TFAP2C activates PTGES through the NOTCH3 signaling pathway to affect gefitinib resistance in lung adenocarcinoma

Abstract

Lung adenocarcinoma (LUAD) is one of the primary culprits of cancer-related deaths. Current treatment modalities for LUAD have certain limitations, necessitating innovating effective LUAD treatment strategies. Prostaglandin E synthase (PTGES) and TF activating protein 2C (TFAP2C) in the process of drug resistance in LUAD are less studied and need further in-depth research. This study aimed to investigate the specific molecular mechanisms of PTGES and TFAP2C in gefitinib resistance in LUAD. The results indicated that PTGES and TFAP2C were considerably overexpressed in LUAD tissues and cells. Chromatin immunoprecipitation and dual luciferase assay validated that TFAP2C targeted the PTGES promoter region. In addition, gene set enrichment analysis results demonstrated the notable enrichment of PTGES in the NOTCH3 signaling pathway. Overexpression of PTGES remarkably enhanced the viability of PC-9/GR (gefitinib-resistant) cells and their response to gefitinib resistance, which was reversed by the addition of a NOTCH3 inhibitor. Furthermore, overexpressing PTGES upon the TFAP2C silence restored the great inhibition effect conferred by TFAP2C silence in PC-9/GR cells on cell viability and cell response to gefitinib resistance. This study confirmed that TFAP2C can transcriptionally activate PTGES through the NOTCH3 signaling pathway to enhance the response of LUAD cells to gefitinib resistance, proffering a new approach for the treatment of gefitinib resistance in LUAD cells.

Introduction

Lung cancer (LC) is one of the global primary culprits of cancer-related deaths, with studies suggesting that approximately 40% of diagnosed cases are lung adenocarcinoma (LUAD).⁽¹⁾ As the most common subtype in non-small cell lung cancer (NSCLC), LUAD has an increasing incidence in both smokers and non-smokers.^(2,3) Leucine-rich repeat kinase 2 has been reported to be highly expressed in LC, and its high expression promotes LC progression by regulating the TLR4/NF-κB signaling pathway and the NLRP3 inflammasome.⁽⁴⁾ As an epidermal growth factor receptor (EGFR) inhibitor, gefitinib is an effective therapy for EGFR mutant NSCLC patients since its development.⁽⁵⁾ However, its drawback is that it can easily induce drug resistance in patients, which limits its applicability in LUAD treatment. Therefore, mining effective ways to overcome gefitinib resistance is of urgent need. In this project, we first downloaded the mRNA expression data of LUAD from The Cancer Genome Atlas (TCGA) database, and selected prostaglandin E synthase (PTGES) as the research object to dig out its molecular mechanism related to gefitinib resistance in LUAD.

Prostaglandin E2 (PGE2) has been proven to be a pivotal mediator of inflammation, pain, and fever. PGE2 is one of the abundant prostaglandins in the process of arachidonic acid (AA) synthesis, and PTGES is an inducible enzyme in the isomerization process of PGE2.⁽⁶⁾ Abnormal expression of PTGES is tightly linked to cancer progression. Delgado-Goñi et al.⁽⁷⁾ put forward that PTGES overexpression is negatively related to the survival period of patients with melanoma when exploring metabolism-related genes, indicating that PTGES is expected to be a clinical target in prognosis. Wang et al.⁽⁶⁾ pointed out the upregulation of PTGES in lung tumor tissues, and knocking down PTGES can remarkably repress the invasion and migration ability of LC cells. Furthermore, in Gprc5a-ko mice, the PTGES/PGE2 signaling pathway can recruit myeloid-derived suppressor cells (MDSCs) in vivo, facilitating the growth and metastasis of lung tumors through immunosuppressive effects.⁽⁶⁾ Consequently, PTGES may be a promising target for cancer therapy. However, its specific molecular function in LUAD occurrence and progression has not been well elucidated. To expand the PTGES-related molecular regulatory network, in this investigation, we employed the hTFtarget webpage tool to predict the upstream transcription factor (TF) of PTGES. The TF activating protein 2C (TFAP2C) that exhibited a high Pearson correlation with PTGES was chosen as the research object for the exploration of the impact of this regulatory relationship on LUAD drug resistance.

