Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.
Colorectal cancer (CRC) is a heterogeneous disease and one of the most prevalent malignancies worldwide. Previous research has demonstrated that mitophagy is crucial to developing colorectal cancer. This study aims to examine the association between mitophagy-related genes and the prognosis of CRC patients. Gene expression profiles and clinical information of CRC patients were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate Cox regression and the least absolute shrinkage and selection operator (LASSO) regression analysis were applied to establish a prognostic signature using mitophagy related genes. Kaplan–Meier and receiver operating characteristic (ROC) curves were used to analyze patient survival and predictive accuracy. Meanwhile, we also used the Genomics of Drug Sensitivity in Cancer (GDSC) database and Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to estimate the sensitivity of chemotherapy, targeted therapy and immunotherapy. ATG14 overexpression plasmid was used to regulate the ATG14 expression level in HCT116 and SW480 cell lines, and cell counting kit-8, colony formation and transwell migration assay were performed to validate the function of ATG14 in CRC cells. A total of 22 mitophagy-driven genes connected with CRC survival were identified, and then a novel prognostic signature was established based on 10 of them (AMBRA1, ATG14, MAP1LC3A, MAP1LC3B, OPTN, VDAC1, ATG5, CSNK2A2, MFN1, TOMM22). Patients were divided into high-risk and low-risk groups based on the median risk score, and the survival of patients in the high-risk group was significantly shorter in both the training cohort and two independent cohorts. ROC curve showed that the area under the curves (AUC) of 1-, 3- and 5-year survival were 0.66, 0.66 and 0.64, respectively. Multivariate Cox regression analysis confirmed the independent prognostic value of the signature. Then we constructed a Nomogram combining the risk score, age and M stage, which had a concordance index of survival prediction of 0.77 (95% CI 0.71–0.83) and more robust predictive accuracy. Results showed that CD8+ T cells, regulatory T cells and activated NK cells were significantly more enriched in the high-risk group. Furthermore, patients in the high-risk group are more sensitive to targeted therapy or chemotherapy, including bosutinib, elesclomol, lenalidomide, midostaurin, pazopanib and sunitinib, while the low-risk group is more likely to benefit from immunotherapy. Finally, in vitro study confirmed the oncogenic significance of ATG14 in both HCT116 and SW480 cells, whose overexpression increased CRC cell proliferation, colony formation, and migration. In conclusion, we developed a novel mitophagy-related gene signature that can be utilized not only as an independent predictive biomarker but also as a tool for tailoring personalizing treatment for CRC patients, and we confirmed ATG14 as a novel oncogene in CRC.
Background: Colorectal cancer (CRC) is a heterogeneous disease and one of the most common malignancies in the world. Previous studies have found that mitophagy plays an important role in the progression of colorectal cancer. This study is aimed to investigate the relationship between mitophagy-related genes and the prognosis of patients with CRC.Methods: Gene expression profiles and clinical information of CRC patients were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate Cox regression and the least absolute shrinkage and selection operator (LASSO) regression analysis were used to establish the prognostic signature composed of mitophagy related genes. Kaplan-Meier curve and receiver operating characteristic (ROC) curve were used to analyze patient survival and verify the predictive accuracy of the signature, respectively. Construction of a nomogram prognostic prediction model was based on risk scores and clinicopathological parameters. Using the Genomics of Drug Sensitivity in Cancer (GDSC) database and Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to estimate the sensitivity of chemotherapy, targeted therapy and immunotherapy. Results: A total of 44 mitophagy-driven genes connected with CRC survival were identified, and prognostic signature was established based on the expression of 10 of them (AMBRA1, ATG14, MAP1LC3A, MAP1LC3B, OPTN, VDAC1, ATG5, CSNK2A2, MFN1, TOMM22). Patients were divided into high-risk and low-risk groups based on the median risk score, and the survival of patients in the high-risk group was significantly shorter than that of the low-risk group among the TCGA cohort (median OS 67.3 months vs not reached, p=0.00059) and two independent cohorts from GEO (median OS in GSE17536: 54.0 months vs not reached, p=0.0082; in GSE245: 7.7 months vs not reached, p=0.025). ROC curve showed that the area under the curves (AUC) of 1-, 3- and 5-year survival were 0.66, 0.66 and 0.64, respectively. Multivariate Cox regression analysis confirmed the independent prognostic value of the signature. Then we constructed a nomogram combining the risk score, age and M stage, which had a concordance index of survival prediction of 0.77 (95% CI=0.71-0.83) and more robust predictive sensitivity and specificity. Results showed that CD8+ T cells, regulatory T cells and activated NK cells were significantly more abundant in the high-risk group. Furthermore, patients in the high-risk group were more sensitive to potential targeted therapies, including Motesanib, ATRA, Olaparib, Selumetinib, AZD8055 and immunotherapy. Conclusion: In conclusion, we constructed and validated a novel mitophagy related gene signature that can be used as an independent prognostic biomarker for CRC, and may lead to better stratification and selection of precise treatment for CRC patients.
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