level of CCL5 but increased the mRNA levels of FGF2 and GDF15, which was consistent with the conditional profile shown in Fig. 5D. In

EGFRL858R × TP53+/− mice, the loss of p53 increased the mRNA level of CCL5 and decreased the mRNA level of GDF15, which was also consistent with the conditional profile. However, the loss of

p53 increased the mRNA level of FGF2 (Fig. 5E, b, right panel), which was inconsistent with the results shown in Fig. 5D. Taken together, these results suggest that p53-mediated CCL5 and

GDF15 are secreted by cancer cells to regulate the TME. P53-MEDIATED CCL5 AND GDF15 REGULATE M2-MACROPHAGE POLARIZATION TO CONTROL CANCER PROGRESSION What is the effect of the p53-mediated

TAM on cancer progression? First, we found that E2 treatment of A549 cells dramatically decreased p53 expression and increased CD44 expression in lung cancer cells and M2 macrophages,

indicating that E2-mediated inhibition of p53 expression may lead to cancer malignancy (Fig. 6A). We used CD206 and CD68 as the M2-macrophage markers to study the effect of p53-induced CCL5

on M2-macrophage polarization (Fig. 6B). Conditioned medium collected from H1299L858R cells with GFP-p53 overexpression decreased M2-macrophage polarization, but adding recombinant CCL5

protein to the conditioned medium reversed this effect (Fig. 6B), indicating that p53-mediated inhibition of CCL5 could prevent the differentiation of M2 macrophages. In addition, the effect

of CCL5 and GDF15 on the migration of M2-macrophage and H1299 cancer cells was studied by culturing the cells with the conditioned medium collected from H1299L858R cells with or without

GFP-p53 overexpression (Fig. 6C). The data indicated that GFP-p53 overexpression inhibited the migration of M2 macrophages and H1299 lung cancer cells, and the addition of anti-GDF15

antibodies or recombinant CCL5 protein blocked the effect of GFP-p53 expression on cancer cell migration (Fig. 6C). When we used conditioned medium from H1299L858R cells with p53 expression

to treat H1299L858R cancer cells, CD44 expression in cancer cells was inhibited, suggesting that 53 mediated the TAM to inhibit cancer stemness (Fig. 6D). The effect of p53-mediated changes

in conditioned medium on the morphology of macrophages was also observed (Fig. 6E). The data indicated that lamellipodia around M2 macrophages were observed in control and GFP-overexpressing

cells but were lost in GFP-p53-overexpressing cells, suggesting that p53 in lung cancer cells regulates secreted proteins to affect the morphology of macrophages, which is related to tumor

infiltration (Fig. 6E). Finally, to study the association between M2 macrophages and sex-dependent lung cancer, samples were collected from male and female mice with EGFRL858R-induced lung

cancer and used to study M2 macrophages that were stained with anti-YM1 antibodies and then scored from 1 to 4 based on the YM1 signal (Fig. 7A, a, b). The data indicated that the scores of

female mice were higher than those of male mice, indicating that more M2 macrophages were found in female lung cancer mice (Fig. 7A). The level of CCL5 was increased in female lung cancer

mice but was decreased after the ovary was removed (Fig. 7B, left panel). In addition, the level of GDF15 was slightly decreased in female lung cancer mice but was slightly increased after

the ovary was removed (Fig. 7B, right panel). In addition, the mRNA levels of TP53 and GDF15 were increased, and the mRNA levels of DNMT1 and CCL5 were decreased in ovariectomized female

mice, which is consistent with the results of this study (Fig. 7C). Finally, the levels of p53, DNMT1, GDF15, and CCL5 in lung cancer patients were studied (Fig. 8 and Supplementary Table

3). Samples from ten lung cancer patients were used to study the levels of p53, DNMT1, CCL5, and GDF15 and then scored from 0 to 3 based on protein signals (Fig. 8A). The data indicated that

the scores of CCL5 and DNMT1 were higher than those of p53 and GDF15 (Fig. 8A) in the clinical samples from lung cancer patients, which is consistent with our study. In addition, the levels

of CCL5 and DNMT1 in female patients were higher than those in male patients, but there was no significant difference in GDF15 and p53 levels between the male and female cohorts (Fig. 8A).