TFAP2C is a member of the AP2 TF family, participating in cycle regulation, cell proliferation, and apoptosis process,⁽⁸⁾ thus taking an instrumental part in the tumor occurrence and progression. TFAP2C activates the TGFBR1-PAK1 signaling pathway to facilitate the proliferation ability and cell cycle progression of lung tumor cells as well as enhance the malignancy and invasiveness of tumor cells.⁽⁹⁾ Furthermore, TFAP2C has also been extensively studied in terms of tumor cell resistance. Research by Wang et al.⁽¹⁰⁾ uncovered that TFAP2C can facilitate the stemness and metastasis of colorectal cancer (CRC) cells by upregulating ROCK1 and ROCK2 to repress the Hippo signaling pathway. Knocking down TFAP2C expression in bladder cancer (BCa) cells can boost sensitivity to cisplatin treatment by modulating the EGFR and NF-κB signaling pathways.⁽¹¹⁾ However, the influence of TFAP2C on the level of gefitinib resistance in LUAD cells is still not clearly understood.

This project mainly evaluated the gefitinib resistance level of LUAD cell lines with PTGES abnormally expressed through CCK-8 assay and cell cloning formation experiment. Additionally, we employed bioinformatics methods as a tool to predict the upstream regulatory factor TFAP2C of PTGES. Moreover, the molecular experimental analysis of the impact of the TFAP2C/PTGES regulatory axis on the gefitinib resistance of LUAD cells was carried out. The project will proffer new theoretical foundations and ideas for the study of resistance-related drugs in LUAD cells.

Materials and Methods

Bioinformatics analysis

We downloaded the mRNA expression data of LUAD from TCGA database (https://portal.gdc.cancer.gov/). Combining edgeR differential analysis (|logFC|>1, FDR<0.05), we screened out multiple differentially expressed genes (DEGs) and determined the research objects of this project based on previous literature and DEGs. Based on the TCGA_LUAD dataset, gene set enrichment analysis (GSEA) analysis was carried out to predict the signaling pathways greatly enriched by the target genes. In addition, the upstream potential TFs of the target gene were predicted using the hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget#!/). The differently expressed TFs were yielded through the interaction with the DEGs. Potential TFs were subjected to Pearson correlation analysis with target genes. We selected the TF with high correlation as the research object.

Cell cultivation and reagent purchase

Human LUAD cells A549 (BNCC337696), NCI-H1975 (BNCC340345), PC-9 (BNCC340767), human bronchial epithelial cells BEAS-2B (BNCC359274), and human embryonic kidney cells 293T (BNCC353535) were all purchased from the BeNa Culture Collection (BNCC, Beijing, China). Gefitinib-resistant LUAD strain PC-9/GR was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 293T, BEAS-2B, PC-9, and PC-9/GR cells were cultivated in DMEM-H medium. NCI-H1975 cells were cultivated in the RPMI-1640 medium. In an F-12K medium, we cultivated A549 cells. All media contained 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA), 100 ‍U/ml penicillin, and 100 ‍mg/L streptomycin (Thermo Fisher Scientific). The medium for 293T cells also contained 2 ‍mM l-glutamine. Cells were kept in an incubator at 37°C with 5% CO₂. We added 0.1 ‍μM (final concentration) of gefitinib (Sigma-Aldrich, St. Louis, MO) to the drug-resistant PC-9/GR cells to maintain their resistance. Subsequently, we treated drug-resistant PC-9/GR cells with BMS-986115 (8.5 nM) (MedChemExpress, Monmouth Junction, NJ) and different concentrations of gefitinib (0 ‍μM, 4 ‍μM, 8 ‍μM, 12 ‍μM, and 16 ‍μM).

