Functional And Mechanistic Studies Of NEDD9in Regulating The Malignant Behavior Of Epithelial Ovarian Cancer

Ovarian cancer is one of the most common gynecological malignan-cies and is the leading cause of cancer-related deaths in women worldwide.The most common form of ovarian cancer (≥80%), thought to derive fromovarian surface epithelium, is called epithelial ovarian carcinoma (EOC).The high mortality rates related to ovarian cancer are thought to be causedby advanced disease at diagnosis (approximately70-75%), which resultsfrom the rather asymptomatic nature of early stage disease and the lack ofan adequate early detection screening method, ultimately leads towidespread intra-abdominal metastases and peritoneal implantation ordistant metastasis. Although there are initial responses to cytoreductivesurgery combined with platinum-taxane–based chemotherapy, mostpatients with advanced disease eventually relapse and develop drugresistance, and the5-year survival rate is generally less than30%. Theinvasion and metastasis of ovarian cancer and chemotherapy drugresistance is the difficulty in the clinical treatment of ovarian cancer.Therefore Novel biomarkers with high sensitivity and specificity are urgently required as effective diagnostic and therapeutic targets in EOC.Neural precursor cell–expressed, developmentally down-regulated9(NEDD9), one of Cas family proteins, is a scaffolding protein that residesat focal adhesions. It plays an important role in mediating signaltransduction, and can regulate protein complexes controlling migration andchemotaxis, apoptosis, cell cycle, and differentiation. Growing evidencehas identified NEDD9as a prometastatic gene and a marker of poorprognosis in a number of malignant tumors including melanoma, breastcancer, and head and neck squamous cell carcinoma. Whole-genomeexpression profiling has identified the NEDD9gene as one of theup-regulated genes in advanced stages of papillary serous ovarian cancer.However, NEDD9protein expression has not been detected in EOC. Thepotential role of the NEDD9protein in EOC remains unclear, despite itsknown association with other various human cancers. Therefore, In thepresent study, we aimed to examine the role and mechanism of NEDD9inmalignant behaviors in EOC by the following three parts, thereby providingnew targets for early diagnosis and gene targeting treatment of EOC. PART ONETHE EXPRESSION AND SIGNFICANCE OF NEDD9IN EPITHELIAL OVARIAN CANCERObjective: To investigate the expression of NEDD9and thecorrelation between NEDD9expression and clinicopathologic parametersand prognosis in EOC.Methods:(1) Immunohistochemical staining was performed to detect theexpression of NEDD9in129EOC,18borderline tumors,22benign tumorsand15normal ovaries paraffin tissue samples. The association of NEDD9expression with clinicopathologic parameters in EOC patients wasanalysed.(2) Western blot analysis was also conducted to detected NEDD9protein expression in freshly frozen tissues, including26EOC tissues,10borderline tumor tissues,14benign tumor tissues and10normal ovariantissues.(3) The correlation between NEDD9expression and survival wasevaluated using the Kaplan-Meier method. Multivariate analysis forsurvival was performed using the Cox hazard model(4) Western blotting was performed to examine the expression ofNEDD9in various ovarian cancer cell lines (SKOV3, OVCAR-3,3AO,A2780). And immunofluorescence was performed to examine thelocalization of NEDD9in ovarian cancer cell lines. Results:(1) Immunohistochemical staining show that all EOC specimens werepositive for NEDD9expression (high NEDD9expression:69.0%, lowNEDD9expression:31.0%); in contrast, NEDD9immunoreactivity wasnegatively or weakly observed in the normal ovaries (6.7%, P＜.001),benign tumors (13.6%, P＜0.001), and borderline tumors (16.7%, P＜0.001). NEDD9overexpression was significantly associated withadvanced-stage tumors (FIGO classes III-IV; P＜0.001), high-gradecarcinoma (grades2-3; P＜0.001), and suboptimal primary cytoreductivesurgery (residual disease＜1cm; P=0.028). Nevertheless, no significantcorrelations were found between NEDD9expression status and the age atdiagnosis, serum Ca-125levels, ascites, or histologic type (all P＞0.05).(2) Western blotting shaowed that NEDD9protein expression in EOC(1.46±0.26) was higher than that in borderline (0.68±0.12; P＜0.01) andbenign tumors (0.52±0.11; P＜0.01) and in normal ovaries (0.52±0.06; P＜0.01).