DISCUSSION The poor prognosis of women with lung cancer has recently become an important clinical issue. In this study, we found that E2 inhibited p53 to control not only in cancer cells but

also cells in the tumor-associated microenvironment. We found that E2-induced DNMT1 and p53 formed a negative feedback loop, thereby increasing CCL5 and decreasing GDF15 expression,

subsequently enhancing M2-macrophage polarization and ultimately leading to poor prognosis in females with lung cancer (Fig. 8B). Previous studies have revealed that E2 treatment induces

rapid activation of the EGFR pathway, suggesting that nonnuclear ER translocation regulates the EGFR pathway to influence lung cancer progression [37]. Moreover, 67% of EGFR

mutation-positive tumors have high nuclear ERβ expression (versus 37% in EGFR wild-type tumors) [38]. Based on these previous studies, ER-mediated and EGFR-mediated signaling pathways may

positively coregulate the cancer-related gene expression during lung cancer progression. In this study, we found that E2 treatment could induce DNMT1 expression, subsequently increasing DNA

methylation in the promoter of p53. Because E2 actives EGFR signaling and the TP53 promoter sequence contains ER-binding motifs, ERs may regulate the transcriptional activity of DNMT1 by

activation of the EGFR signaling pathway and direct recruitment to its promoter. According to previous studies, the TP53 mutation rate is higher in women with lung cancer than in men and is

related to a poor prognosis in these individuals [32, 33]. In this study, we found high TP53 mutation rates only in females with late-stage lung cancer, not in those with early-stage lung

cancer. We also found that estrogen significantly inhibited p53 expression and was an important factor in inducing the high TP53 mutation rate in late-stage lung cancer. p53, which is a

tumor suppressor, maintains genomic integrity to avoid cancer heterogeneity during cancer progression [39, 40]. In this study, we found that the survival rates of EGFRL858R male mice were

higher than those of female mice, but this difference was abolished in EGFRL858R × TP53+/− mice, indicating that estrogen-mediated inhibition of p53 in female mice is critical for poor

prognosis. Although we have studied the role and mechanism by which p53 affects tumor burden in vivo, we only used eight EGFRL858R and EGFRL858R × TP53+/− mice to study the survival rate.

More mice will be used to confirm this finding in the future. We also found that many p53 target genes were altered by estrogen treatment, which is consistent with previous studies in which

p53 was revealed to be a tumor suppressor. In addition to the effect of p53 within cancer cells, when the TP53 was knocked out in mice with EGFRL858R-induced lung cancer, not only did the

size of the tumor increase, but marked immune cell infiltration was also observed around cancer cells, indicating that p53, as a tumor suppressor, inhibited not only cancer cells directly

but also M2-macrophage differentiation. The TAM is involved in cancer progression (i.e., cancer malignancy and drug resistance) [41,42,43,44]. Many proteins, such as cytokines secreted from

nearby cells (e.g., macrophages, fibroblasts, and other immune cells, including T cells and B cells), regulate cancer cell progression and macrophage polarization. Our data indicated that

estrogen-mediated inhibition of p53 in H1299L858R cancer cells changed the protein composition (FGF2, GDF15, IGFBP2, CCL5, PTX3, VEGF, and MMP9) of conditioned medium. GDF15, CCL5, and FGF2

were regulated only in H1299L858R cells but not in H1299 cancer cells, suggesting that EGFR signaling and p53 coregulate these three secreted proteins. Fibroblast growth factor type 2 (FGF2)

can bind to heparin and has broad mitogenic and angiogenic activities. Our protein array data showed that GFP-p53 positively regulated FGF2 secretion by cells, and positively regulated FGF2

in cancer cells. However, FGF2 expression was also increased in TP53-knockout mice with EGFRL858R-induced lung cancer, which is inconsistent with the results in cancer cells. p53 may

regulate FGF2 differently under different conditions, and the detailed regulatory mechanism needs to be further clarified. Growth differentiation factor 15 (GDF15) has been reported to be

involved in various physiologic or pathologic pathways including cancer [45]. GDF15 is one of the ligands that bind to TGF-β receptors to modulate the SMAD pathway and regulate gene

expression [46]. Previous studies have indicated that EZH2-mediated inhibition of GDF15 represses NSCLC proliferation [47]. In this study, we first showed that p53 positively regulated GDF15

