The Underlying Effects And Mechanism Of Rad50 And CD47 In Ovarian Cancer

Ovarian carcinoma is one of the most lethal gynecologic malignancies,of which the incidence rate is increasing in the recent years.It's hard to be detected for ovarian cancer patients at its onset,for the patients often have no obvious symptoms.As a result,70%patients could only be treated in the late stage,and 70%can not be cured.The mortality rate of ovarian cancer patients is very high and five-year survival rate has been hovering around 25%-30%.It's known that serous cystadenocarcinoma accounts for 40%of ovarian malignancies,which owns the characteritics of easily peritoneal metastasis and poor prognosis.The patients usually couldn't be treated effectively owing to its malignant behaviors.Still,the underlying molecular mechanism responsible for those remains unclear.Consequently,effective candidates for early screening and treatment are in great need.We believe the research on molecular level is still the key point and hot spot to improve the prognosis of ovarian cancer patients.In this research,specimens of high grade serous ovarian carcinoma(HGSOC)and fimbriae tube(FT)were investigated by western blot(WB)and immunohistochemistry(IHC)to elucidate the expression of Rad50 and CD47 protein during serous ovarian carcinogenesis and to investigate the association between Rad50 or CD47 expression and clinical pathological parameters.Using high-throughput proteomics and tissue microarray(TMA)technology,we found the expression of Rad50 and CD47 in HGSOC tissues are both higher than that in FT tissues.Rad50 is one of the components of the multi-protein complex Mrell-Rad50-NBS1(MRN)which is the major sensor of DNA double-strand break(DSB).It has been implicated in both major mechanisms of DNA DSB repair(non-homologous end joining,NHEJ and homologous recombination,HR),DNA fork restart,telomere maintenance,meiosis and signaling to the cell cycle checkpoints.In normal cells,the integrity of the genome is ensured by the efficient DNA damage repair network.At present,it's known that some famous DNA damage repair protein,such as p53 and BRAC1,are tumor suppressors,which play an important role in the progression of HGSOC.However,increasing studies reported DNA damage repair proteins are highly overexpressed in some tumors and they were found they could predict the prognosis of patients,such as patients of melanoma.Based on its oncogene status,some DNA repair proteins were verified to promote tumor metastasis.Consequently,I guess Rad50 may be associated with the proliferation and metastasis of serous ovarian cancer and be related to the prognosis of patients.In this study,we discuss the role of Rad50 in the development and metastasis of HGSOC and explore the potential molecular mechanism.The discovery of the role of Rad50 in this field would definitely help us understand better the molecular mechanism of ovarian cancer.CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction.In addition to serving as a potent signal to aid escape from immune surveillance,CD47 also plays a role in cellular proliferation,apoptosis,adhesion and migration.The main contents of the research are as the following:Part ?:The underlying effects and mechanism of DNA damage repair protein Rad50 in ovarian cancer cells growth and proliferationPart ?:The underlying effects and mechanism of Rad50 in ovarian cancer cells metastasisPart ?:The underlying effects and mechanism of CD47 in ovarian cancerPart ?:The underlying effects and mechanism of DNA damage repair protein Rad50 in ovarian cancer cells growth and proliferationBackground and Object:Ovarian cancer is the most lethal cancer of the gynecologic malignancies and threatens the female health all around the world.High-grade serous ovarian carcinoma(HGSOC)is featured with poor biological behaviors.The underlying molecular mechanisms responsible for its onset and malignant behaviors are always the hotspots to study.As a consequence,effective candidates for early screening and molecular targeted treatment are in great need.We believe finding them is the key point to improve the therapeutic effect and prognosis of ovarian cancer patients.Ovarian cancer is increasingly considered as a genetical disease,and researchers have found many molecules that play important roles in their development.Rad50,the molecule we studied,is a DNA damage repair protein that was testified to be closely related to chemical resistance.However,the function of Rad50 to the progression of HGSOC remains unknown.The aim of this study was to investigate the relationship between the expression of Rad50 in HGSOC and the prognosis of HGSOC patients by analyzing the clinical specimens with clinical information.To verify its oncogene status,we carried out molecular and cellular biology experiments to explore whether the proliferation ability could change when Rad50 was altered in ovarian cancer cells.Finally,we investigated the potential mechanism.Material and Methods:The HGSOC specimens