C1orf116 inhibits acquired resistance to EGFR inhibitors in EGFR mutant lung adenocarcinoma by suppressing the ATM/ATR pathways

Introduction

Lung cancer has been the deadliest malignant tumor globally in recent years, with lung adenocarcinoma (LUAD) being the most prevalent subtype, accounting for approximately 40%, and its incidence shows a year-on-year increasing trend (1). Epidermal growth factor receptor (EGFR)-sensitive mutations are one of the important driving factors leading to the emergence and progression of LUAD (2). The mutation rate in female patients with LUAD is over 60% (3). EGFR tyrosine kinase inhibitor (EGFR-TKI) has become the first-line drug for EGFR-mutant LUAD, including erlotinib, gefitinib, afatinib and osimertinib (2,4). Studies have shown that EGFR-TKIs have good efficacy in treating advanced EGFR-mutant LUAD patients, with an objective response rate of 60–70% (5-7). Erlotinib, as an EGFR-TKI, has shown significant efficacy in LUAD patients with EGFR mutations (8). However, with its wide clinical application, and a rising number of patients are developing resistance to targeted therapy, posing a significant challenge to TKI treatment (9). Therefore, there is an urgent need to explore potential mechanisms to overcome acquired TKI resistance.

The DNA damage response (DDR) is essential for repairing DNA damage and preserving genomic stability (10). Tumor cells have the ability to evade apoptosis by upregulating DNA damage repair mechanisms, consequently developing resistance to chemotherapy agents (11). Identifying new key regulatory factors involved in the DDR pathway of tumor cells and weakening tumor cell resistance to chemotherapy drugs by blocking these regulatory pathways is an important strategy to improve cancer treatment outcomes (12,13). Mutations that disrupt key DNA damage repair genes, such as BRCA1/2, ataxia-telangiectasia mutated (ATM), and RAD51, are recognized as triggers for tumorigenesis. BRCA1/2 and ATM are crucial components of the homologous recombination (HR) pathway, a significant mechanism involved in repairing DNA double-strand breaks (DSBs) (14,15).

The C1orf116 gene is emerging as a significant factor in tumor development. A study found that C1orf116 is a key regulator in the epithelial to mesenchymal transition (EMT) process, which plays a critical role in tumor metastasis (16). Furthermore, low expression of C1orf116 has been linked to unfavorable outcomes in patients with lung and prostate cancer. Additionally, C1orf116 expression is lower in metastatic tumors compared to primary prostate cancer, and its expression decreases in high-grade lung cancer (17). When analyzing the GSEA6981 and GSE121623 datasets, C1orf116 was significantly down-regulated in HCC827/ER cells. Therefore, based on the potential function of C1orf116 in tumors and its significant down-regulation in drug-resistant cells, we selected C1orf116 as the research target to explore its role in EGFR-TKI resistance.

In the present study, we investigated the differentially expressed genes (DEGs) between HCC827 and erlotinib-resistant HCC827 cells. Next, the impact and associated molecular mechanisms of C1orf116 on the development of acquired resistance to erlotinib were explored in lung cancer cells. Furthermore, overexpression (OE) of C1orf116 significantly reversed resistance to erlotinib in HCC827/ER erlotinib-resistant cells by reversing DNA damage repair phosphorylated (p)-ATM/ataxia-telangiectasia and Rad3-related (ATR) signaling pathway. Our findings provided a potential target to reverse EGFR-TKIs resistance. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-265/rc).

Methods

Screening of analysis of the Gene Expression Omnibus (GEO) database

The gene expression profiles GSE69181 and GSE121634 datasets from GEO database were analyzed using the edgeR package. The fold change (FC) in gene expression was calculated with a cutoff of FC ≥1 and a P<0.05. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted to explore the activated pathways of DEGs using the online tool DAVID Bioinformatics Resources 6.8 (http://david.ncifcrf.gov). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Generation of HCC827/ER and PC9/ER cells

HCC827 and PC9 cells (human LUAD cell line) were obtained from the American Type Culture Collection (ATCC, Cat# CRL-2755). Erlotinib-resistant cell lines (HCC827/ER and PC9-ER) were acquired according to a previously published method (18). Initially, HCC827 and PC9 cells were cultured with erlotinib at escalating doses from 0.005 to 2.5 µM in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) over a period of 6 months. The establishment of the HCC827/ER and PC9-ER cell line was confirmed after 6 months of continuous erlotinib exposure, during which the cells exhibited resistance to erlotinib concentrations up to 5 µM. Following this, the experiment was terminated, and the cells were preserved as stocks for future experiments. The erlotinib-resistant cells were thereafter cultured in 5 µM erlotinib for all subsequent experiments unless specified otherwise. Both HCC827/ER and HCC827 cells were then treated with varying concentrations of erlotinib (0.01, 0.1, 0.5, 1, 2.5, and 5 µM) in RPMI 1640 medium supplemented with 10% FBS, with controls maintained in erlotinib-free RPMI 1640 medium supplemented with 10% FBS.