to repress the migration abilities of M2 macrophages and lung cancer cells. In addition, C–C motif chemokine ligand 5 (CCL5) is a secreted protein involved in immunoregulatory and

inflammatory processes that functions as a chemoattractant for blood monocytes, memory T-helper cells, and eosinophils [48,49,50]. Previous studies have indicated that IL6 mediates

macrophage infiltration after irradiation by upregulating CCL5 expression in NSCLC. Our study showed that GFP-p53 increased GDF15 expression but decreased CCL5 expression in H1299L858R

cells, thereby repressing M2-macrophage differentiation and subsequently preventing cancer malignancy. E2-mediated EGFR signaling and p53 expression are critical for the poor prognosis in

females with lung cancer. DNA methylation is important for maintaining genomic integrity and gene expression [51, 52]. Disorders associated with DNA methylation are involved in various

diseases, including cancer [53]. A previous study indicated that DNMT1 could methylate the promoter of TP53 to inhibit its expression [34, 54]. We showed that estrogen treatment of lung

cancer cells could induce DNMT1 expression, thereby inducing hypermethylation of the TP53 promoter and leading TP53 downregulation. Our study also showed that other DNMTs, DNMT2, DNMT3a, and

DNMT3b were slightly increased, which might also contribute to the hypermethylation of the TP53 promoter. Although there was no significant difference in TP53 mutations between male and

female lung cancer patients in the early stage, estrogen-induced DNMT expression to inhibit p53 expression, which might be one of the critical reasons for the induction of genomic

instability, subsequently increasing TP53 mutation rates in the late stage of lung cancer in female patients. In the future, blocking estrogen or DNMT activities may be beneficial for the

survival rates of female lung cancer patients. MATERIALS AND METHODS CELL CULTURE AND TRANSFECTION Human lung adenocarcinoma epithelial cell line A549, PC14, H1299, H1299L858R, and human

monocyte THP-1 cell lines from ATCC were cultured with RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco™, Waltham, MA, USA), 100 μg/ml streptomycin

and 100 U/ml penicillin G sodium (Gibco). E2-A549 cells were obtained by treating A549 cells with 1 μg/ml E2 for 3 weeks. All cells were incubated at 37 °C with 5% CO2. For transfecting

plasmid, PolyJet (SignaGen Laboratories, Frederick, MD, USA) was used according to the manufacturer’s instructions. IMMUNOHISTOCHEMISTRY Paraffin-embedded human and mice lung cancer tissues

were obtained from the National Cheng Kung University Hospital Tissue Bank and doxycycline-induced EGFRL858R mice, respectively, and the tissues were cut into 5-μm sections.

Immunohistochemistry was performed using a Novolink™ Polymer Detection Systems (Leica Biosystems) following the manufacturer’s instructions. Antigen retrieval was performed using citrate

buffer (pH 6.0, Scytek). Primary antibodies, anti-p53 (A5761, ABclonal, 1:50), anti-CCL5 (A5630, ABclonal, 1:50), anti-YM1 (Abcam, 1:50), anti-DNMT1 (A16729, ABclonal, 1:50) and anti-GDF15

(A0185, ABclonal, 1:50), were used to incubate with tissue samples for overnight at 4 °C. Sections were photographed by Olympus BX-51 microscope. ANIMAL CARE AND ANIMAL MODELS The

experiments related with animals were approved by the Institutional Animal Care and Use Committee (IACUC:108005) at National Cheng Kung University (NCKU). These transgenic mice were

generated in National Laboratory Animal Center (NLAC, Taiwan, Tainan). After breeding, two-month-old transgenic mice were used to study lung cancer development. Caging was provided suitable

space and accommodates appropriate population densities that allowed animals’ sufficient freedom of movement. To provide amounts of food that must be for transgenic mice normal growth and

maintenance of normal body weight. These transgenic mice, including EGFRL858R and EGFRL858R × TP53+/− mice, were accessed to fresh and uncontaminated drinking water. Transgenic mice were

also observed and cared at least for two to three times per week. All methods involving animals were performed in accordance with the relevant guidelines and regulations. COLLECTION OF

SPECIMENS FROM LUNG CANCER PATIENTS All human study has been conducted in accordance with the guidelines and regulations. The study using human specimens was approved by the Clinical

