RAD51 Sustains Mitochondrial And Redox Homeostasis In Human Ovarian Cancer Cells

Ovarian cancer is the most lethal gynecological malignancy.Because of lack of biomarkers and symptoms at early stage,most patients are diagnosed at advanced stage.Patients usually have poor prognosis because of late diagnosis,relapse and chemoresistance.The overall 5-year survival is only 30%.This severly threaten the health of women.Abnormal homologous recombination repair(HRR),high level of reactive oxygen species(ROS)and upregulation of antioxidant genes are characteristic features of ovarian cancer.However,the molecular mechanisms governing the redox homeostasis in ovarian cancer cells remain to be fully elucidated.Therefore,it is pressing to explore the molecular mechanisms governing redox homeostasis and seek effective therapeutic targets for the treatment of ovarian cancer.The high response rate of ovarian cancer to platinum analogues is believed to be due to a high prevalence of defective HRR.HRR plays a critical role for error-free repair of DNA double-strand breaks.The recombinase activity of the RAD51 protein forms the catalytic core of the HRR pathway for the repair of DSBs.In addition,RAD51-mediated HR plays a critical role in facilitating efficient DNA replication in mammalian cells.Many studies have shown that RAD51 is overexpressed in a variety of tumors and the high-expression of RAD51 is associated with poor prognosis.RAD51 is also associated with drug resistance in tumors,and down-regulation of RAD51 can promote sensitivity of tumors to radiotherapy and chemotherapy.Because RAD51 is the key catalytic protein in HRR,it is reasonable to assume that the chemo-or radioresistance of cancer cells conferred by RAD51 overexpression may be related to an increased DNA repair capacity.However,BRCA1,another key player in HRR,was recently shown to promote antioxidant defense by upregulating Nrf2.This suggests that DNA repair proteins may also possess antioxidant effect.Recent studies indicate that nuclear DNA damage can trigger mitochondrial response,and increased production of mitochondrial reactive oxygen species may exacerbate nuclear DNA damage.ROS are essential for many cellular processes.While it is easy to understand that an increased antioxidant capacity may be necessary for the ovarian cancer cells to cope with the increased oxidative stress,whether the HRR proteins may indirectly contribute to the maintenance of redox homeostasis remains to be determined.Would defective HRR lead to mitochondrial response?In the present study,we investigated the role of RAD51 in the mitochondrial and redox homeostasis in human ovarian cancer cells.To investigate the expression of RAD51 in ovarian cancer,we firstly analyzed RAD51 expression in ovarian cancers using data from OncomineTM databank.mRNA level of RAD51 was detected in normal ovary,peritoneal tissues and ovarian cancer.We found that RAD51 was overexpressed in ovarian cancer.Next we measured the mRNA levels of RAD51 in high grade serous ovarian cancer(HGSOC,n=46)and Fallopian tube epithelia(FT,n=20)by qPCR.RAD51 mRNA levels are generally higher in HGSOC than in FT.We then examined RAD51 expression in HGSOC(n=228)and FT(n=41)by immunohistochemistry.The immunostaining intensity of RAD51 was significantly higher in HGSOC than in FT.Furthermore,Kaplan-Meier plotter analysis showed that high RAD51 expression is associated with poor prognosis in ovarian cancer patients.These results indicate that RAD51 is generally overexpressed in ovarian cancer and is associated with poor prognosis.We then examined the biological significance of high expression of RAD51 in ovarian cancer.We knocked down RAD51 by transfecting ovarian cancer cells with siRNA or shRNA.We examined the proliferation-inhibitory effect of RAD51 depletion by colony formation assay and EdU incorporation and evaluated cell cycle distribution and apoptosis by flow cytometry.We found that RAD51 knockdown reduced proliferation of ovarian cancer cells and caused G2/M arrest in vitro.However,no increase in apoptosis was detected after RAD51 knockdown.Next,we investigated the role of RAD51 in tumor growth in vivo.The average tumor size in RAD51 knockdown group was much smaller than in the control groups,and there was also a significant decrease in the abundance of Ki-67 positive cells in the tumor xenografts,suggesting that RAD51 knockdown impedes tumor growth in vivo.Our previous results suggest that RAD51 overexpression can promote the proliferation ability of ovarian cancer cells under oxidative stress,and overexpression of RAD51 can reduce ROS levels induced by hydrogen peroxide.This suggest that overexpression of RAD51 in ovarian cancer cells may possess antioxidant effect Therefore,we measured the ROS levels in ovarian cancer cells in which RAD51 was depleted by siRNA or inhibited by B02.We observed that either RAD51 knockdown or RAD51 inhibitor treatment caused a significant increase in ROS in the ovarian cancer cell lines tested.There was also a significant increase