ERCC6L, a DNA helicase, is involved in cell proliferation and associated with survival and progress in breast and kidney cancers

Abstract

By analyzing 4987 cancer transcriptomes from The Cancer Genome Atlas (TCGA), we identified that excision repair cross-complementation group 6 like (ERCC6L), a newly discovered DNA helicase, is highly expressed in 12 solid cancers. However, its role and mechanism in tumorigenesis are largely unknown. In this study, we found that ERCC6L silencing by small interring RNA (siRNA) or short hairpin RNA (shRNA) significantly inhibited the proliferation of breast (MCF-7, MDA-MB-231) and kidney cancer cells (786-0). Furthermore, ERCC6L silencing induced cell cycle arrest at G0/G1 phase without affecting apoptosis. We then performed RNA sequencing (RNA-seq) analysis after ERCC6L silencing and identified that RAB31 was markedly downregulated at both the transcriptional and translational levels. Its downstream protein, phosphorylated MAPK and CDK2 were also inhibited by ERCC6L silencing. The xenograft experiment showed that silencing of ERCC6L strikingly inhibited tumor growth from the 7th day after xenograft in nude mice. In addition, higher ERCC6L expression was found to be significantly associated with worse clinical survival in breast and kidney cancers. In conclusion, our results suggest that ERCC6L may stimulates cancer cell proliferation by promoting cell cycle through a way of RAB31-MAPK-CDK2, and it could be a potential biomarker for cancer prognosis and target for cancer treatment.

Keywords: MAPK; cancer; excision repair cross-complementation group 6 like; proliferation.

Figure 1. ERCC6L expression in 12 cancer types from The Cancer Genome Atlas (TCGA)

All p values are less then 4.5E-11 except for KICH (p = 0.0003).

Figure 2. Effect of of ERCC6L knockdown on cell proliferation, apoptosis and cell cycle

A. Effect of ERCC6L knockdown on cell proliferation at different time points in MCF-7 cells. B. Effect of of ERCC6L knockdown on cell apoptosis in MCF-7 cells. MCF7 cells were stained with Annexin V. C-D. Effect of ERCC6L knockdown on cell cycle in MCF-7 cells. Effect of ERCC6L knockdown on cell cycle significantly arrested at G0/G1 phase compared with the control in MCF-7 cells. The vertical axis shows the percentage of cells at different phases of cell cycle. E. Effect of ERCC6L knockdown on cell proliferation at different time points in MDA-MB-231 cell lines. F. Effect of ERCC6L knockdown on cell proliferation at different time points in 786-0 cells. G. Effect of ERCC6L knockdown on cell cycle in MDA-MB-231 cells. H. Effect of ERCC6L knockdown on cell cycle in 786-0 cells. ERCC6L were silenced by siRNA in MCF-7 cells, and were silenced by two different shRNAs (sh#1-ERCC6L and sh#2-ERCC6L) in both MDA-MB-231 and 786-0 cell lines. Error bars represent mean ± SD. *p<0.05 compared to the control group. **p<0.01 compared to the control group. ***p<0.001 compared to the control group.

Figure 3. RNA-seq analysis after ERCC6L knockdown

A. Heatmap of genes with differential expression in MCF-7 cells after transfection. B. Validation of differentially expressed genes (DEGs) by qRT-PCR. β-actin was used as the internal control. C. Validation of reported cancer-associated DEGs (RAB31, MZT2B, FN1, and ARPP19) by western blot. β-actin was used as the internal control. D. Mitogen-activated protein kinase (MAPK), cyclin dependent kinase 2 (CDK2) and cyclin D1 (CCND1) protein levels after ERCC6L knockdown in MCF-7 cells. *p<0.05 compared to the si-control group. **p<0.01 compared to the si-control group. ***p<0.001 compared to the control group.

Figure 4. Effect of ERCC6L silencing on tumor growth and associations of ERCC6L expression with clinical survival and progress of breast and kidney cancer

A. Tumor masses were harvested from MDA-MB-231-sh-Control and MDA-MB-231-sh#2 ERCC6L after tumors had grown for one month. B. Tumor weight after knockdown of ERCC6L. C. Changes in tumor volume after knockdown of ERCC6L in nude mice. D. Kaplan-Meier survival analysis in KIRC from TCGA. E. Kaplan-Meier survival analysis in BRCA from TCGA. F. Expression of ERCC6L at stage I, stage II, and stage III in breast cancer. G. Association of ERCC6L expression with survival at stage I in breast cancer. H. Association of ERCC6L expression with survival at stage II in breast cancer. I. Association of ERCC6L expression with survival at stage III in breast cancer. The clinical samples were devided into two groups according ERCC6L mRNA expression levels. High expression indicates that the mRNA value is greater than the median value of all tumors samples; Low expression indicates that the mRNA value is lower than the median value of all tumors samples. *p<0.05 compared to the sh-control group. **p<0.01 compared to the sh-control group. ***p<0.001 compared to the sh-control group.