Cell transfection

We purchased si-TFAP2C, si-NC, si-PTGES, oe-NC, and oe-PTGES plasmids from RiboBio (Guangzhou, China). The gefitinib-resistant PC-9/GR cells to be transfected were seeded in a 6-well plate (cell density: 1 × 10⁵ cells/ml). When the cell density reached 70~80%, we transfected si-NC, si-TFAP2C, si-PTGES, oe-NC, and oe-PTGES plasmids respectively into the above PC-9/GR cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). Moreover, the cells were kept in a 37°C, 5% CO₂ incubator for 48 ‍h. We gathered the transfected cells for subsequent experiments.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

We employed Trizol reagent (Invitrogen, Waltham, MA) to extract total RNA from the cell suspension, and reversely transcribed the extracted RNA to obtain cDNA according to the instructions of the reverse transcription kit PrimeScript^TM RT Master Mix (Takara Bio, Kusatsu, Japan). Subsequently, we employed SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) to carry out q-PCR on the Applied Biosystems^TM 7500 real-time fluorescent quantitative PCR system. The gene expression levels were normalized using the 2^−ΔΔCt method, with GAPDH as the internal control, for comparing the relative expression levels of TFAP2C and PTGES mRNA. Three replicate experiments were done for each group. Table 1 is a list of primer sequence for qRT-PCR.

Table 1.

Primers for qRT-PCR

Gene	Primer sequence
GAPDH	Forward Primer-GTCTCCTCTGACTTCAACAGCG
	Reverse Primer-ACCACCCTGTTGCTGTAGCCAA
TFAP2C	Forward Primer-GAAGAGGACTGCGAGGATCG
	Reverse Primer-GCTGATATTCGGCGACTCCA
PTGES	Forward Primer-AGGCTGCGGAAGAAGGTATG
	Reverse Primer-GGCCATGTTTCCCTATCCCG

Western blot (WB)

With RIPA (Beyotime, Shanghai, China) lysis buffer containing the phosphatase inhibitors and protease inhibitors, we lysed cell suspension and extracted proteins in cells. The protein concentration in the lysate was assessed beforehand by using a BCA protein detection kit (Beyotime). The total protein was then isolated by SDS-PAGE gel and transferred to the PVDF membrane. The membrane was sealed in TBST containing 5% skim milk at room temperature for 1 ‍h. The Membrane was incubated with the primary antibody against NOTCH3 (ab300527) overnight at 4°C, followed by 1 ‍h of incubation with the secondary antibody, goat anti-rabbit IgG H&L conjugated with horseradish peroxidase (HRP) (ab7090) at room temperature. We visualized protein bands by using an enhanced electrochemiluminescence (ECL) kit (P0018M; Beyotime) and imaged them with a ChemiScope 6000 chemiluminescence imaging system (Clinx, Shanghai, China). ImageJ was utilized for quantitative analysis to calculate the relative expression levels of the target protein in comparison with the internal control protein GAPDH (ab181602). All antibodies mentioned were purchased from Abcam (Cambridge, UK).

CCK-8 assay for cell viability

We employed the CCK-8 cytotoxicity assay kit (APExBIO, Houston, TX) to detect the cell viability of drug-resistant PC-9/GR cells. First, the cell suspension transfected with plasmids for 48 ‍h was added to a 96-well plate (5,000 cells for each well) and incubated in a cell culture incubator overnight at 37°C, 5% CO₂. Subsequently, in the following 48 ‍h, we treated the cell suspension with different concentrations of gefitinib (0 ‍μM, 4 ‍μM, 8 ‍μM, 12 ‍μM, and 16 ‍μM) in the culture medium. The addition of 10 ‍μl CCK-8 solution in each well was followed by an additional 2 ‍h of cultivation. The 96-well plate was then placed in a microplate reader to measure the optical density at 450 ‍nm. The IC₅₀ value was calculated using Graphpad 8.0 software.