Research Ethics Committee at National Cheng Kung University Medical Center (Tainan, Taiwan; IRB: A-ER-107-039). After surgical resection at National Cheng Kung University Hospital, specimens

of patients with lung adenocarcinomas were collected for Immunohistochemical analysis or western blotting. The pathological data were analyzed by clinical pathologists. Informed consent was

obtained from all subjects. STATISTICAL ANALYSIS All samples were used for statistical analysis. The difference between two groups was analyzed by Student’s _t_ test. The _P_ value, which

is smaller than 0.05, was considered statistically significant. SEM is used to calculate and plot error bars from raw data. Overall survivals were estimated by means of the Kaplan–Meier

method and compared using the log-rank test. REFERENCES * Hsu LH, Liu KJ, Tsai MF, Wu CR, Feng AC, Chu NM, et al. Estrogen adversely affects the prognosis of patients with lung

adenocarcinoma. Cancer Sci. 2015;106:51–59. Article  CAS  PubMed  Google Scholar  * Fidler-Benaoudia MM, Torre LA, Bray F, Ferlay J, Jemal A. Lung cancer incidence in young women vs. young

men: a systematic analysis in 40 countries. Int J Cancer. 2020;147:811–9. Article  CAS  PubMed  Google Scholar  * Jemal A, Miller KD, Ma J, Siegel RL, Fedewa SA, Islami F, et al. Higher lung

cancer incidence in young women than young men in the United States. N. Engl J Med. 2018;378:1999–2009. Article  PubMed  PubMed Central  Google Scholar  * Turgeon MO, Perry NJS,

Poulogiannis G. DNA damage, repair, and cancer metabolism. Front Oncol. 2018;8:15. Article  PubMed  PubMed Central  Google Scholar  * Lakin ND, Jackson SP. Regulation of p53 in response to

DNA damage. Oncogene. 1999;18:7644–55. Article  CAS  PubMed  Google Scholar  * Chang C-J, Chao C-H, Xia W, Yang J-Y, Xiong Y, Li C-W, et al. p53 regulates epithelial-mesenchymal transition

and stem cell properties through modulating miRNAs. Nat Cell Biol. 2011;13:317–23. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kim H, Choi JA, Kim JH. Ras promotes transforming

growth factor-β (TGF-β)-induced epithelial-mesenchymal transition via a leukotriene B4 receptor-2-linked cascade in mammary epithelial cells. J Biol Chem. 2014;289:22151–60. Article  CAS 

PubMed  PubMed Central  Google Scholar  * Wang SP, Wang WL, Chang YL, Wu CT, Chao YC, Kao SH, et al. p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug. Nat

Cell Biol. 2009;11:694–704. Article  CAS  PubMed  Google Scholar  * Zhang J, Lei Y, Gao X, Liang Q, Li L, Feng J, et al. p53 attenuates the oncogenic Ras-induced epithelial-mesenchymal

transition in human mammary epithelial cells. Biochem Biophys Res Commun. 2013;434:606–13. Article  CAS  PubMed  Google Scholar  * Kim T, Veronese A, Pichiorri F, Lee TJ, Jeon YJ, Volinia S,

et al. p53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1 and ZEB2. J Exp Med. 2011;208:875–83. Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang C,

Ge Q, Zhang Q, Chen Z, Hu J, Li F, et al. Targeted p53 activation by saRNA suppresses human bladder cancer cells growth and metastasis. J Exp Clin Cancer Res: CR. 2016;35:53. Article 

PubMed  PubMed Central  CAS  Google Scholar  * Ye J, Wei X, Shang Y, Pan Q, Yang M, Tian Y, et al. Core 3 mucin-type O-glycan restoration in colorectal cancer cells promotes

MUC1/p53/miR-200c-dependent epithelial identity. Oncogene. 2017;36:6391–407. Article  CAS  PubMed  Google Scholar  * Uehara I, Tanaka N. Role of p53 in the regulation of the inflammatory

tumor microenvironment and tumor suppression. Cancers 2018;10:219. * Kawauchi K, Araki K, Tobiume K, Tanaka N. p53 regulates glucose metabolism through an IKK-NF-kappaB pathway and inhibits

cell transformation. Nat Cell Biol. 2008;10:611–8. Article  CAS  PubMed  Google Scholar  * Webster GA, Perkins ND. Transcriptional cross talk between NF-kappaB and p53. Mol Cell Biol.

