Radiosensitizing Effect Of Reprimo(RPRM)in Non-small-cell Lung Cancer Cells And The Underlying Mechanisms

Purpose:Reprimo(RPRM),which was first isolated from mouse embryonic fibroblasts with wild type p53 exposed to X-irradiation,is a DNA damage-inducible gene.RPRM has been believed to be a tumor-suppressor gene since its discovery.Previous studies have shown that RPRM is hypermethylated in some malignant tumors including gastric cancer,breast cancer,colorectal cancer and other malignant tumors,resulting in its low expression or even deletion in these cancers.And RPRM deficiency promotes cancer cell proliferation,invasion and migration,thus affecting the prognosis of cancer.On the other hand,overexpression of RPRM in cancer cells induces a series of responses such as G2/M cell cycle arrest,inhibition of cell proliferation and migration,and increase in apoptosis,etc.All of these results suggest a potential important role of RPRM in the initiation,progression and treatment of cancer.However,the investigation on this gene is still very limited so far.It remains unclear how RPRM regulates the dynamic balance of cells and tissues,including the signaling pathwa'ys of proliferation,migration and cell survival.And the mechanism underlying its DNA methylation in tumor cells is also unknown.Recent studies have found that epigenetic aberration,such as abnormal methylation of DNA,is associated with reduced radiosensitivity of tumors.Then does hypermethylated RPRM affect radiosensitivity of tumors?Furthermore,RPRM can be activated by DNA damage,and it participates in the regulation of cell growth and apoptosis,then does it play a role in DNA damage response(DDR)?All of these questions need to be answered.Therefore,it is of great theoretical significance in understanding the mechanisms of various life activities as well as the process of carcinogenesis to investigate the function of RPRM.In addition,clarifying whether RPRM radiosensitizes cancer cells and whether RPRM participates in DNA damage repair response has important theoretical and practical value for better understanding the mechanisms of radiation resistance of tumors,finding novel targets for radiosensitization of cancers in radiotherapy,and exploring the further mechanism of DDR.Methods:In this study,non-small-cell lung cancer(NSCLC)cells were used to investigate the function of RPRM.In the first part of this study,the expression of RPRM in three different non-small-cell lung cancer cell lines,namely H460,A549 and H1299,was detected by real-time quantitative PCR and Western blotting.After that,stable RPRM overexpression and RPRM knockdown in H460 cells were achieved by plasmid transfection and lentivirus infection.In order to explore the radiosensitizing effect of RPRM on cancer cells,clonogenic assay and micronucleus formation assay were performed.In the second part of this study,the mechanisms of RPRM underlying its radiosensitizing effect were studied.First,the effect of RPRM on radiation-induced DNA damage was examined by comet assay.Then in order to clarify the role of RPRM in DNA damage repair pathway,real-time quantitative PCR,protein immunoblotting and immunofluorescent microscopy were used to determine the expression of DNA damage repair-related proteins in H460 cells overexpressing RPRM and the negative control cells after X-irradiation.To further study the mechanism of RPRM underlying its regulatory effect on DNA damage repair pathway,the subcellular localization and translocation of RPRM were identified by immunofluorescent microscopy,separation of nuclear and cytoplasmic components,and immunoprecipitation experiments.The regulatory effect of RPRM on the subcellular localization of ATM after irradiation was verified by protein immunoblotting and immunofluorescent microscopy.After inhibiting the transport of ATM from nucleus to cytoplasm with nuclear inhibitor Leptomycin B,the role of RPRM in regulating ATM subcellular localization was determined by using fluorescence intensity analysis of nuclear and cytoplasmic ATM.Additionally,the expression of ATM in irradiated cancer cells in the presence and absence of MG 132,a proteasome inhibitor,was assessed by Western blot to clarify the role of RPRM in regulating ATM degradation.In the third part of this study,the effects of RPRM overexpression on tumor growth and radiosensitivity were studied using xenograft model in vivo.H460 cells overexpressing RPRM and the negative control cells were subcutaneously injected into nude mice,respectively.The