(3) Kaplan-Meier analysis showed that patients with a high level ofNEDD9expression had a shorter OS and PFS compared with those with alow level of NEDD9expression(P＜0.01). Multivariate analysis indicatedthat NEDD9overexpression (P=0.033), advanced stage (P＜0.001), andhigh-grade carcinoma (P=0.01) were independent predictors of poorsurvival. (4) Western blotting showed that the expression level of NEDD9varied in ovarian cancer cell lines, and immunofluorescence showed thatNEDD9was predominantly localized in the cytoplasm and membrane.Conclusion: NEDD9is overexpressed and associated with advanced-stage tumors, high-grade carcinoma, suboptimal primary cytoreductive andunfavorable prognosis in EOC, these indicated NEDD9may play aimportant role in the development of EOC. Different levels of NEDD9expression in four cell lines was possible related to invasion and metastasisin EOC. PART TWOCONSTRUCTION OF LENTIVIVIRAL VECTORS FORKNOCKDOWN NEDD9AND GENERRATION OFSTABLE TRANSFECTED SKOV3CELL LINEObjective: To construct lentiviral vector for knockdown NEDD9gene,and to generate a stable transfected SKOV3cell line for following studies.Methods:(1) Four NEDD9specific shRNA（NEDD9shRNA-1,2,3,4） and one negative control sequence were designed and synthesized. The fidelityof the DNA sequence was confirmed by bidirectional sequencing.(2) The recombinant plasmid and Lipofectamine2000were co-transfected into293T cells, supernatants were collected, concentrated, andmeasured titer.(3) SKOV3cells were transfected with lentivirus at MOI=40,transfected cells were cultured for72h and were harvested for Real-timePCR analysis.(4) NEDD9shRNA, which has best silence effect, was transfected togenerate a stable transfected SKOV3cell line according to themanufacturer’s protocol, and the expression of NEDD9mRNA and proteinwere analyzed by Real-time PCR and Western blotting.Results:(1) Four NEDD9shRNA and negative control sequence weredesigned and synthesized successfully. DNA sequencing showed that thelentiviral vectors plasmids for knockdown NEDD9were constructedsuccessfully.(2)72h after transfection, PCR analysis showed that NEDD9shRNA-4has the best silencing effect in transfected SKOV3cell line.(3) A stable transfected SKOV3cell line was successfully generatedby puromycin, and Real-time PCR and Western blotting analysis showedthat the expression of NEDD9were significantly inhibited. Conclusion: the lentiviral vector for high knockdown NEDD9wasconstructed successfully, and a stable transfected SKOV3cell line weregenerated successfully. PART THREECONSTRUCTION OF LENTIVIVIRAL VECTORS FORKNOCKDOWN NEDD9AND SCREENING OF STABLETRANSFECTED SKOV3CELL LINEObjective: To observe the effect of NEDD9silencing on adhesion,migration, invasion and proliferation in SKOV3cells, and to explore themechanism of NEDD9in regulating the malignant behaviors of epithelialovarian cancer.Methods:(1) Adhesion assay was used to detecte the change of cell adhesion inSKOV3cells before and after silencing NEDD9.(2) Transwell assay was used to detecte the change of cell migrationand invasion in SKOV3cells before and after silencing NEDD9.(3) MTT assay and plate clone formation assay were used to detecte the change of cell proliferation in SKOV3cells before and after silencingNEDD9.(4) Western blot analysis were used to examine the expression level ofE-cadherin、MMPs and the activity of ERK1/2and p38MAPK signalingpathway in SKOV3cells before and after silencing NEDD9.Results:(1) Adhesion assay showed that NEDD9silencing in SKOV3cellsenhanced the homogeneous adhesion and reduced heterogeneous adhesion(vs SKOV3-CON and SKOV3-NC, P＜0.05).(2) Transwell assay showed that NEDD9silencing in SKOV3cellsinhibited the cell migration and invasion:Transwell migration assay showed that the number of migratory cellsin SKOV3-KD had a significant reduction compared with that inSKOV3-CON and SKOV3-NC (P＜0.05).Transwell matrigel invasion assay showed that the number of invasivecells in SKOV3-KD had a significant reduction compared with that inSKOV3-CON and SKOV3-NC (P＜0.05).(3) MTT assay and plate clone formation assay showed that NEDD9silencing in SKOV3cells inhibited cell proliferation and colony formationMTT assay showed that afer cultured0-7days, cell proliferation in SKOV3-KD was significantly inhibited compared with that inSKOV3-CON and SKOV3-NC (P＜0.05).Plate clone formation assay showed that the number of cell colonies inSKOV3-KD significantly decreased compared with that in SKOV3-CONand SKOV3-NC (P＜0.05).(4) Western blot analysis indicated that the level of E-cadherin wasup-regulated, MT1-MMP, MMP-2and phospho-ERK1/2were down-regulated with NEDD9silencing in SKOV3cells. In contrast, No changewas found in the level of MMP-9, ERK1/2, p38MAPK and phospho-p38MAPK with NEDD9silencing in SKOV3cells.Conclusion: NEDD9silencing in SKOV3cells enhanced thehomogeneous adhesion and reduced heterogeneous adhesion, inhibitedcell migration, cell invasion, cell proliferation and colony formation invitro. NEDD9may regulate the malignant behavior of epithelial ovariancancer through the activation of the ERK1/2signaling pathways.