Colony formation assay

We seeded 200 cells in each well of a 12-well plate. 2 ‍ml of DMEM-H medium containing 10% FBS was added to each well. The cells were further maintained in a 5% CO₂ incubator for 14 days, with real-time observation of cell colony formation. When obvious clones appeared in the wells, the cultivation ceased. We rinsed cells with phosphate-buffered saline (PBS) three times, immobilized them with 4% paraformaldehyde for 15 ‍min, and then stained them with 1% crystal violet for 30 ‍min. After three rinses with PBS, the cell cloning situation was observed under a microscope. We calculated the number of colonies that contained 50 cells at least and took photos, with three replicate groups conducted.

Dual-luciferase reporter assay

Firstly, we constructed pGL3-LCN2-WT and pGL3-LCN2-MUT plasmids. The 3'UTR region of the LCN2 fragment containing the TFAP2C binding sites and the 3'UTR region of the LCN2 fragment with the mutant binding sites were inserted into the pGL3 vector. The PGL3-WT-PTGES/PGL3-MUT-PTGES and si-TFAP2C/NC plasmids were transfected into 293T cells by utilizing Lipofectamine 2000 (Invitrogen) transfection reagent. After 48 ‍h of transfection, we measured the luciferase enzyme activity of each group of cells by utilizing the Dual-Luciferase Reporter Assay System (Promega, Madison, WI), with each independent experiment repeated three times.

Chromatin immunoprecipitation (CHIP)

PC-9/GR cells were seeded into 6-well plates (cell density: 1 × 10⁵ cells/ml), with 1% formaldehyde solution added to crosslink the proteins and DNA in the cells. 1 × glycine was added to terminate the crosslinking. Cells were lysed with RIPA buffer (Beyotime) to extract cell nuclei, and then chromatin fragments were obtained by sonication. Agarose magnetic beads (Millipore, Burlington, MA) and IP-grade Anti-TFAP2C antibody (ab218107; Abcam) were added to the cell lysate and incubated overnight at 4°C. The agarose magnetic beads were collected, heated, and boiled to de-crosslinking. By using a DNA purification kit (Invitrogen), the remaining DNA was purified and recovered. Anti-IgG antibody (ab6715; Abcam) was utilized as a negative control. Finally, the enrichment of DNA fragments was assessed by qPCR. CHIP-qPCR primers are listed in Table 2.

Table 2.

Primers for CHIP-qPCR

Gene	Primer sequence
PTGES	Forward Primer-TTTGAGTCCCTCCAAAGGGC
	Reverse Primer-CCCATCAAGGGGACATTTGC

Statistical analysis

All data obtained in this project were presented as mean ± SD, with three replicates for each group. GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA) was applied in data analysis and graphing. The t test determined the differences between the two groups. The analysis of variance (ANOVA) assessed the differences among multi-groups. Statistical significance was defined as p<0.05.

Results

PTGES is highly upregulated in LUAD

In this investigation, we first downloaded the expression data of LUAD mRNA from TCGA database, obtaining multiple DEGs. PTGES was chosen as the research object based on the previous literature.⁽⁶⁾ The t test analysis uncovered a remarkable elevation in PTGES expression in tumor tissues compared to adjacent tissues (Fig. 1A). Furthermore, through qPCR analysis, we also observed elevated mRNA levels of PTGES in LUAD cell lines A549, NCI-H1975, and PC-9 compared to human bronchial epithelial cells BEAS-2B (Fig. 1B). Taken together, PTGES is upregulated in LUAD tissues and cells, exerting an instrumental influence on LUAD development.

Fig. 1.

The expression of PTGES in LUAD tissues and cells. (A) Analysis of PTGES expression in LUAD tissues and adjacent normal tissues in the TCGA database, with green representing normal tissues and red representing tumor tissues. (B) qPCR detected the mRNA expression of PTGES in human bronchial epithelial cells BEAS-2B and LUAD cells A549, NCI-H1975, and PC-9. *p<0.05.