Lentiviral expression vectors and stable expression cells for C1orf116

The human C1orf116 coding sequence (CDS) was cloned into the expression vector pCDH-CMV-MCS-puro and amplified using the following specified primers: C1orf116 forward 5'-GACCTTTGCCAATGAAGTCTCC-3' and reverse 5'-TTGGGGCCTTAGGGATAGAAAT-3'. Lentiviral vectors carrying either C1orf116 or empty inserts were generated using established protocols. The lentiviral particles were then introduced into HCC827/ER cells by supplementing the growth medium with the virus. HCC827/ER and PC9/ER cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 5 µM erlotinib. For C1orf116 knockdown, cells were transfected with short hairpin RNA targeting C1orf116 (sh-C1orf116) or shRNA-negative control (sh-NC) lentiviral vectors (GenePharma, Shanghai, China). Cells stably expressing C1orf116 were selected by treating them with 1.0 µg/mL of puromycin for 3 days.

Quantitative real time polymerase chain reaction (qRT-PCR)

Total RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, complementary DNA (cDNA) was synthesized from the RNA, and the resulting cDNA regions of interest were amplified using TB Green^TM Fast qPCR Mix (Takara, Kyoto, Japan). The 2^−ΔΔCt method was employed for calculating relative gene expression levels. The primer sequences used were as follows: GAPDH forward 5'-GAGAAGGCTGGGGCTCATTT-3', reverse 5'-AGTGATGGCATGGACTGTGG-3'; C1orf116 forward 5'-GACCTTTGCCAATGAAGTCTCC-3', reverse 5'-TTGGGGCCTTAGGGATAGAAAT-3'.

Drug sensitivity assay

Cells were treated with doxorubicin (ADM), cisplatin (cDDP), or etoposide (VP16) at gradient concentrations (0–10 µM, 48 h). Cell viability was assessed using CCK-8 assays (Dojindo, Kumamoto, Japan), and half-maximal inhibitory concentration (IC₅₀) values were calculated via GraphPad Prism 9.0.

Transwell assay

Cells (1×10⁵ per well) were seeded into the upper chamber of the Transwell insert, and serum-containing medium was added to the lower chamber. After incubation for 24 h, the cells were fixed with 4% paraformaldehyde for 10 min and then stained with 0.1% crystal violet for 10 min. The Transwell chambers were gently rinsed, and the number of invaded cells was observed and counted.

RNA-sequencing (RNA-seq) and data analysis

RNA-seq was carried out by Shanghai Reverse Ear Biotechnology Co., Ltd. (Shanghai, China). Initially, total RNA was extracted from Vector and C1orf116-OE HCC827/ER cells using Trizol (Invitrogen, Carlsbad, CA, USA) and treated with DNase I (Invitrogen). The RNA quality and purity were assessed using an Agilent 2100 Bioanalyzer. Subsequently, sequencing was performed on the Illumina Xplus platform.

DEGs were conducted utilizing the R package ‘DESeq2’ (version 1.40.2) comparing the Vector and C1orf116 OE groups. Genes displaying |log₂FC| ≥1 and adjusted P value <0.05.

Western blotting assay

The lysis buffer (Beyotime Biotechnology, Shanghai, China), which was supplemented with protease and phosphatase inhibitors (Beyotime Biotechnology), was used to lyse the cells on ice. After centrifugation at 16,000 ×g for 10 minutes at 4 ℃, the supernatant obtained from the cell lysate was used for immunoblotting analysis. The antibodies used targeted the following proteins: anti-ATM (Proteintech, Wuhan, China; Cat# 2531, RRID: AB_330330), anti-p-ATM (Proteintech, Cat# 2531, RRID: AB_330330), anti-Chk2 (Proteintech, Cat# 2531, RRID: AB_330330), anti-E-cadherin (Proteintech, Cat# 20874-1-AP), N-cadherin (Proteintech, Cat# 22018-1-AP), anti-Vimentin (Proteintech, Cat# 10366-1-AP), BRCA1 (sc-6954, Santa Cruz, Dallas, TX, USA), MDC1 (ab11169, Abcam, Cambridge, UK), 53BP1 (ab21083, Abcam) and β-actin (A5441, Sigma, Louis, MO, USA). The bands were then visualized using a gel documentation system (Bio-Rad, Hercules, CA, USA).

Animal experiments

Female BALB/c nude mice (5–6 weeks old, weighing 18–20 g) were procured from the SPF Animal Center at SLAC Animal (Shanghai, China). All animal care and experimental procedures were conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. KY2024-738-02). Tumor measurements were performed every 3 days.