1999;19:3485–95. Article  CAS  PubMed  PubMed Central  Google Scholar  * Weisz L, Damalas A, Liontos M, Karakaidos P, Fontemaggi G, Maor-Aloni R, et al. Mutant p53 enhances nuclear factor

kappaB activation by tumor necrosis factor alpha in cancer cells. Cancer Res. 2007;67:2396–401. Article  CAS  PubMed  Google Scholar  * Dixit D, Sharma V, Ghosh S, Mehta VS, Sen E.

Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition. Cell Death

Dis. 2012;3:e271. Article  CAS  PubMed  PubMed Central  Google Scholar  * Scala G, Federico A, Palumbo D, Cocozza S, Greco D. DNA sequence context as a marker of CpG methylation instability

in normal and cancer tissues. Sci Rep. 2020;10:1721. Article  CAS  PubMed  PubMed Central  Google Scholar  * Edwards JR, Yarychkivska O, Boulard M, Bestor TH. DNA methylation and DNA

methyltransferases. Epigenetics Chromatin. 2017;10:23. Article  PubMed  PubMed Central  CAS  Google Scholar  * Peng W-X, Koirala P, Zhang W, Ni C, Wang Z, Yang L, et al. lncRNA RMST Enhances

DNMT3 Expression through Interaction with HuR. Mol Ther. 2020;28:9–18. Article  CAS  PubMed  Google Scholar  * Vernier M, McGuirk S, Dufour CR, Wan L, Audet-Walsh E, St-Pierre J, et al.

Inhibition of DNMT1 and ERRα crosstalk suppresses breast cancer via derepression of IRF4. Oncogene. 2020;39:6406–20. Article  CAS  PubMed  PubMed Central  Google Scholar  * Oshiro MM, Watts

GS, Wozniak RJ, Junk DJ, L Munoz-Rodriguez J, Domann FE, et al. Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression. Oncogene. 2003;22:3624–34. Article  CAS 

PubMed  Google Scholar  * Nabilsi NH, Broaddus RR, Loose DS. DNA methylation inhibits p53-mediated survivin repression. Oncogene. 2009;28:2046–50. Article  CAS  PubMed  Google Scholar  * Wu

X-Y, Chen H-C, Li W-W, Yan J-D, Lv R-Y. DNMT1 promotes cell proliferation via methylating hMLH1 and hMSH2 promoters in EGFR-mutated non-small cell lung cancer. J Biochem. 2020;168:151–7.

Article  CAS  PubMed  Google Scholar  * Zhang L, Tian S, Zhao M, Yang T, Quan S, Song L, et al. SUV39H1-mediated DNMT1 is involved in the epigenetic regulation of Smad3 in cervical cancer.

Anti-cancer Agents Medicinal Chem. 2021;21:756–65. Article  CAS  Google Scholar  * Gao X, Cai Y, Wang Z, He W, Cao S, Xu R, et al. Estrogen receptors promote NSCLC progression by modulating

the membrane receptor signaling network: a systems biology perspective. J Transl Med. 2019;17:308. Article  PubMed  PubMed Central  CAS  Google Scholar  * Hsu LH, Chu NM, Kao SH. Estrogen,

estrogen receptor and lung cancer. Int J Mol Sci. 2017;18:1713. * Young M-J, Chen Y-C, Wang S-A, Chang H-P, Yang W-B, Lee C-C, et al. Estradiol-mediated inhibition of Sp1 decreases

miR-3194-5p expression to enhance CD44 expression during lung cancer progression. J Biomed Sci. 2022;29:3. Article  CAS  PubMed  PubMed Central  Google Scholar  * Hsu TI, Wang MC, Chen SY,

Yeh YM, Su WC, Chang WC, et al. Sp1 expression regulates lung tumor progression. Oncogene. 2012;31:3973–88. Article  CAS  PubMed  Google Scholar  * Hsu TI, Wang YC, Hung CY, Yu CH, Su WC,

Chang WC, et al. Positive feedback regulation between IL10 and EGFR promotes lung cancer formation. Oncotarget. 2016;7:20840–54. Article  PubMed  PubMed Central  Google Scholar  * Wei C-L,

Wu Q, Vega VB, Chiu KP, Ng P, Zhang T, et al. A global map of p53 transcription-factor binding sites in the human genome. Cell. 2006;124:207–19. Article  CAS  PubMed  Google Scholar  * Gealy

R, Zhang L, Siegfried JM, Luketich JD, Keohavong P. Comparison of mutations in the p53 and K-ras genes in lung carcinomas from smoking and nonsmoking women. Cancer Epidemiol, Biomark Prev.