PTGES enhances the sensitivity of LUAD cells to gefitinib

The above bioinformatics analysis and cell experiments confirmed that PTGES was upregulated in LUAD tissues and cells, so we attempted to dig out the effect of PTGES on the viability and gefitinib resistance of drug-resistant LUAD cells. Firstly, the LUAD gefitinib-resistant PC-9/GR cells were clustered into groups: si-NC, si-PTGES, oe-NC, and oe-PTGES. We utilized qPCR to determine the mRNA expression level of PTGES, unearthing a remarkable decrease in the si-PTGES group in comparison with the control group but a relative elevation in the oe-PTGES group, thus indicating successful transfection of PTGES into drug-resistant PC-9/GR cells (Fig. 2A). The CCK-8 experiment demonstrated that the viability of PC-9/GR cells in the si-PTGES group was considerably reduced, while it was considerably elevated in the oe-PTGES group in comparison with the control group, further confirming the evident promoting role of PTGES in PC-9/GR cell growth (Fig. 2B). The cell cloning experiment also supported this conclusion. As displayed in Fig. 2C, the number of cell colonies in the si-PTGES group was considerably reduced compared to the control group, while it was considerably elevated in the oe-PTGES group compared to the oe-NC group. Subsequently, we treated PC-9/GR cells with different concentration gradients of gefitinib (0 ‍μM, 4 ‍μM, 8 ‍μM, 12 ‍μM, and 16 ‍μM) for 48 ‍h. As evidenced by the CCK-8 assay, the IC₅₀ of the si-PTGES group was considerably lower compared to si-NC, while in the oe-PTGES group, it was considerably higher than that of oe-NC group (Fig. 2D). The above experimental indicated that silencing PTGES can boost the drug sensitivity of PC-9/GR cells to gefitinib, leading to a decrease in cell viability. Conversely, PTGES overexpression reduces drug sensitivity and increases cell viability, demonstrating a positive correlation of PTGES expression with the viability and resistance of LUAD cells.

Fig. 2.

The knockdown of PTGES enhances the sensitivity of PC-9/GR cells to gefitinib. (A) qPCR measured the mRNA levels of PTGES in four groups of PC-9/GR cells (si-NC/si-PTGES, oe-NC/oe-PTGES). (B) CCK-8 assay determined the cell viability of four groups of PC-9/GR cells. (C) Cell colony formation experiment assessed the proliferation ability of four groups of PC-9/GR cells. (D) CCK-8 detected the inhibitory effect of gefitinib on the growth of PC-9/GR cells, with IC₅₀ values calculated. *p<0.05.

PTGES facilitates the sensitivity of LUAD cells to gefitinib through the NOTCH3 pathway

