Crif1 Interacting With CDK2 Promotes Radiation Resistance

Background and objective:As a high-energy physical damage factor,radiation damages the structure and function of biological macromolecules mainly through the direct effect of radioactive energy accumulation and the indirect effect of free radicals.DNA is the most important target of radiation injury.DNA double-strand breaks（DSBs）are the pivotal lethal factor of radiotherapy.DNA Damage Response（DDR）regulates DNA damage repair,cell cycle arrest,and apoptosis by complex cascade reaction network,and ultimately determines the survival of cells exposed to radiation.Radiotherapy mainly induces DNA damage,especially DSBs,to kill tumor cells,through direct or indirect effects of ionizing radiation.The radiotherapy effect is determined by the radiosensitivity of tumor cells and the protection of tumor microenvironment.The key to affecting the radiosensitivity of tumor cells is the activation of cell cycle checkpoint and the repair of DNA damage.Osteosarcoma（OS）is the most common primary malignant tumor in bone originating from interstitial cells.Local tumor control is vital for the prognosis,and tumor resection is still the gold standard of clinical treatment.For unresectable OS or cases that are not completely resected,as well as bone resection and complex remodeling after tumor resection,radiotherapy is one therapeutic option for achieving local tumor control.However,the radioresistance of OS requires a high dose of radiation to achieve an effective result,which produces high rates of side effects and limits the application of radiation in the treatment of OS.Study of the molecular mechanisms of radioresistance in OS to identify novel molecular targets could help overcome this obstacle,improve local tumor control,broaden the therapeutic window,and extend patient survival.The radioresistance of osteosarcoma is associated with a strong repair ability following sub-lethal-dose radiation.This repair ability is closely related to the prior activation of the DNA damage response（DDR）,and inhibiting the DDR enhances the radiosensitivity of tumor cells.Studies have confirmed that the inhibition of p21^ras,Wee1 and NBS1 can effectively improve the radiosensitivity of osteosarcoma cells.Following radiation damage,p21^ras and Wee1 help activate the DNA damage checkpoint,likely by inhibiting the activity of cyclin dependent kinase 2（CDK2）.CDK2 phosphorylates NBS1 at Ser432,thereby initiating DNA repair.Radiation enhances the phosphorylation level of CDK2 on Thr160,which is required for ATP binding to CDK2 and promotes DNA repair via the phosphorylation of NBS1 at Ser432.Radiation can also inhibit the dephosphorylation of Thr14 and Tyr15 of CDK2 and lead to cell cycle arrest.We postulate that some biomarkers may mediate CDK2 phosphorylation states in DNA repair and cell cycle arrest,respectively.CR6-interacting factor-1（Crif1）,which contains a nuclear localization signal（NLS）and a mitochondrial targeting sequence（MTS）,is a negative regulator of cell growth and cell cycle progression.Our previous study found that Crif1 is involved in leukemic cell cycle G0/G1 arrest and that these effects may be mediated via the bone marrow microenvironment through an interaction with CDK2.After radiation,Crif1 shows elevated expression,implying its association with radioresistance of bone marrow mesenchymal stem cell.All of the above evidence prompted us to hypothesize that Crif1 plays a role in the radioresistance of OS.Here,we report the high level of expression of Crif1 in OS,demonstrate that Crif1promotes radioresistance by regulating the activity of CDK2 and Crif1 knockdown enhances the radiosensitivity of OS in a xenograft mouse model.Thus,we identify Crif1 as a potential biomarker of radioresistance and target for radiosensitization in osteosarcoma.Methods:1.Clinical data analysis:OS tissues and tumor-adjacent tissues used for western blotting,Real-time quantitative PCR and immunofluorescence were collected from 13 patients