1999;8:297–302. CAS  Google Scholar  * Toyooka S, Tsuda T, Gazdar AF. The TP53 gene, tobacco exposure, and lung cancer. Hum Mutat. 2003;21:229–39. Article  CAS  PubMed  Google Scholar  *

Kang JH, Kim SJ, Noh DY, Park IA, Choe KJ, Yoo OJ, et al. Methylation in the p53 promoter is a supplementary route to breast carcinogenesis: correlation between CpG methylation in the p53

promoter and the mutation of the p53 gene in the progression from ductal carcinoma in situ to invasive ductal carcinoma. Lab Investig. 2001;81:573–9. Article  CAS  PubMed  Google Scholar  *

Peterson EJ, Bögler O, Taylor SM. p53-mediated repression of DNA methyltransferase 1 expression by specific DNA binding. Cancer Res. 2003;63:6579–82. CAS  PubMed  Google Scholar  * Folkerd

EJ, Dowsett M. Influence of sex hormones on cancer progression. J Clin Oncol. 2010;28:4038–44. Article  CAS  PubMed  Google Scholar  * Mukku VR, Stancel GM. Regulation of epidermal growth

factor receptor by estrogen. J Biol Chem. 1985;260:9820–4. Article  CAS  PubMed  Google Scholar  * Nose N, Sugio K, Oyama T, Nozoe T, Uramoto H, Iwata T, et al. Association between estrogen

receptor-beta expression and epidermal growth factor receptor mutation in the postoperative prognosis of adenocarcinoma of the lung. J Clin Oncol. 2009;27:411–7. Article  CAS  PubMed  Google

Scholar  * Albrechtsen N, Dornreiter I, Grosse F, Kim E, Wiesmüller L, Deppert W. Maintenance of genomic integrity by p53: complementary roles for activated and non-activated p53. Oncogene.

1999;18:7706–17. Article  CAS  PubMed  Google Scholar  * Kastenhuber ER, Lowe SW. Putting p53 in context. Cell. 2017;170:1062–78. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Wang T, Xiao M, Ge Y, Krepler C, Belser E, Lopez-Coral A, et al. BRAF inhibition stimulates melanoma-associated macrophages to drive tumor growth. Clin Cancer Res. 2015;21:1652–64. Article 

CAS  PubMed  PubMed Central  Google Scholar  * Vouri M, Hafizi S. TAM receptor tyrosine kinases in cancer drug resistance. Cancer Res. 2017;77:2775–8. Article  CAS  PubMed  Google Scholar  *

Aras S, Zaidi MR. TAMeless traitors: macrophages in cancer progression and metastasis. Br J Cancer. 2017;117:1583–91. Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang YC, Wu YS,

Hung CY, Wang SA, Young MJ, Hsu TI, et al. USP24 induces IL-6 in tumor-associated microenvironment by stabilizing p300 and β-TrCP and promotes cancer malignancy. Nat Commun. 2018;9:3996.

Article  PubMed  PubMed Central  CAS  Google Scholar  * Wischhusen J, Melero I, Fridman WH. Growth/differentiation factor-15 (GDF-15): from biomarker to novel targetable immune checkpoint.

Front Immunol. 2020;11:951–951. Article  CAS  PubMed  PubMed Central  Google Scholar  * Min KW, Liggett JL, Silva G, Wu WW, Wang R, Shen RF, et al. NAG-1/GDF15 accumulates in the nucleus and

modulates transcriptional regulation of the Smad pathway. Oncogene. 2016;35:377–88. Article  PubMed  CAS  Google Scholar  * Lu X, He X, Su J, Wang J, Liu X, Xu K, et al. EZH2-mediated

epigenetic suppression of GDF15 predicts a poor prognosis and regulates cell proliferation in non-small-cell lung cancer. Mol Ther Nucleic Acids. 2018;12:309–18. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Murooka TT, Rahbar R, Platanias LC, Fish EN. CCL5-mediated T-cell chemotaxis involves the initiation of mRNA translation through mTOR/4E-BP1. Blood.