tumors were irradiated locally only once when the volume of tumors reached about 100 mm3.After that,the volume of the tumors was measured and monitored continuously.Results:The expression of RPRM varied in different non-small-cell lung cancer cells:the expression level of RPRM was low in H460 and A549 cells with wild type p53,but high in H1299 cells with deficient p53.After H460 cells being exposed to X-irradiation,RPRM was activated in a dose-dependent and time-dependent manner.Compared with the negative control group,the survival fraction decreased significantly and the micronucleus formation increased obviously in RPRM-overexpressing H460 and A549 cells irradiated with X-rays,indicating that RPRM overexpression radiosensitized cancer cells.On the other hand,RPRM knockdown H460 cells showed greater plating efficiency and lower micronucleus formation after irradiation compared with the relevant control.These data indicated that RPRM does affect the radiosensitivity of cancer cells.Further studies showed that the DNA damage was more severe in H460 cells overexpressing RPRM shortly after irradiation manifest as longer DNA tail and larger tail moment in the comet assay results.Furthermore,8 hours after irradiation,while the damaged DNA of the control cells was almost completely repaired,there was still some residue unrepaired DNA damage left in cells overexpressing RPRM,suggesting that RPRM might inhibit the capability of DNA damage repair of cancer cells.In order to verify the effect of RPRM on DDR,we examined the activation and expression of DDR-related proteins.The results showed that the expression of ATM,a core protein of DDR,decreased significantly in H460 cells overexpressing RPRM 2 hours after irradiation,which led to the inhibition of its activation.Activation of its downstream DDR-related proteins such as y-H2AX,53BP1,RAD51 and p53 were thus inhibited,resulting in defective DDR in cells with RPRM overexpression.After proteasome inhibitor MG 132 was added to inhibit protein degradation,the expression of ATM in H460 cells overexpressing RPRM increased significantly,suggesting that RPRM decreased the expression of ATM by promoting its degradation.We further explored the mechanism underlying ATM degradation promoted by RPRM.We showed for the first time that RPRM could rapidly translocate from cytoplasm to nucleus under the guidance of IPO-11 in cells exposed to X-irradiation.Moreover,the nuclear translocation of RPRM promoted ATM translocation from nucleus to cytoplasm,followed by ATM degradation in cytoplasm,thus interfering with its important role in DNA damage repair response.We also confirmed the interaction between RPRM and ATM by immunoprecipitation assay,which provided reliable evidence for the regulation of ATM translocation from nucleus to cytoplasm by RPRM.On the other hand,after inhibiting ATM translocation,the radiosensitivity of cells decreased significantly,suggesting that ATM translocation from nucleus to cytoplasm was essential to the radiosensitizing effect of RPRM.The radiosensitizing effect of RPRM has also been confirmed in vivo.Compared with the control group,tumors with high expression of RPRM grew slower,and shrank more rapidly after irradiation even to the extent that there was no visible mass.This result agreed with the in vitro data described above.These results indicate that RPRM inhibits DNA damage repair response by promoting ATM translocation and degradation,which makes damaged DNA unable to be repaired effectively,thus increasing the radiosensitivity of cancer cells.Conclusion:For the first time,we revealed a novel role of RPRM in radiosensitizing non-small-cell lung cancer cells.The underlying mechanisms involve the nuclear translocation of RPRM mediated by a nuclear transporter called IPO-11 under DNA damage induction.The nuclear RPRM thus binds to ATM and promotes ATM translocation from nucleus to cytoplasm and its degradation in cytoplasm,thus inhibiting the activation of ATM in response to DNA damage,resulting in impaired activation of downstream DNA damage repair-related proteins.Thus the DNA damage repair capability of the cells overexpressing RPRM decreases,leading to increased radiosensitivity of cancer cells.This study for the first time confirmed that RPRM plays an important role in regulating ATM,the core protein in DNA damage repair pathway.It provides a novel radiosensitizing target,thus may have important implications in clinical adjuvant radiotherapy and prognosis of lung cancer.