who underwent tumorectomy at the Department of Orthopedics,The Second Affiliated Hospital,Third Military Medical University,and the Cancer Centre,Daping Hospital and Research Institute of Surgery,Third Military Medical University,from 2013-2015.Paraffin embedded archived OS tissues used for H&E staining,immunohistochemistry（IHC）and survival rate analysis were collected from 30 patients at the Department of Pathology,The Second Affiliated Hospital,Third Military Medical University,and the Department of Pathology&Clinical Biobank,Daping Hospital and Research Institute of Surgery,Third Military Medical University,from 2013-2014.At the same time,age,gender,tumor location,the main treatment program,whether the recurrence of metastasis and survival period were collected.2.Molecular mechanism research:（1）.Real-time quantitative PCR and Western blotting were used to detect mRNA and protein expression of Crif1 in U2OS,HOS,Saos-2 and K7M2 cells.（2）.The U2OS cells were transfected with Crif1 sh RNA,U2OS cells were transfected with NT shRNA（Non-targeting shRNA）as the control group.（1）Inhibition efficiency of Crif1 was confirmed by real-time quantitative PCR and Western blotting.（2）Cell proliferation and cell cycle were detected by CCK-8 assay and cell cycle analysis.（3）The apoptotic rate was detected by Annexin V-APC Apoptosis Analysis Kit and TUNEL assay after cells exposed to radiation.（4）After radiation,cell cycle,cell proliferation,DNA replication,DNA repair,mitochondrial structure and mitochondrial membrane potential were investigated.（3）.In the U2OS cells transfected with Crif1 shRNA or NT sh RNA,the effect of inhibition of Crif1 expression on CDK2 subcellular localization and kinase activity after irradiation was detected.（1）The subcellular localization of Crif1 and CDK2 was investigated by immunofluorescence before and after radiation.（2）The interaction between Crif1 and CDK2 was detected by immunoprecipitation and GST-pulldown.（3）The protein expression of CDK2、Cyclin E、CDK2-p T160、CDK2-pT14、CDK2-pY15、NBS1、NBS1-pS432、Cdc25A was detected by Western blot in 0,1,2,4,6,8,12 and 24 hr after cells exposed to radiation.（4）The distribution of Crif1,CDK2,CDK2-p T160 and CDK2-pT14 in the nucleus and cytoplasm was detected by Western blot and immunofluorescence after radiation.（5）EdU andγH2AX positive cells were detected by Click-i T?Ed U Alexa Fluor?555 Imaging Kit and immunofluorescence after radiation.3.In vivo animal experiments:U2OS-NT sh RNA or U2OS-sh Crif1 cells（5×10⁶）were subcutaneously injected into the dorsal lateral flank of the Balb/c nu/nu mice for Xenografts.When the tumor volume reached 200 mm3,the tumor-bearing mice were given a single 5 Gy dose of radiation to the tumor area.Twelve hours after exposure to the radiation,the tumors were collected from 3mice each in the shCrif1 and NT shRNA groups.The samples were paraffin-embedded,sectioned,and analyzed by H&E staining,IHC and TUNEL assay.The remaining mice were weighed every other day and the tumor volumes were measured using a Vernier caliper for 42 days after the radiation.Results:1.The clinical study found that the Crif1 expression increased in osteosarcoma tumor tissue,and the expression was a negative correlation with osteosarcoma patient overall survival.The results of IHC showed that Crif1 protein levels were significantly higher in tumor tissues than those in corresponding adjacent non-tumor tissues.Immunofluorescence was performed to further confirm that Crif1 was overexpressed in OS tissues,and the protein was primarily located in the cytoplasm.In 13 human OS samples,the Crif1 protein was highly expressed in all OS samples by western blot analysis,and Crif1 mRNA was highly expressed in 10 cases in these tumor tissues（76.9%）compared to those in normal tissue.Furthermore,strong staining of Crif1 was present in 60%of paraffin embedded archived OS tissues,and patients with high Crif1 expression had a poorer overall survival（P=0.030）.2.CDK2 activity regulated by Crif1 promoted radiation resistance of osteosarcoma Cells.