Targeted Inhibition Of Artemis Enhanced Radiosensitivity In Tumor Cells

Background:Radiotherapy is very effective in local control of cancerous tumors, but its curative potential is often limited by intrinsic radioresistance of most malignant tumor cells. Overcoming this radioresistance is clinically important and became important research focus. A major determinant of tumor response to radiation is tumor cell radiosensitivity which dependent on its capability of repairing DNA damage. Furthermore, based on understanding of radiation tumor biology, there is a differential DNA repair mechanisms between tumor and normal cells. Therefore, reducing the capacity of cancer cells to repair DNA damage could sensitize tumors to radio/chemotherapy. In DNA damaging induced by ionizing radiation (IR), DNA double-strand breaks (DSBs) are considered the most cytotoxic type of DNA lesion and are lethal threat to cells. If misrepaired, DSBs may lead to chromosomal translocations and cell death. The major pathway for the repair of IR-induced DSBs in mammalian cells is non-homologous end-joining (NHEJ). Coupled with DSBs repairing, many signaling cascade is initiated mainly by protein kinase ATM to inducing cell cycle arrest and facilitating DNA repair. The main proteins required for NHEJ in mammalian cells are the Ku70/80 heterodimer, the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Artemis. XRCC4. DNA ligase?and XLF (XRCC4-like factor, also called Cernunnos). Defects in any of these molecules may disturb DNA repair efficiency, and increase cellular sensitivity to radiation. Thus, specific inhibitors of this key pathway are very useful tools to modulate tumor radiosensitivity. Though there is some known DNA repair protein/enzyme inhibitors targeted the DNA-PKcs or ATM that can enhanced tumor radiosensitivity, this field is clearly not saturated and ripe for further study and innovation. Artemis is one of the best characterized molecules in the NHEJ family. It was originally identified as a key regulator for V(D)J recombination due to its activities on hairpin opening and overhang processing. Artemis deficiency causes dysfunction of V(D)J recombination and results in radiosensitive severe combined immunodeficiency (RS-SCID) in human. Artemis-deficient human cells displayed hypersensitivity to many DNA damaging agents, like etoposide and bleomycin, and IR. Based on current knowledge, the biological function of Artemis in DSB repair can be summarized in the following two aspects:(1) serves as a processing enzyme to remove lesions or modify secondary structures, and foster DNA end resection and repair by the NHEJ pathway; (2) acts as a multifunctional protein involved in regulating cell cycle checkpoints, chromatin remodeling, p53 expression, and cellular stress response. Thus, such as other molecules in DSB repair pathways, Artemis is also clearly viable target. Selective targeting of Artemis has the potential to sensitize tumors to radio/chemotherapy. However, how modification of Artemis affects cell radiosensitivity has not been largely investigated. Here, this study demonstrated that tumor cell radiosensitivity can be modulated by disruption of Artemis function. 1 The study of Artemis expression in human colorectal cancer and the correlation with tumor radiosensitivity.For the establishment of strategy about Artemis as a target for radiosensitization, it is very necessary to study Artemis expression in cancer and the correlation with tumor radiosensitivity. We assess the expression of Artemis in cancer and adjacent normal tissues by quantitative PCR in 50 cases of human colorectal carcinoma samples and found that expression of Artemis in 28% of colon cancer is a higher in cancer tissue than adjacent normal tissues. Then using quantitative PCR and colony-forming assays, we assessed the relationship between Artemis expression levels of tumor cells and radiosensitivity in several colorectal cancer cell lines. Thus, we established the necessity and potential about Artemis as a target for radiosensitization.2 Lentivirus-mediated RNAi silencing targeting Artemis enhanced tumor radiosensitivityIt was reported that a hypomorphic Artemis cell line that have reduced but detectable Artemis protein are as radiosensitive as Artemis null cell lines. Thus, reduced the expression of Artemis could lead to increase radiosensitivity in cancer cells. Using lentivirus-mediated siRNA. we knockdown Artemis in human colorectal cancer cell line. RKO. Colony-forming assay. MTT assay and in vivo assay with nude mice were done to verify the increased sensitivity to IR and other DNA damaging agent in Artemis-knockdown RKO cells.?-H2AX foci assay was done to verify the impaired DSB rejoining in Artemis-knockdown RKO cells. We found the delayed DSBs rejoining in Artemis-knockdown RKO cells corresponding to Artemis-deficient cells. Through a variety of apoptosis detection methods and Western blot, we study the impact of silencing Artemis on apoptosis signaling pathways induced by DNA-damaging agent in RKO cells. Increased apopotosis happened in Artemis-knockdown RKO cells and p53/p21 signaling pathway is involved in this events.3 The dominant negative mutant Artemis enhances tumor cell radiosensitivityThe dominant negative mutation is the other way to disrupt protein function, which is more specific than siRNA and have a wider scope of application. Some of Artemis mutant has the dominant inactivation effect have been reported.Based on unique role of Artemis in radiation-induced DNA damage and its structure-function analyses, we constructed the mutant fragment of Artemis (D37N-413aa) in the absence of phosphorylation site and nuclease activity. The mutant Artemis was overexpressed in radioresistant cervical carcinoma cell line. HeLa cells. Cell survival was measured by the clonogenic assay. The yH2AX foci assay was used to monitor DNA repair after irradiation. We found that expression of the mutant Artemis delayed DNA DSB rejoining after irradiation, thereby enhanced radiosensitivity of HeLa cell. Co-immunoprecipitation and Western blot analysis were performed to study protein interaction and activation of Artemis. The results indicates that this mutant Artemis bind to DNA-PKcs and ATM, inhibited activation of endogenous Artemis, the key molecule for DNA repair and cell radiosensitivity. Conclusion1?There is significant heterogeneity about Artemis expression in different tissues. Artemis expression was higher in part of colorectal cancer tissue than adjacent normal tissue. High levels of Artemis could contribute to radioresistance in tumor cells. Therefore, we propose that in the high expressed Artemis and radioresistant colorectal cancer, strategies of targeted inhibition Artemis which can enhance tumor radiosensitivity and reduced normal side-effect has importance clinical significance.2?We developed lentiviral RNAi vectors with high efficiency silencing Artemis and silenced Artemis protein in tumor cells. Silencing Artemis protein increased the tumor cells sensitivity to IR and DNA-damaging agent in vitro and in vivo. The mechanism involved p53/p21 signaling pathway related apoptosis. The study provide new strategies for improve the treatment in radioresistant or chemoresistant colorectal cancer.3?The dominant negative mutant Artemis fragment enhanced tumor cell radiosensitivity through blocking activity of endogenous Artemis and DNA repair. It is another way to modulate tumor cell radiosensitivity via targeting Artemis. This novel mechanism of radiosensitivity furtherly suggests the potential role of Artemis in cancer therapy.