were obtained from primary ovarian cancer patients receiving no surgery or chemotherapy previously and the FT tissues were used as normal controls.Western blot(WB)was used to detect the differet expression of Rad50 between the two groups.The appropriate ovarian cancer cell lines were selected and the RAD50 gene was overexpressed or knocked out using the lentivirus packaging system to construct the stable cell lines.In order to study the role of Rad50 in vitro,we performed plate colony formation,soft agar colony formation and MTT assays to investigate clonogenic ability,anchorage-independent growth and proliferation ability.The cell cycle and apoptosis situations were analyzed by flow cytometry.We also used WB to detect the changes of some cell-cycle related proteins(P21Cipl,P27Kipl,Cyclin D1,etc.)according to their influence on the cellular biological function.In the end,we used the stable Rad50-overexpressing and Rad50-knockdown ovarian cancer cells to do xenograft experiments.We recorded and compared the proliferation rate of transplanted tumor and the final volume,which made a further confirm that Rad50 is a proto-oncogene.Results:The expression of Rad50 in HGSOC samples was higher than that in normal control group and the IHC of TMAs revealed that the high Rad50 expression was significantly correlated with poor overall survival.Furthermore,Rad50 expression was related to lymph nodes metastasis and the greater omentum metastasis.Rad50 ectopic expression significantly changed the proliferation ability of ovarian cancer cells.By MTT,colony formation and soft agar colony formation assays,we testified that when Rad50 was significantly increased,the abilities of cell proliferation and colony formation were enhanced.Moreover,Rad50 depletion induced G0/G1 phase arrest in SKOV3 and G0/G1,S phase arrest in A2780 cells which could explain the drastic reduction in the proliferation rates of these EOC cells observed earlier.Additionally,we verified Rad50 suppression significantly increased the expression of p21Cipl and p27Kipl in the cell lines using WB.However,no changes of apoptosis situations were observed.Finally,in vivo experiments,we also demonstrated that Rad50-knockown cell lines had decreased tumorigenesis.Growth curves for tumor volumes in xenografts were measured and showed the growth rate of tumors derived from RAD50-OVE-A2780 cells was faster than those in control group and the shRAD50/NC-A2780 group obtained the opposite result.The final weights of the tumors exerted the same tendency with the growth curve.Conclusion:Rad50,the DNA damage repair protein,can promote the growth and proliferation of ovarian cancer;the high expression of Rad50 predicts poor prognosis of HGSOC patients.Part ?:The underlying effects and mechanism of Rad50 in ovarian cancer cells metastasis.Background and Object:An increasing trendency in mortality was observed for ovarian cancer patients all around the world.HGSOC patients have very poor prognosis,not only due to the chemical resistance and the late stage at diagnosis,but also owing to the severe malignant behaviors,such as invasive ability,which makes it easier to spread over the pelvic and abdominal captivities,then it's hard to be cured.Epithelial-mesenchymal transition(EMT),a critical process to facilitate cancer cells to form tumor metastases,plays a pivotal role in tumor progression in many malignances including ovarian cancer.The process of EMT was regulated by multiple pathways,of which NF-?B pathway is a very important path.It was found that the DNA repair protein Rad50 could activate NF-?B pathway by binding to CARD9.The purpose of this study is to verify the relationship between Rad50 and ovarian cancer invasion and migration and explore the underlying mechanisms.The link between DNA repair protein and the metastasis of ovarian cancer is the focus of our work.Many DNA repair proteins showed oncogenic potential and can promote tumor metastasis in some preious reports,such as in melanoma.We investigate the role of Rad50 in the metastasis of ovarian cancer.And we believe Rad50 can be a potential candidate for tumor screening,as well as the molecular target in the treatment of ovarian cancer.Materials and Methods:Stable cell lines established in the first part were continuely used in the migration and invasion assay by counting the number of adherent cells passing through the transwell chamber.Western blot was used to detect the changes of EMT-related proteins.WB and immunofluorescence were performed to detect NF-?B pathway,and WB analysis of nuclear and cytosolic extracts was used to further verify the NF-?B component distribution in the cells when Rad50 altered.We used Co-IP to testify whether Rad50 and CARD9 interact with each other in EOC cells and used siRNAs to downregulate CARD9 and then overexpress Rad50 to observe the changes of cancer cells.Finally,in vivo nude mice metastasis assay,ovarian cells were intraperitoneally injected or injected into the lateral tail vein.All mice were