To illuminate the specific mechanism of PTGES in LUAD gefitinib resistance, we conducted GSEA on PTGES. In Fig. 3A, PTGES was observed to be remarkably concentrated in the NOTCH3 pathway. Subsequently, the drug-resistant PC-9/GR cells were clustered into si-NC/si-PTGES and oe-NC/oe-PTGES to measure the impact of PTGES on the expression level of NOTCH3 protein in different groups of cells. WB experiment uncovered that the NOTCH3 protein content in the si-PTGES group was remarkably lower than that in the control group, while in the oe-PTGES group, it was remarkably higher than that of control group oe-NC (Fig. 3B), indicating that knocking down PTGES in drug-resistant PC-9/GR cells effectively reduced the expression level of NOTCH3 protein and downregulated NOTCH3 signaling pathway transduction while overexpressing PTGES upregulated NOTCH3 protein and provoked NOTCH3 signaling pathway transduction. BMS-986115 is an effective inhibitor of Notch utilized in cancer research. To further verify the effect of PTGES on NOTCH3 signaling, we constructed the following three groups of cells: oe-NC + DMSO, oe-PTGES + DMSO, and oe-PTGES + BMS-986115. CCK-8 assay demonstrated that the tumor cell viability in the oe-PTGES + DMSO group was considerably elevated in comparison with the control group, which was greatly reversed back to the control level by adding the NOTCH3 inhibitor BMS-986115 (Fig. 3C). The cloning formation experiment revealed that the cell proliferation ability of the oe-PTGES + DMSO group was remarkably increased, which was decreased by the addition of BMS-986115 on this basis (Fig. 3D). Subsequently, cells from each group were treated with different concentrations of gefitinib (0 ‍μM, 4 ‍μM, 8 ‍μM, 12 ‍μM, and 16 ‍μM). In Fig. 3E, the IC₅₀ of the oe-PTGES + DMSO group cells was considerably increased compared to the control group, and it returned to the control group level after adding BMS-986115 on this basis. In conclusion, PTGES can affect the sensitivity of LUAD cells to gefitinib by triggering NOTCH3 pathway.

Fig. 3.

PTGES regulates the NOTCH3 signaling pathway to affect the resistance of LUAD cells to gefitinib. (A) GSEA analyzed PTGES enrichment based on LUAD mRNA expression level from TCGA database. (B) WB detected the protein expression of NOTCH3 in four groups of cells (si-NC/si-PTGES, oe-NC/oe-PTGES). (C) CCK-8 assessed the cell viability of three groups of cells (oe-NC + DMSO, oe-PTGES + DMSO, oe-PTGES + BMS-986115). (D) Colony formation experiment measured the colony formation of three groups of cells (oe-NC + DMSO, oe-PTGES + DMSO, oe-PTGES + BMS-986115). (E) CCK-8 assay evaluated the growth inhibition of three groups of cells (oe-NC + DMSO, oe-PTGES + DMSO, oe-PTGES + BMS-986115) by gefitinib, with IC₅₀ values calculated. *p<0.05.

TFAP2C is the potential upstream regulatory molecule of PTGES

The above experiments unearthed that PTGES affected the sensitivity of LUAD cells to gefitinib resistance by controlling the NOTCH3 signaling pathway. However, the upstream regulatory mechanism was not clear, so we predicted the potential upstream TFs of PTGES through the hTFtarget webpage tool by intersecting with the upregulated genes in LUAD to obtain 13 differentially expressed potential TFs. Combining with the literature, we selected TFAP2C as the research object (Fig. 4A). TFAP2C was compared with PTGES for Pearson correlation analysis (Fig. 4B), which revealed a high correlation between the two. Analysis from the TCGA database demonstrated that TFAP2C in LUAD tumor tissues was remarkably upregulated than that in normal tissues (Fig. 4C), implying that it could be one of the factors affecting LUAD. Furthermore, based on qPCR, a notable elevation in the mRNA level of TFAP2C in LUAD cells A549, NCI-H1975, and PC-9 was observed compared to human bronchial epithelial cells BEAS-2B (Fig. 4D). Subsequent CHIP and dual-luciferase reporter experiments were carried out to verify whether there was a binding relationship between the two. As displayed in Fig. 4E, the CHIP experiment uncovered that the DNA enrichment level of the Anti-TFAP2C group was substantially elevated compared to the Anti-IgG group, suggesting that TFAP2C was capable of binding to the promoter region of PTGES. Furthermore, the dual luciferase reporter assay demonstrated that si-TFAP2C remarkably reduced the activity of firefly luciferase in the PGL3-WT-PTGES group (Fig. 4F). Given all results, TFAP2C can target binding to PTGES, thereby functioning as an upstream TF to provoke PTGES transcription.

Fig. 4.