Statistical analysis

The statistical analyses were conducted with the aid of SPSS software. For comparing two groups, Student’s t-test was employed, whereas one-way analysis of variance (ANOVA) was utilized when the comparison involved multiple groups. The findings were articulated in terms of mean ± standard deviation (SD). Significance levels indicated by P<0.05 were deemed to be statistically significant.

Results

C1orf116 was decreased in erlotinib-resistant LUAD cells and associated with LUAD patient’s poor prognosis

To evaluate DEGs and the mechanisms behind resistance to erlotinib among the HCC827 and erlotinib-resistant cells HCC827 (HCC827/ER), we analyzed DEGs in between the HCC827/ER and HCC827 cells from GSE69181 and GSE121634. The total of 764 DEGs was identified in GSE69181, of which 552 and 212 were high/low expressed in HCC827/ER cells, respectively (Figure 1A). Moreover, transcriptome analysis of the GSE121634 dataset revealed that a total of 3,626 DEGs were identified, among 1,705 up-regulation genes and 1,921 down-regulation genes in HCC827/ER cells (Figure 1B). Subsequently, we conducted a functional enrichment analysis of these DEGs, revealing a significant cluster of genes that play roles in mitotic cell cycle phase transition, positive regulation of cell adhesion and endoplasmic reticulum, and etc. (Figure S1A,S1B). Subsequent analysis of KEGG pathways revealed that highly enriched pathways, including the PI3K-AKT signaling pathway and MAPK signaling pathway (Figure 1C,1D). Interestingly, C1orf116 was significantly down-regulated in HCC827/ER cells from GSE6918 and GSE121634 (Figure 1E). Furthermore, low expression of C1orf116 was notably related to reduced OS in the LUAD-The Cancer Genome Atlas (TCGA) database (Figure 1F). Collectively, these findings suggest a plausible involvement of C1orf116 in the emergence of resistance to EGFR-TKIs of LUAD. Considering the established role of C1orf16 in tumor biology, particularly its involvement in EMT and metastasis, as well as its significant down-regulation in resistant cells, we prioritized C1orf116 for further functional and mechanistic studies to explore its potential role in EGRF-TKI resistance.

Figure 1 DEGs was identified in between parental cells and erlotinib-resistant cells. (A) Volcano plot was generated to display the genes detected by RNA-seq in between HCC827 and HCC827/Erlotinib-resistant (HCC827/ER) cells from GSE69181 datasets. (B) Volcano plot was created to show the genes detected by RNA-seq from GSE121634 datasets. (C) KEGG pathway enrichment analysis was conducted on the up-regulated and down-regulated genes from GSE69181 dataset. (D) KEGG pathway enrichment analysis was performed on up-regulated or down-regulated genes from GSE121634 dataset. (E) The expression of C1orf116 was analyzed form GSE69181 dataset. (F) Kaplan-Meier survival curve analysis of C1orf116 expression levels in LUAD patients. P=0.02. **, P<0.01; ***, P<0.001. CI, confidence interval; DEGs, differentially expressed genes; ECM, extracellular matrix; ER, erlotinib-resistant; HR, hazard ratio; IL-17, interleukin 17; KEGG, Kyoto Encyclopedia of Genes and Genomes; LUAD, lung adenocarcinoma; NS, not significant; OS, overall survival; RNA-seq, RNA-sequencing; SE, standard error.

C1orf116 decreased cell proliferation, invasion, migration and EMT in erlotinib-resistant LUAD cells

To determine if C1orf116 has a functional impact on LUAD cells’ resistance to ERGF-TKI, we developed erlotinib-resistant HCC827/ER. As shown in Figure 2A, these HCC827/ER cells were unresponsive to erlotinib treatment. Next, we generated stable C1orf116 OE HCC827/ER cell line using a lentivirus vector, and confirmed the mRNA level of C1orf116 via RT-qPCR (Figure 2B). The impact of C1orf116 OE on cell malignancy was assessed using multiple assays. The Cell Counting Kit-8 (CCK8) assay (Figure 2C) and the cell clone formation assay (Figure 2D,2E) demonstrated that C1orf116 OE significantly hindered the proliferation of HCC827/ER cells. Flow cytometry analysis of apoptosis showed that increasing C1orf116 levels led to an enhanced rate of apoptosis in HCC827/ER cells (Figure 2F,2G). Moreover, the transwell (Figure 2H,2I) and wound healing assays (Figure 2J,2K) indicated that the invasive and migratory abilities of cells in the C1orf116 OE group were diminished compared to the control group. Collectively, these results revealed that C1orf116 acts as a tumor suppressor in vitro for HCC827/ER cells.