in the level of 8-OHdG in RAD51 knockdown cells.Moreover,there was a significant increase in 8-OHdG staining intensity in the tumor xenografts formed by RAD51 knockdown ovarian cancer cells.To test whether the increased oxidative stress as a consequence of RAD51 depletion may also mediate the anti-proliferative effect,we examined whether the anti-proliferative effect of RAD51 knockdown could be rescued by antioxidant N-acetylcysteine(NAC).The proliferation abilities of RAD51-depleted ovarian cancer cells,as measured by colony formation,cell viability and EdU incorporation,were significantly rescued by NAC.Importantly,the levels of γ-H2AX caused by RAD51 knockdown were significantly reduced by NACMitochondria are one of the major sources of intracellular ROS.We next examined whether mitochondria were the source of ROS in RAD51 knockdown cells.The level of mitochondrial superoxides,as measured by MitoSOX,was significantly increased in RAD51 inhibited ovarian cancer cells.Of note,MitoQ(a mitochondria-targeted antioxidant)treatment significantly attenuated the elevation of ROS in RAD51 knockdown cells.Furthermore,the mitochondrial membrane potential(MMP)was decreased in RAD51 knockdown cells.The maximal oxygen consumption rate(OCR)was significantly increased in RAD51-depleted ovarian cancer cells.RAD51 knockdown also increased the glycolytic capacity in A2780 and HEY cells.These results suggested that RAD51 depletion results in mitochondria dysfunction.We next determined whether the increased OCR in RAD51-depleted cancer cells was associated with increased accumulation of mitochondria.Depletion of RAD51 in A2780 cells displayed a significant increase in mitochondrial content,as quantified by flow cytometry of cells stained with Mitotracker.RAD51 inhibition similarly induced an increased buildup of mitochondria.We next investigate the mechanism by which RAD51 inhibition leads to an increase of mitochondria contents.It has been reported that cells arrested at G2/M phase usually harbor more mitochondria and exhibit a higher level of ROS than at other phases of the cell cycle Because CHK1 is known to mediate G2/M arrest and the ovarian cancer cells are arrested at G2/M when RAD51 are depleted,we speculated that CHK1 may mediate the G2/M arrest and the generation of mitochondrial oxidative stress in response to RAD51 depletion.Indeed,the protein level of phosphorylated CHK1,which marks activated CHK1,was greatly increased when RAD51 was depleted.Importantly,inhibition of CHK1 abolished G2/M arrest caused by RAD51 depletion.Moreover,the mitochondrial accumulation caused by RAD51 depletion was abrogated by the CHK1 inhibitor and siRNA.Consistently,increase in the level of MitoSOX and ROS were attenuated by the inhibition of CHK1.We next determined the proliferation of A2780 cells in response to RAD51 depletion,CHK1 inhibition or the combined treatment of the two.While RAD51 depletion or CHK1 inhibition alone each resulted in a reduction in the EdU-positive cells,the two in combination led to no synergistic effect in the proliferation of ovarian cancer cells,suggesting that the alleviation of mitochondrial oxidative stress by CHK1 inhibition may have prevented an additive inhibitory effect on proliferation.Recent studies showed that RAD51 can localize in human mitochondria and play an important role in the maintenance of the mitochondrial genome integrity.It has been reported that depletion of RAD51 results in a significant decrease in mtDNA copy number following oxidative stress.We therefore examined whether mitochondrial oxidative stress induced by RAD51 knockdown was related to the maintenance of mtDNA genome integrity.We examined the mtDNA copy number of RAD51 knockdown cells and found mtDNA copy number decreased significantly in RAD51 knockdown cells.Surprisingly,the levels of ROS,MitoSOX and Mitotracker were all increased in rhoO cells by RAD51 depletion.These results revealed that mitochondrial oxidative stress caused by RAD51 knockdown can occur independent of mtDNA.Our results revealed that RAD51 is upregulated in ovarian cancer.High RAD51 level reduces the level of DNA damage and the associated CHK1 activation,ultimately leading to cancer cell survival and malignancy.Low RAD51 level compromises HRR,causing more DNA damage,activating CHK1 and arresting cells at G2/M,and consequently leading to mitochondrial oxidative stress,which may aggravate nuclear DNA damage and reinforce a vicious feedforward loop.The increased oxidative stress inhibits cancer cell survival and malignancy.Our findings indicate that some DNA repair genes can indirectly promote cancer progression by sustaining redox homeostasis in cancer cells.Highlights of this study:1.RAD51 is overexpressed in ovarian cancer and is associated with poor prognosis.2.Depletion of RAD51 leads to increased mitochondrial superoxide production and oxidative DNA damage.3.Increased production of mtROS requires CHK1-mediated G2/M arrest.4.mtROS increase caused by RAD51 depletion is indqjendent of mtDNA integrity.