The prediction of the upstream TF of PTGES and the validation of their interaction relationship. (A) hTFtarget webpage tool predicted the potential upstream TF of PTGES. (B) Pearson correlation analysis of TFAP2C and PTGES. (C) TCGA database analyzed the expression of TFAP2C in LUAD tissues and adjacent normal tissues, with blue representing normal tissues and red representing tumor tissues. (D) qPCR detected TFAP2C mRNA expression in human bronchial epithelial cells BEAS-2B and LUAD cells A549, NCI-H1975, and PC-9. (E) The CHIP experiment verified the DNA binding relationship between TFAP2C and the PTGES promoter region. (G) Dual luciferase assay validated the binding relationship between TFAP2C and PTGES. *p<0.05.

TFAP2C transcriptionally upregulates PTGES and facilitates LUAD cell resistance to gefitinib by activating the NOTCH3 signal

Previously, we validated the enrichment of PTGES in the NOTCH3 signaling pathway and the impact exerted by PTGES on LUAD cell resistance to gefitinib through this pathway. Additionally, using the bioinformatics method, we predicted TFAP2C as the upstream TF. Moreover, we attempted to figure out whether TFAP2C in LUAD cells regulated the NOTCH3 signaling pathway by provoking PTGES and thereby affecting cell resistance to gefitinib. Firstly, we set up cell groups: si-NC + oe-NC, si-TFAP2C + oe-NC, and si-TFAP2C + oe-PTGES. According to qPCR, a notable decrease in PTGES expression in cells of the si-TFAP2C + oe-NC group was observed, which was reversed after transfection with oe-PTGES (Fig. 5A). CCK-8 assay uncovered a notable reduction in cell viability in the si-TFAP2C + oe-NC group, which was restored in the si-TFAP2C + oe-PTGES group (Fig. 5B). As evidenced by Fig. 5C, the colony formation experiments presented a remarkable decrease in the number of colonies formed by the si-TFAP2C + oe-NC group, which was reversed to the initial level after oe-PTGES transfection. Finally, cells from each group were treated with different concentrations of gefitinib (0 ‍μM, 4 ‍μM, 8 ‍μM, 12 ‍μM, and 16 ‍μM), with IC₅₀ determined by the CCK-8 assay. The IC₅₀ value of the si-TFAP2C + oe-NC group was considerably decreased in comparison with the control group, while it increased to the control level after oe-PTGES transfection (Fig. 5D). The above experiments further proved that TFAP2C in LUAD cells can facilitate cell sensitivity to gefitinib by provoking PTGES to modulate the NOTCH3 signaling pathway.

Fig. 5.

TFAP2C in LUAD cells modulates the NOTCH3 signaling pathway by activating PTGES, thereby affecting the sensitivity of cells to gefitinib. (A) qPCR detected the mRNA level of PTGES in three groups of cells (si-NC + oe-NC, si-TFAP2C + oe-NC, si-TFAP2C + oe-PTGES). (B) CCK-8 assay measured cell viability in the three groups. (C) The colony formation assay assessed the colony formation of three groups of cells. (D) CCK-8 assay evaluated the growth inhibition of gefitinib on three groups of cells, with IC50 values calculated. *p<0.05.

Discussion

Since drug resistance is a main cause of cancer progression and death in cancer patients, mining safe and effective strategies to reduce cancer cell resistance remains a challenge. Gefitinib, as an epidermal growth factor receptor inhibitor (EGFR-TKI), can effectively repress tumor growth and metastasis. Former studies have pointed out that certain methods can effectively boost the sensitivity of LUAD cells to gefitinib.^(12–14) Inspired by this background, this project aimed to elucidate the potential molecular mechanisms for treating gefitinib-resistant LUAD cells. We first obtained multiple DEGs from the TCGA database and selected the PTGES gene as the research object based on previous literature to study its potential applications in tumor progression.