Figure 2 C1orf116 inhibits cell proliferation, invasion and migration in HCC827/ER cells. (A) The cell viability was determined by MTS assay. (B) The level of C1orf116 mRNA was detected by using qRT-PCR assay. (C) Cell proliferation was evaluated by CCK8 assay. (D) Cell growth was analyzed through clone formation assays (crystal violet staining, ×100). (E) The number of cell colonies was counted. (F) Cell apoptosis analyzed by FACS (X axis: Annexin V-FIFT; Y axis: PI). (G) The percentage of apoptotic cells is presented. (H) The transwell assay was employed to assess cell invasion (crystal violet staining, ×100). (I) The number of migrated cells was determined. (J) A wound healing assay was conducted to investigate cell migration (crystal violet staining, ×100). (K) The area of wound closure was counted. *, P<0.05; **, P<0.01; ***, P<0.001. CCK8, Cell Counting Kit-8; FACS, fluorescence activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxypheny1)-2-(4-sulfopheny1)-24H-tetrazolium; NC, negative control; OE, overexpression; qRT-PCR, quantitative real-time polymerase chain reaction; PI, propidium iodide.

To further explore the mechanism of C1orf116 in erlotinib-resistant LUAD cells, particularly whether it is involved in tumor invasion and metastasis through affecting the EMT process, the expression of EMT-related markers was further examined. Western blot assay revealed that the expression levels of epithelial marker E-cadherin were significantly upregulated in C1orf116-overexpressing HCC827/ER cells, while the expression levels of the mesenchymal markers N-cadherin and vimentin were significantly downregulated in C1orf116-overexpressing HCC827/ER cells (Figure S2). This result suggests that C1orf116 may influence the invasive and migratory abilities of tumor cells by regulating the EMT process, thereby partially affecting the development of erlotinib-resistant LUAD cells.

OE of C1orf116 increased erlotinib sensitivity in erlotinib-resistant LUAD cells

To investigate the association between C1orf116 and acquired resistance to erlotinib, the responsiveness of erlotinib in C1orf116 OE cell lines was assessed using a CCK8 assay. The results revealed that HCC827/ER C1orf116 OE cells displayed significantly increased sensitivity to erlotinib (Figure 3A). C1orf116 OE significantly inhibited the size of the colons in HCC827/ER treated with erlotinib (Figure 3B). The cell apoptosis rate was significantly increased in HCC827/ER-C1orf116 OE cells treated with erlotinib (Figure 3C,3D). Additionally, the invasion (Figure 3E,3F) and migration (Figure 3G,3H) of cells were reduced in HCC827/ER-C1orf116 OE cells treated with erlotinib.

Figure 3 C1orf116 increased erlotinib sensitivity in erlotinib-resistant LUAD cells. (A) Cell viability measured by CCK-8 assay. (B) Colony formation assay in HCC827/ER cells treated with the indicated erlotinib concentrations (cells stained with 0.1% crystal violet, ×10); representative images (left) and quantification (right). (C) Representative flow-cytometry plots of apoptosis (X axis: Annexin V-ITC; Y axis: PI). (D) Quantification of apoptotic cells; data are presented as mean ± SD of three independent experiments. (E) Representative Transwell invasion images (cells stained with 0.1% crystal violet, ×100). (F) Quantification of invaded cells; data are presented as mean ± SD from five randomly selected visual fields. (G) Representative wound-healing images (×10). (H) Quantification of wound closure; data are presented as mean ± SD from five randomly selected visual fields. *, P<0.05; **, P<0.01; ***, P<0.001. CCK8, Cell Counting Kit-8; LUAD, lung adenocarcinoma; OE, overexpression; SD, standard deviation; PI, propidium iodide.

To verify whether the role of C1orf116 in EGFR-mutant LUAD resistance is universal, we further validated it in other EGFR-mutant LUAD models. In addition to the HCC827/ER cell line, we also selected the PC9/ER cell line. Stable cell lines overexpressing C1orf116 were constructed, and their impact on erlotinib sensitivity was assessed. The results showed that in the PC9/ER cell line, OE of C1orf116 similarly significantly enhanced the sensitivity of cells to erlotinib, manifested by inhibited cell proliferation (Figure S3A,S3B), increased apoptosis (Figure S3C,S3D), and reduced invasion (Figure S3E,S3F) and migration capabilities (Figure S3G,S3H). These supplementary experimental results further confirm the important role of C1orf116 in regulating EGFR-TKI resistance and suggest that its mechanism of action may be universal. Overall, these findings demonstrate the pivotal role of C1orf116 as a regulatory molecule in influencing acquired resistance to erlotinib in LUAD.

OE of C1orf116 increased erlotinib sensitivity in vivo

Subsequently, we investigated the impact of C1orf116 on erlotinib sensitivity in an in vivo setting. The upregulation of C1orf116 enhanced responsiveness to erlotinib, as evidenced by inhibited tumor size (Figure 4A,4B), reduced tumor volume (Figure 4C) and tumor weight (Figure 4D). These results demonstrated that enforced expression of C1orf116 significantly suppressed tumor progression and heightened sensitivity to erlotinib treatment, resulting in decreased tumor size and weight.

