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. 2022 Jun 8;20(1):264.
doi: 10.1186/s12967-022-03446-z.

C2orf40 inhibits metastasis and regulates chemo-resistance and radio-resistance of nasopharyngeal carcinoma cells by influencing cell cycle and activating the PI3K/AKT/mTOR signaling pathway

Zuozhong Xie  1   2 , Wei Li  1 , Jingang Ai  1 , Jun Xie  3 , Xiaowei Zhang  4
Affiliations

C2orf40 inhibits metastasis and regulates chemo-resistance and radio-resistance of nasopharyngeal carcinoma cells by influencing cell cycle and activating the PI3K/AKT/mTOR signaling pathway

Zuozhong Xie et al. J Transl Med. .

Abstract

Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor of epithelial origin in head and neck with high incidence rate in Southern China. C2orf40 has been identified as a tumor suppressor gene in many cancers. However, the roles of C2orf40 in nasopharyngeal carcinoma has not been studied.

Methods: In this study, a bioinformatics analysis was performed to identify the differentially expressed genes in NPC. The quantitative methylation levels was detected using pyrosequencing. qRT-PCR, western blotting, immunohistochemistry and immunofluorescence were used to detect the expression level of related RNA and proteins. Cell proliferation was detected using CCK-8 assay, and colony formation capability was detected using colony formation assays. Cell migration and invasion were analyzed using wound-healing and Transwell assays, respectively. The apoptosis level of cells was assessed using TUNEL staining. Endogenous DNA damage and repair were assessed by the comet assay. Cell cycle analyses carried out by flow cytometry. Finally, We used a xenograft nude mouse to verify the roles of C2orf40 in chemoresistance and radioresistance in vivo.

Results: We found that the C2orf40 expression was significantly downregulated in NPC tissues and inversely associated with a poor prognosis. In vivo and in vitro functional experiments confirmed that overexpression of C2orf40 significantly inhibited the migration and invasion of NPC cells, and promoted their sensitivity to radiotherapy and chemotherapy of NPC cells. Mechanically, the expression level of C2orf40 was negatively correlated with the expression levels of CCNE1 and CDK1. Overexpression of C2orf40 induced cell cycle arrest of NPC cells at G/M phase. In addition, C2orf40 can down-regulated the expression levels of homologous recombination-related proteins (BRCA1, BRCA2, RAD51, and CDC25A) and inhibited the activity of the PI3K/AKT/mTOR signaling pathway.

Conclusion: The results clarified the biological functions and mechanisms of C2orf40, as a tumor suppressor gene, in NPC, and provided a potential molecular target for improving the sensitivity of NPC to radiotherapy and chemotherapy.