2008;111:4892–901. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kameyoshi Y, Dörschner A, Mallet AI, Christophers E, Schröder JM. Cytokine RANTES released by thrombin-stimulated

platelets is a potent attractant for human eosinophils. J Exp Med. 1992;176:587–92. Article  CAS  PubMed  Google Scholar  * Proost P, De Meester I, Schols D, Struyf S, Lambeir AM, Wuyts A,

et al. Amino-terminal truncation of chemokines by CD26/dipeptidyl-peptidase IV. Conversion of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection. J Biol Chem.

1998;273:7222–7. Article  CAS  PubMed  Google Scholar  * Meng H, Cao Y, Qin J, Song X, Zhang Q, Shi Y, et al. DNA methylation, its mediators and genome integrity. Int J Biol Sci.

2015;11:604–17. Article  CAS  PubMed  PubMed Central  Google Scholar  * Petryk N, Bultmann S, Bartke T, Defossez P-A. Staying true to yourself: mechanisms of DNA methylation maintenance in

mammals. Nucleic Acids Res. 2021;49:3020–32. Article  CAS  PubMed  Google Scholar  * Ehrlich M. DNA hypermethylation in disease: mechanisms and clinical relevance. Epigenetics.

2019;14:1141–63. Article  PubMed  PubMed Central  Google Scholar  * Cheng JC, Auersperg N, Leung PCK. Inhibition of p53 represses E-cadherin expression by increasing DNA methyltransferase-1

and promoter methylation in serous borderline ovarian tumor cells. Oncogene. 2011;30:3930–42. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful for

the support of clinical specimens from the Human Biobank, Research Center of Clinical Medicine, National Cheng Kung University Hospital. This work was supported by the grants

(109-2320-B-006-025-MY3 and 109-2320-B-006-055) obtained from the Ministry of Science and Technology, Taiwan. This research was supported in part by Higher Education Sprout Project, Ministry

of Education to the Headquarters of University Advancement at National Cheng Kung University (NCKU). AUTHOR INFORMATION Author notes * These authors contributed equally: Yung-Ching Chen,

Ming-Jer Young. AUTHORS AND AFFILIATIONS * Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan Yung-Ching Chen * Department of Biotechnology and Bioindustry

Sciences, National Cheng Kung University, Tainan, Taiwan Ming-Jer Young, Hui-Ping Chang, Chia-Yu Liu & Jan-Jong Hung * Division of Thoracic Surgery, Department of Surgery, College of

Medicine National Cheng Kung University, Tainan, Taiwan Chia-Chi Lee & Yau-Lin Tseng * Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

Yi-Ching Wang * The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Wen-Chang Chang * Graduate Institute

of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan Jan-Jong Hung Authors * Yung-Ching Chen View author publications You can also search for this author

inPubMed Google Scholar * Ming-Jer Young View author publications You can also search for this author inPubMed Google Scholar * Hui-Ping Chang View author publications You can also search

for this author inPubMed Google Scholar * Chia-Yu Liu View author publications You can also search for this author inPubMed Google Scholar * Chia-Chi Lee View author publications You can

also search for this author inPubMed Google Scholar * Yau-Lin Tseng View author publications You can also search for this author inPubMed Google Scholar * Yi-Ching Wang View author

publications You can also search for this author inPubMed Google Scholar * Wen-Chang Chang View author publications You can also search for this author inPubMed Google Scholar * Jan-Jong

Hung View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS CYC, YMJ, CHP, LCY, and LCC performed experiments. YMJ did the bioinformatics

analysis. TYL, WYC, and CWC provided the clinical cohorts and supervised the research. HJJ design the experiments and prepared the manuscript. CORRESPONDING AUTHOR Correspondence to Jan-Jong

Hung. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

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inhibition of DNMT1 decreases p53 expression to induce M2-macrophage polarization in lung cancer progression. _Oncogenesis_ 11, 25 (2022). https://doi.org/10.1038/s41389-022-00397-4 Download

citation * Received: 11 November 2021 * Revised: 07 April 2022 * Accepted: 12 April 2022 * Published: 19 May 2022 * DOI: https://doi.org/10.1038/s41389-022-00397-4 SHARE THIS ARTICLE Anyone

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