Figure 4 Overexpression of C1orf116 increased erlotinib sensitivity in vivo. (A) HCC827/ER cell derived xenografts model were established and treated with two different regimens for 28 days (n=5). (B) Overexpression of C1orf116 reduced HCC827/ER and enhanced sensitivity to erlotinib. (C) Tumor growth curve of HCC827/ER cell derived xenograft model. (D) Tumor weight of CC827/ER cell derived xenograft model. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; OE, overexpression.

C1orf116 knockdown promotes multidrug resistance (MDR) and upregulates DNA damage repair proteins in erlotinib-resistant LUAD cells

To further validate the role of C1orf116 in EGFR-TKI-resistant cells, we stably knocked down C1orf116 using shRNA in both HCC827/ER and PC9/ER cells and assessed its impact on MDR. As shown in Figure S4A, sh-C1orf116 markedly reduced C1orf116 mRNA levels compared to the sh-NC control group. In HCC827/ER cells, C1orf116 depletion significantly increased the IC50 values for doxorubicin (ADM), cDDP, and VP16 (Figure S4B). Similar findings were observed in PC9/ER cells (Figure S4C). C1orf116 loss activates the DDR pathway. Western blot analysis revealed that C1orf116 knockdown significantly upregulated the expression of key DDR proteins BRCA1, MDC1 and 53BP1 (Figure S4D-S4G). Collectively, these data indicated that C1orf116 deficiency enhanced DDR activity via upregulation of BRCA1, MDC1 and 53BP1 to promote MDR in EGFR-TKI-resistant cells. This further supports C1orf116 as a potential therapeutic target for reversing MDR.

RNA-seq revealed that C1orf116 regulated DNA damage repair

Our subsequent objective was to elucidate the downstream signaling pathways regulated by C1orf116 in mediating EGFR-TKI resistance. To investigate the influence of C1orf116 OE on erlotinib’s mechanism of action, RNA-Seq analyses were conducted on HCC827/ER-NC and C1orf116-OE cells to identify alterations. The volcano plot displays the top 10 significantly increased and decreased genes in HCC827/ER of NC and C1orf116 OE group (Figure 5A). The heatmap showed significantly differentially expression genes in NC and C1orf116 OE groups (Figure 5B). Subsequently, C1orf116 impacts DNA packaging complex and mismatch repair complex binding in response to DNA damage (Figure 5C). KEGG enrichment analysis of the significant differential expression genes revealed DNA replication, EGFR-TKI resistance, mismatch repair as significant pathways (Figure 5D).

Figure 5 C1orf116 regulated DNA damage repair. (A) Volcano plot representing DEGs between the C1orf116-OE group and the NC group. Red dots indicate up-regulated genes, blue dots represent down-regulated genes, and gray dots denote genes with no significant change. (B) Heat map of DEG. (C) GO enrichment analysis of DEGs, categorized into three main aspects: BP, CC, and MF. (D) KEGG pathway enrichment analysis of DEG. BP, biological process; CC, cellular component; DEG, differentially expressed gene; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; NC, negative control; NS, not significant; OE, overexpression.

Low expression of C1orf116 promoted the upregulation of ATM/ATR pathway signaling

Further, gene set enrichment analysis (GSEA) enrichment analysis revealed that low expression of C1orf116 increased DNA repair, Mismatch repair, and DNA replication (Figure 6A,6B). The DDR mechanism encompasses a series of coordinated checkpoints and repair pathways that oversee the regulation of checkpoints, apoptosis induction, and DNA repair processes to safeguard DNA integrity within human cell systems. Central to this signal transduction response were the key proteins ATM and ATR. The ATM/ATR signaling in HCC827/ER cell lines was further validated by Western blot. OE of C1orf116 significantly inhibited the p-ATM, p-ATR, p-Chk2 protein expression (Figure 6C,6D). Our results indicated that C1orf116 downregulates the expression of ATM/ATR signaling pathway and enhances sensitivity to erlotinib in HCC827/ER cells.

Figure 6 Low expression of C1orf116 promoted the upregulation of ATM/ATR pathway signaling. (A) GSEA HALLMARK pathway enrichment analysis. (B) GSEA KEGG pathway enrichment analysis. (C) The protein expression of p-ATR, ATR, p-ATM, ATM, p-Chk2, Chk2 were detected by using Western blot. (D) The protein level of p-ATM/ATM, p-ATR/ATR, p-Chk2/Chk2 in HCC827/ER of NC and C1orf116 OE were analyzed. ***, P<0.001. ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiectasia and Rad3-related; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; OE, overexpression; p, phosphorylated.