euthanized at the end of week 5 and were dissected for a further laparotomy.Results:Rad50 overexpression induced cell morphological changes and promoted migration and invasion activities of ovarian cancer cells.Rad50-knockdown cells exhibited epithelial morphology compared to their control cells.On the other side,Rad50-overexpressing cells exhibited relatively fibroblastic morphology.Rad50 overexpression could promote migration and invasion abilities of EOC cells and those were caused by EMT facilitated by Rad50,while once Rad50 suppressed,MET occurred.Furthermore,we demonstrated that the expression of Rad50 affects the activity of NF-?B pathway in ovarian cancer cells and accordingly changes the expression of EMT-related proteins.A higher level of nuclear accumulation of P65 in Rad50-overexpressining cells was evident by WB analysis and opposite result was observed in Rad50-knockdown cells.Co-IP assay showed that Rad50 binded to CARD9.This binding promotes the activation of NF-?B and activates the EMT-related molecules,thus inducing the occurrence of EMT.Finally,the nude mice experiments further verified Rad50 can affect metastasis in vivo.Conclusion:The overexpression of Rad50 in ovarian cancer cells can activate the NF-?B pathway by binding CARD9,and then regulate the changes of EMT-related molecules,which lead to the change of EMT.Finally,the migration and ivasion abilities were increased.These two parts focus on the effects of Rad50 on the biological behaviors of ovarian cancer cells,including growth and metastasis,and we also explored the underlying mechanisms.Main innovative points are as follows:1.We focus on the effect of Rad50 on the malignant behaviors of EOC cells and explore the potential mechanisms,not limited to the traditional research based on the chemical resistance.2.We first advocated that Rad50 was a protein with oncogenic potential in HGSOC.It provides a new point about the research of DNA repair protein.3.We first found Rad50 overexpression could promote the metastasis of ovarian cancer,and the mechanism is Rad50 could affect the expression of EMT-related proteins.4.We first found Rad50 could bind to CARD9 to activate NF-?B pathway and then initiate EMT process.We made a supplement about the mechanism of its function to promote cancer metastasis.5.We first verified Rad50 an oncogenic protein in EOC cells using in vivo experiments.Part ?:The underlying effects and mechanism of CD47 in ovarian cancerBackground and Object:CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated phagocysis.CD47 is overexpressed in various human malignancies.However,the expression and functional signifcance of CD47 in HGSOC has not been completely understood.In this study,we reported the expression of CD47 in HGSOC and its relation with the prognosis of HGSOC patients.Functional investigations revealed that CD47 overexpression in ovarian cancer cells signifcantly promoted migration and invasion.Materials and Methods:The expression of CD47 was detected by qRT-PCR,WB and IHC using the collected HGSOC/FT specimens and ovarian cancer/normal related cell lines.We selected A2780 cells to construct CD47-overexpressing cell lines,and selected A2780 and SKOV3 cells to downregulate CD47 by transfection with siRNA.We performed colony formation assay to investigate clonogenic ability and use flow cytomety to detect the cell cycle distribution in EOC cells after changing the expression of CD47.Transwell assay was used to detect the migration ability.And then the the expression of EMT-related protein was detected by WB and immunofluorescence staining.Results:CD47 is highly expressed in epithelial hyperplasia of the FT.In the same FT specimen,CD47 was highly expressed in multiple layer area than single layer area.The mRNA and protein levels of CD47 in HGSOC were significantly higher than that in FT group.Survival curve showed that patients with higher CD47 expression had a significantly shorter overall survival time than did patients with low CD47 expression.Elavated CD47 expression was associated with high level of CA125.Transwell assay showed EOC cells with CD47 overexpression enhanced the migration and invasion potentials and CD47-knockown cells decreased the metastasis ability.WB showed CD47 overexpression increased level of N-cadherin and decreased level of E-cadherin.On the contrary,the attenuation of CD47 reduced the expression of N-cadherin and the expression of E-cadherin increased obviously.CD47 didn't affect the proliferation and cell cycle distribution of ovarian cancer cells.Conclusion:Our results showed that CD47 was commonly overexpressed in hyperplasia of the FT and HGSOC.High expression of CD47 was associated with poor prognosis and high level of CA125 in HGSOC patients.Moreover,CD47 induced EMT through modulation of E-cadherin and N-cadherin.However,we found that CD47 did not influence on proliferation and cell cycle progression.Our fndings indicate that CD47 is a strong candidate biomarker and therapeutic target in HGSOC.