PTGES is upregulated in various cancers such as melanoma⁽⁷⁾ and pancreatic cancer,⁽¹⁵⁾ serving as a biological target. However, its biological function and potential value in drug-resistance mechanisms of LUAD are not yet mature. Herein, we observed that PTGES was upregulated in LUAD tissues and cells. Its overexpression boosted the cell viability and proliferation ability of gefitinib-resistant LUAD PC-9/GR cells. In addition, overexpressing PTGES also elevated the IC₅₀ value of gefitinib-resistant PC-9/GR cells, thereby facilitating the resistance of PC-9/GR cells to gefitinib. This is in line with the results of Terzuoli et al.,⁽¹⁶⁾ where microsomal prostaglandin E synthase-1 (mPGES-1) (also known as PTGES) is overexpressed in gefitinib-resistant NSCLC cells, with its inhibition remarkably elevating the sensitivity of gefitinib-resistant cells to cisplatin and the reactivity to gefitinib. Combining the results of this project, PTGES holds promise to be a future potential target for alleviating drug resistance in LUAD patients. The signal of the NOTCH pathway is mediated by activating four NOTCH receptors (NOTCH1–4), each of which can modulate unique biological processes. Changes in the NOTCH3 signaling pathway play an instrumental part in the progression and pathogenesis of lung diseases, including chronic obstructive pulmonary disease and LC.⁽¹⁷⁾ Through GSEA, we observed that PTGES was greatly gathered in the NOTCH3 signaling pathway. Experimental results also indicated that overexpressing PTGES facilitated the cell viability and proliferation ability of gefitinib-resistant PC-9/GR cells, while adding the NOTCH3 signaling pathway inhibitor BMS-986115 restored it to the control level. Collectively, PTGES in this project can affect the response of LUAD cells to gefitinib by mediating the NOTCH3 pathway.

However, since the upstream regulatory mechanism of PTGES was not clear, we predicted TFAP2C as the upstream TF of PTGES through the hTFtarget webpage tool. TFAP2C is a member of the AP-2 family, and AP-2 proteins have been proven to play a pivotal part in developing drug resistance in cancer therapy.⁽¹⁰⁾ Studies have pointed out that TFAP2C can transcribe and trigger LINC00922, affecting the drug resistance mechanism of doxorubicin (DOX)-resistant osteosarcoma through the TFAP2C/LINC00922/miR-424-5p feedback loop.⁽¹⁸⁾ TFAP2C can transcribe and activate MALAT1 to enhance the resistance of docetaxel (DTX) chemotherapy in LUAD.⁽¹⁹⁾ The above examples all proved that TFAP2C acts as a TF of drug resistance-related genes in different cancers. Our research also supported this view. We unearthed that TFAP2C targeted and regulated the downstream gene PTGES related to gefitinib resistance, thereby affecting the resistance of LUAD to gefitinib. We thus inferred that TFAP2C was implicated in multiple drug-resistant pathways in cancer, suggesting the possibility of TFAP2C as a target for overcoming tumor drug resistance.

In summary, our research indicated that PTGES and TFAP2C are upregulated in LUAD. Mechanistically, TFAP2C can modulate the NOTCH3 signaling pathway affecting the sensitivity to gefitinib and cell viability of LUAD by activating PTGES. We identified a new mechanism pathway for anti-resistant LUAD cells, which may be promising as a potential therapeutic target for LUAD. However, the shortcoming of this investigation lies in the lack of in vivo experiments. Therefore, in the future, we will further illuminate the impact of this pathway on LUAD by applying this mechanism in an in vivo study.

Author Contributions

JX contributed to the study design, wrote the article, and gave the final approval of the version to be submitted. JC conducted the literature search and revised the article. LZ acquired the data. JH performed data analysis and drafted.

Data Availability Statement

The data and materials in the current study are available from the corresponding author on reasonable request.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not for profit sectors.

Ethics Approval and Consent to Participate

Ethical approval is not required for this study in accordance with local or national guidelines.

Consent to Participate Statement

Patient consent were not required in accordance with local or national guidelines.

Conflict of Interest

No potential conflicts of interest were disclosed.