Discussion

The development of resistance to EGFR-TKIs poses a significant obstacle in managing LUAD with EGFR mutations (19,20). Our study offers fresh perspectives on how C1orf116 influences resistance to EGFR-TKIs, underscoring its possible role as a therapeutic target. The findings demonstrate that C1orf116 is significantly downregulated in erlotinib-resistant LUAD cells and is related to poor prognosis in LUAD patients. This suggests that C1orf116 could potentially function as a predictive biomarker of resistance to EGFR-TKIs.

Previous research has indicated that diminished levels of C1orf116 promote cancer cell proliferation, migration, and invasion, leading to progression to advanced stages of tumor development and decreased survival rates (16,17). Our results indicate that OE of C1orf116 in erlotinib-resistant cells significantly inhibits cell proliferation, invasion, and migration, while also increasing the sensitivity of these cells to erlotinib. Both in vitro and in vivo studies revealed this impact, indicating that C1orf116 is pivotal in the emergence of resistance acquired. The mechanisms underlying these effects appear to involve the regulation of DDR pathways, particularly the ATM/ATR signaling pathway. While some phenotypic effects of C1orf116 OE (reduced proliferation/migration) were commonly observed in cancer studies (16,17), its specific downregulation in resistant cells, selective effects on the ATM/ATR pathway, and clinical correlations suggest a particular role in EGFR-TKI resistance.

The DDR comprises an intricate web of pathways designed to identify and mend DNA damage, thus preserving genomic integrity (21,22). In cancer cells, enhanced DDR can contribute to resistance to various therapies, including EGFR-TKIs (23-25). Our RNA-seq data revealed that C1orf116 OE affects the level of genes involved in DNA repair processes, including those related to DNA replication and mismatch repair. This suggests that C1orf116 may modulate the DDR by influencing the expression of key genes in these pathways. Poly (ADP-ribose) polymerase (PARP) inhibitors (olaparib and niraparib) are mainly used for the treatment of ovarian cancer, breast cancer, etc., with BRCA gene mutations (26,27). PARP inhibitors work by inhibiting the activity of the PARP enzyme, blocking single-strand DNA repair, which results in the buildup of DNA damage, ultimately leading to cell demise (28,29).

Furthermore, our GSEA and Western blot analyses confirmed that C1orf116 downregulates the expression of the ATM/ATR signaling pathway. The ATM/ATR pathway is a critical mediator of the DDR, and its activation can result in the halt of cell cycle progression and the initiation of DNA repair processes, thereby promoting resistance to DNA-damaging agents (30,31). By inhibiting this pathway, C1orf116 could potentially increase the responsiveness of cancer cells to EGFR-TKIs, making them more susceptible to the cytotoxic effects of these drugs. DDR is the cellular response to genotoxic stress, which includes processes such as DNA repair, cell cycle arrest, and apoptosis (32,33). ATM and ATR protein inhibitors, such as AZD0156 and LY2606368, are also used (34). ATM and ATR are crucial kinases in cellular responses to DNA damage. Inhibiting them can disrupt DNA damage signaling and repair processes, enhancing the effectiveness of radiotherapy and chemotherapy (35). DNA damage repair inhibitors have broad potential applications in cancer treatment, particularly showing significant efficacy in treating cancers with specific gene mutations or defects, such as ovarian and breast cancers with BRCA gene mutations (36). Moreover, they can be used in combination with other anticancer drugs to enhance treatment effectiveness and overcome drug resistance.

The role of C1orf116 in cancer progression and metastasis has been previously implicated in other studies. For instance, it has been reported as having a significant part in the process of epithelial-mesenchymal transition (EMT), a process crucial for tumor metastasis (17). Our findings align with these reports, as we observed that C1orf116 OE significantly reduced the invasive and migratory capabilities of erlotinib-resistant LUAD cells, and reversed EMT markers, restoring epithelial characteristics in resistant cells. This aligns with previous reports linking C1orf116 to EMT regulation (16) and suggests a multifaceted role for C1orf116 in overcoming EGFR-TKI resistance. This further reinforces the idea that C1orf116 functions as a tumor suppressor in LUAD.

While C1orf116 has been identified as a hub gene in pancreatic cancer based on functional similarity among DEGs (17), it was not found to be prognostically significant in that context. This raises the possibility that the function of C1orf116 may be tumor-type specific. In contrast, our study and others have shown that C1orf116 expression is prognostically relevant in LUAD, where low expression is associated with poor survival. This discrepancy may reflect the distinct biological roles of C1orf116 across different cancer types, particularly its involvement in EMT and metastasis in lung and prostate cancers, as previously reported.