（1）.Crif1 m RNA and protein expression were higher in the 4 tested OS cell lines（Human OS U2OS,HOS,and Saos-2 cells;murine OS K7M2）than in non-OS control cancer cells（HuH-7,A549,Jurkat,He La）.The expression pattern of Crif1 in OS cell lines was like which in BMSCs,a Crif1 overexpression cell lines reported by ourselves.（2）.High expression of Crif1 promoted radiation resistance of osteosarcoma cells.Crif1 knockdown cells exhibited significantly more apoptosis than the control group,as shown by FACS analysis and TUNEL assay.In U2OS-sh Crif1 cells,a sharp increase was observed in the proportion of the cells in S phase and in the proportion of EdU-positive cells.Crif1 knockdown resulted in an increase in the number ofγH2AX foci at 6 h post-ionizing radiation（IR）compared with U2OS-NT sh RNA cells.We found that downregulation of Crif1 led to fragmentation of mitochondrial and reduction in mitochondrial membrane potential after IR.（3）.Crif1 participated in the spatial regulation of CDK2 after radiation.We explored localization of Crif1 and CDK2 in OS cell lines and found some degree of co-localization of Crif1 and CDK2.The co-localization of aggregated Crif1 and CDK2 was found in the nuclei post-IR.Decreased expression of Crif1 led to significantly reduced nuclear translocation of CDK2 after IR.Co-immunoprecipitation and GST-pulldown showed that Crif1 was associated with CDK2 in OS cells and Crif1 could directly bind CDK2.（4）.The interaction between Crif1 and CDK2 promoted the DNA damage repair after radiation.After radiation,CDK2-pT160 was significantly increased and aggregated in nucleuses,a rapid enhancement of the Nbs1-pS432 and a decrease in the number ofγH2AX foci at 6 h post-IR were observed in U2OS cells.In contrast,in the Crif1 knockdown cells,CDK2-pT160 was primarily distributed in the cytoplasm,the enhancement of the CDK2Thr160 and Nbs1-pS432 was delayed,and an increase in the number ofγH2AX foci at 6 h post-IR was observed.（5）.The interaction between Crif1 and CDK2 provided time for DNA repair by activation G1 checkpoint.In NT shRNA cells,the G1 checkpoint was activated by phosphorylation of CDK2 on Thr14 and CDK2-pT14 aggregation in nucleuses.In contrast,in the Crif1 knockdown cells,CDK2 Thr14 phosphorylation was observed to be decreased and mainly located in the cytoplasm,the proportion of EdU-andγH2AX double-positive cells was much higher than those in the NT shRNA group.3.Crif1 promotes the radioresistance of OS in vivo.The nude mouse model of OS was established by subcutaneous injection of U2OS-NT sh RNA and U2OS-sh Crif1 cell lines.The obtained tumor mass was confirmed as OS by H&E staining,and the expression of Crif1 was confirmed by IHC to be down-regulated in the tumor formed by U2OS-sh Crif1 cells.After the animals received radiotherapy,frozen sectioning of the tumor tissue was performed,followed by TUNEL staining.The results showed that,compared with the NT shRNA control group,the number of TUNEL-positive nuclei in the U2OS-shCrif1 group was substantially increased.The combination treatment of radiation and Crif1 knockdown led to significant tumor regression,whereas the inhibition of tumor growth by IR alone was limited.Six weeks after radiotherapy,the tumors were resected,the sizes of tumors were far smaller in the U2OS-shCrif1 group than in the NT sh RNA control.Conclusion:In the present study,our data showed that the high expression of Crif1 in OS promote the radioresistance of osteosarcoma.Crif1 was essential for post-IR CDK2 nuclear translocation and responsible for Thr14/Thr160 phosphorylation.The phosphorylation of CDK2 Thr160 facilitated DNA damage repair by promoting the formation of MRN complex.The inhibition of dephosphorylation CDK2 Thr14 activated the G1 checkpoint and provided time for DNA damage repair.Crif1 knockdown enhanced the radiosensitivity of osteosarcoma in vitro and in vivo.Therefore,Crif1 is expected to become a new target in the development of radiosensitizers for osteosarcoma.