Keywords: C2orf40; Metastasis; Nasopharyngeal carcinoma; PI3K/AKT/mTOR signaling pathway; Prognosis.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
Hypermethylation modified C2orf40 is downregulated in nasopharyngeal carcinoma (NPC) cells and it is associated with a poor prognosis. A The expression profile analysis of three GEO datasets (GSE12452, GSE53819, and GSE64634) showed that the C2orf40 expression level in NPC tissues was significantly lower than that in normal nasopharyngeal epithelial tissues. B The C2orf40 expression in NPC tissues (n = 8) and normal nasopharyngeal epithelial tissues (n = 16) was measured by qRT-PCR. C Downregulation of C2orf40 expression was confirmed by Western blotting in four matched NPC and adjacent normal tissues. D‒E The expression level of C2orf40 was analyzed in one immortalized nasopharyngeal epithelial cell line (TERT) and seven NPC cell lines (CNE-1, CNE-2, HONE-1, SUNE-1, HNE-1, HK-1, and 5-8F) by qRT-PCR (D) and Western blotting (E). F The quantitative methylation levels in 24 paired NPC and normal nasopharyngeal epithelial tissues were detected using pyrosequencing. G The NPC cell lines were treated with methyltransferase inhibitor DAC for 48 h, and then, C2orf40 mRNA expression was determined by qRT-PCR. H After collecting clinicopathological and follow-up data and conducting immunohistochemistry, the correlations between C2orf40 expression levels and survival prognosis were analyzed in 94 NPC patients. The experiments were performed in triplicate and repeated twice. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
Overexpression of C2orf40 impairs the migration ability of NPC cells in vitro. A Gene set enrichment analysis (GSEA) was performed using two GEO datasets (GSE12452 and GSE53819) which compared high C2orf40 expression group and low C2orf40 expression group. The results showed the most significant enrichment of the cell migration-related genes in the high C2orf40 expression group. B The migration of HONE-1 and SUNE-1 cells stably overexpressing C2orf40 was assessed by in vitro scratch wound-healing assay. C The effects of C2orf40 overexpression on the migration and invasion abilities of SUNE-1 and HONE-1 cells were investigated via transwell migration and invasion assays. The experiments were performed in triplicate and repeated twice. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
Overexpression of C2orf40 enhances chemo-sensitivity of NPC cells to cisplatin in vitro. A‒B The endogenous expression level of C2orf40 was determined by Western blotting (A) and qRT-PCR (B) after stably overexpressing C2orf40 in SUNE-1 and HONE-1 cells. C SUNE-1 and HONE-1 cells were pretreated with cisplatin for the indicated concentrations, and then, they were subjected to CCK-8 assay. D–F SUNE-1 and HONE-1 cells were exposed to 1 µg/ml cisplatin for 48 h. D The sensitivity of NPC cells to cisplatin was evaluated using colony formation assay. E The apoptosis level of NPC cells was assessed using TUNEL staining. The results were shown as a percentage of TUNEL + cells versus total cells. F Western blot analysis of the expression levels of apoptosis-related genes (C-caspase-3, C-PARP, and Bax) in NPC cells. The β-actin protein was used as loading control. The experiments were performed in triplicate and repeated twice. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
C2orf40 overexpression promotes the sensitivity of NPC cells to radiotherapy in vitro. A GSEA was performed using two GEO datasets (GSE12452 and GSE53819) as mentioned previously. The results demonstrated the enrichment of the gene signatures associated with DNA repair. B‒C HONE-1 and SUNE-1 cells stably overexpressing C2orf40 or controls were submitted to X-ray irradiation (IR). B Concurrent staining for the DNA damage marker γH2AX and endogenous control DAPI revealed that γH2AX expression, as measured by immunofluorescence, was significantly higher after 24 h of 6-Gy IR in NPC cells with C2orf40 overexpression. C Endogenous DNA damage and repair were assessed by the comet assay, indicating that overexpression of C2orf40 impaired the ability of NPC cells to repair DNA damage. D HONE-1 and SUNE-1 cells stably overexpressing C2orf40 or controls were exposed to a range of X-ray doses: 0, 2, 4, 6, and 8 Gy. Then, the clonogenic cell survival was measured to evaluate the radiation sensitivity of NPC cells. E Western blotting showed the γH2AX protein levels in HONE-1 and SUNE-1 cells after exposure to 6-Gy IR for 0, 1, and 12 h, with β-actin as an internal reference. The experiments were performed in triplicate and repeated twice. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
C2orf40 overexpression in NPC cells could induce the cell cycle arrest at G2/M phase. A GO and KEGG pathway enrichment analyses of 544 genes that were negatively correlated with C2orf40 expression (|R|> 0.3, P < 0.05) using Metascape. Data were imported from the three GEO datasets (GSE12452, GSE53819, and GSE64634). B‒C A strong inverse correlation of C2orf40 expression levels with the expression levels of both CCNE1 (B) and CDK1 (C) was noted in the three GEO datasets. D‒E The representative images (D) and statistical analysis (E) of Western blotting of the expression levels of cell cycle-related genes (CDK1, p-CDK1, p-Rb, Rb, CCNE1 and CCNB1) in HONE-1 and SUNE-1 cells. GAPDH was used as an internal reference. F Cell cycle analyses carried out by flow cytometry of HONE-1 and SUNE-1 cells showed a cell cycle arrest at G2/M phase after transiently transfecting C2orf40 plasmids. The experiments were performed in triplicate and repeated twice. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
C2orf40 could down-regulate the expression levels of homologous recombination-associated proteins and inhibit the activation of PI3K/Akt/mTOR signaling pathway. A‒D Correlation analysis revealed that the C2orf40 expression level was negatively correlated with the expression levels of BRCA1 (A), BRCA2 (B), CDC25A (C), and RAD51 (D). The data were obtained from three GEO datasets (GSE12452, GSE53819, and GSE64634). E‒F The representative images (E) and statistical analysis (F) of Western blotting of the expression levels of homologous recombination-associated proteins (BRCA1, BRCA2, RAD51, and CDC25A) in HONE-1 and SUNE-1 cells transiently transfected with C2orf40-overexpressed plasmids. β-actin was used as an internal reference. G‒H The representative images. (G) and statistical analysis (H) of the activation of the PI3K/Akt/mTOR signaling pathway. HONE-1 and SUNE-1 cells were transiently transfected with C2orf40 overexpression vectors or empty vectors, and exposed to 0 or 2 Gy radiation. The experiments were performed in triplicate and repeated twice. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 7
Fig. 7
Overexpression of C2orf40 results in increased chemo-sensitivity and radio-sensitivity of NPC cells in vivo. A‒C An in vivo mouse xenograft tumor model was established by subcutaneous injection of HONE-1 cells into nude mice treated with PBS or cisplatin (4 mg/kg, once every 3 days). A Representative images of xenograft tumors resected from the xenograft model mice. The tumor volume (B) and tumor weight (C) were measured in xenograft tumors from nude mice. D‒F HONE-1 cells were subcutaneously injected into nude mice to establish the transplanted tumor model. When the tumor volume reached 150 ± 25 mm3, a local dose of 2.0 Gy was given 5 times. D Xenograft tumors were resected from mice after 21 days of injection of the cells. E Time–growth curves of tumor xenografts. F Weight of xenograft tumors harvested from mice. Data are presented as the mean ± SD. G Western blotting of the activation of PI3K/Akt/mTOR signaling pathway in xenografts. H Western blotting was used to detect the expression levels of cell cycle-related genes in xenografts. *P < 0.05, **P < 0.01, ***P < 0.001
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