Conclusions

In conclusion, our research presents data indicating that C1orf116 is a pivotal factor in controlling resistance to EGFR-TKIs in LUAD. C1orf116 regulates the ATM/ATR signaling pathway and affects the DDR, offering a potential therapeutic strategy for overcoming resistance. Future studies should focus on elucidating the precise mechanisms by which C1orf116 interacts with the DDR pathways and explore its potential as a therapeutic target in combination with EGFR-TKIs. Additionally, the development of small molecules or other strategies to modulate C1orf116 expression could be a promising avenue for promoting the efficacy of EGFR-TKI therapies in LUAD patients.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-265/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-265/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-265/prf

Funding: This work was supported by grants from Noncommunicable Chronic Diseases-National Science and Technology Major Project (No. 2023ZD0501700), the National Natural Science Foundation of China (No. 82373349), Guangdong Provincial Science and Technology Planning Project (No. 2023B110009), and MOE Changjiang Distinguished Professor Supporting Project (No. KY0120240205).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-265/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal care and experimental procedures were conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. KY2024-738-02).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Alexander M, Kim SY, Cheng H. Update 2020: Management of Non-Small Cell Lung Cancer. Lung 2020;198:897-907. [Crossref] [PubMed]
2. Fu K, Xie F, Wang F, et al. Therapeutic strategies for EGFR-mutated non-small cell lung cancer patients with osimertinib resistance. J Hematol Oncol 2022;15:173. [Crossref] [PubMed]
3. Castellanos E, Feld E, Horn L. Driven by Mutations: The Predictive Value of Mutation Subtype in EGFR-Mutated Non-Small Cell Lung Cancer. J Thorac Oncol 2017;12:612-23. [Crossref] [PubMed]
4. Grant C, Nagasaka M. Neoadjuvant EGFR-TKI therapy in Non-Small cell lung cancer. Cancer Treat Rev 2024;126:102724. [Crossref] [PubMed]
5. Jänne PA, Baik C, Su WC, et al. Efficacy and Safety of Patritumab Deruxtecan (HER3-DXd) in EGFR Inhibitor-Resistant, EGFR-Mutated Non-Small Cell Lung Cancer. Cancer Discov 2022;12:74-89. [Crossref] [PubMed]
6. Zhou F, Guo H, Xia Y, et al. The changing treatment landscape of EGFR-mutant non-small-cell lung cancer. Nat Rev Clin Oncol 2025;22:95-116. [Crossref] [PubMed]
7. Zhao Y, He Y, Wang W, et al. Efficacy and safety of immune checkpoint inhibitors for individuals with advanced EGFR-mutated non-small-cell lung cancer who progressed on EGFR tyrosine-kinase inhibitors: a systematic review, meta-analysis, and network meta-analysis. Lancet Oncol 2024;25:1347-56. [Crossref] [PubMed]
8. Wang XS, Bai YF, Verma V, et al. Randomized Trial of First-Line Tyrosine Kinase Inhibitor With or Without Radiotherapy for Synchronous Oligometastatic EGFR-Mutated Non-Small Cell Lung Cancer. J Natl Cancer Inst 2023;115:742-8. [Crossref] [PubMed]
9. Lin Z, Li J, Zhang J, et al. Metabolic Reprogramming Driven by IGF2BP3 Promotes Acquired Resistance to EGFR Inhibitors in Non-Small Cell Lung Cancer. Cancer Res 2023;83:2187-207. [Crossref] [PubMed]
10. Zhang J, Wu Q, Zhu L, et al. SERPINE2/PN-1 regulates the DNA damage response and radioresistance by activating ATM in lung cancer. Cancer Lett 2022;524:268-83. [Crossref] [PubMed]
11. Cui B, Song L, Wang Q, et al. Non-small cell lung cancers (NSCLCs) oncolysis using coxsackievirus B5 and synergistic DNA-damage response inhibitors. Signal Transduct Target Ther 2023;8:366. [Crossref] [PubMed]
12. Zhou B, Yang Y, Pang X, et al. Quercetin inhibits DNA damage responses to induce apoptosis via SIRT5/PI3K/AKT pathway in non-small cell lung cancer. Biomed Pharmacother 2023;165:115071. [Crossref] [PubMed]
13. Nisar H, Brauny M, Labonté FM, et al. DNA Damage and Inflammatory Response of p53 Null H358 Non-Small Cell Lung Cancer Cells to X-Ray Exposure Under Chronic Hypoxia. Int J Mol Sci 2024;25:12590. [Crossref] [PubMed]
14. Blackford AN, Jackson SP. ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Mol Cell 2017;66:801-17. [Crossref] [PubMed]
15. Liu W, Zheng M, Zhang R, et al. RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy. Adv Sci (Weinh) 2023;10:e2203884. [Crossref] [PubMed]
16. Parsana P, Amend SR, Hernandez J, et al. Identifying global expression patterns and key regulators in epithelial to mesenchymal transition through multi-study integration. BMC Cancer 2017;17:447. [Crossref] [PubMed]
17. Liu S, Cai Y, Changyong E, et al. Screening and Validation of Independent Predictors of Poor Survival in Pancreatic Cancer. Pathol Oncol Res 2021;27:1609868. [Crossref] [PubMed]
18. Nilsson MB, Sun H, Diao L, et al. Stress hormones promote EGFR inhibitor resistance in NSCLC: Implications for combinations with β-blockers. Sci Transl Med 2017;9:eaao4307. Erratum in: Sci Transl Med 2019;11:eaax9987. [Crossref] [PubMed]
19. Song H, Liu D, Wang L, et al. Methyltransferase like 7B is a potential therapeutic target for reversing EGFR-TKIs resistance in lung adenocarcinoma. Mol Cancer 2022;21:43. [Crossref] [PubMed]
20. Li N, Zuo R, He Y, et al. PD-L1 induces autophagy and primary resistance to EGFR-TKIs in EGFR-mutant lung adenocarcinoma via the MAPK signaling pathway. Cell Death Dis 2024;15:555. [Crossref] [PubMed]
21. Srinivas US, Tan BWQ, Vellayappan BA, et al. ROS and the DNA damage response in cancer. Redox Biol 2019;25:101084. [Crossref] [PubMed]
22. Huang RX, Zhou PK. DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduct Target Ther 2020;5:60. [Crossref] [PubMed]
23. Zhang Z, Li Y, Shi R, et al. L3MBTL1, a polycomb protein, promotes Osimertinib acquired resistance through epigenetic regulation of DNA damage response in lung adenocarcinoma. Cell Death Dis 2024;15:649. [Crossref] [PubMed]
24. De Rosa C, De Rosa V, Tuccillo C, et al. ITGB1 and DDR activation as novel mediators in acquired resistance to osimertinib and MEK inhibitors in EGFR-mutant NSCLC. Sci Rep 2024;14:500. [Crossref] [PubMed]
25. Wei W, Shi F, Xu Y, et al. The enrichment of Fanconi anemia/homologous recombination pathway aberrations in ATM/ATR-mutated NSCLC was accompanied by unique molecular features and poor prognosis. J Transl Med 2023;21:874. [Crossref] [PubMed]
26. Li J, Li Q, Zhang L, et al. Poly-ADP-ribose polymerase (PARP) inhibitors and ovarian function. Biomed Pharmacother 2023;157:114028. [Crossref] [PubMed]
27. Das PK, Matada GSP, Pal R, et al. Poly (ADP-ribose) polymerase (PARP) inhibitors as anticancer agents: An outlook on clinical progress, synthetic strategies, biological activity, and structure-activity relationship. Eur J Med Chem 2024;274:116535. [Crossref] [PubMed]
28. Petropoulos M, Karamichali A, Rossetti GG, et al. Transcription-replication conflicts underlie sensitivity to PARP inhibitors. Nature 2024;628:433-41. [Crossref] [PubMed]
29. Bai YR, Yang WG, Jia R, et al. The recent advance and prospect of poly(ADP-ribose) polymerase inhibitors for the treatment of cancer. Med Res Rev 2025;45:214-73. [Crossref] [PubMed]
30. Pilié PG, Tang C, Mills GB, et al. State-of-the-art strategies for targeting the DNA damage response in cancer. Nat Rev Clin Oncol 2019;16:81-104. [Crossref] [PubMed]
31. Durinikova E, Reilly NM, Buzo K, et al. Targeting the DNA Damage Response Pathways and Replication Stress in Colorectal Cancer. Clin Cancer Res 2022;28:3874-89. [Crossref] [PubMed]
32. Gao R, Kalathur RKR, Coto-Llerena M, et al. YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis. EMBO Mol Med 2021;13:e14351. [Crossref] [PubMed]
33. Foo TK, Vincelli G, Huselid E, et al. ATR/ATM-Mediated Phosphorylation of BRCA1 T1394 Promotes Homologous Recombinational Repair and G(2)-M Checkpoint Maintenance. Cancer Res 2021;81:4676-84. [Crossref] [PubMed]
34. Ronco C, Martin AR, Demange L, et al. ATM, ATR, CHK1, CHK2 and WEE1 inhibitors in cancer and cancer stem cells. Medchemcomm 2017;8:295-319. [Crossref] [PubMed]
35. Wu Q, Liu X, Wang LM, et al. Oleandrin enhances radiotherapy sensitivity in lung cancer by inhibiting the ATM/ATR-mediated DNA damage response. Phytother Res 2024;38:4151-67. [Crossref] [PubMed]
36. Grimley E, Cole AJ, Luong TT, et al. Aldehyde dehydrogenase inhibitors promote DNA damage in ovarian cancer and synergize with ATM/ATR inhibitors. Theranostics 2021;11:3540-51. [Crossref] [PubMed]