Functional And Mechanistic Studies Of WAVE1Regulating The Malignant Behavior Of Epithelial Ovarian Cancer

The ovarian malignant cancer, cervical cancer and endometrialcarcinoma are the most commongynecological malignancies.85-90%ofovarian malignant cancer is epithelial ovarian cancer (EOC). EOC is acommongynecological malignancy that is associated with an unfavorableoutcome, and it is the fifth-leading cause of death in females. Worldwide,nearly204,000women are diagnosed, and125,000die due to ovariancancer each year. Seventy-five percent of EOC patients are diagnosed at anadvanced and metastatic stage because of the lack of sensitive screeningtests or early symptoms. To date, the cellular/molecular mechanisms ofEOC metastasis remain poorly understood, even though extensive clinicaland basic research effortshave been undertaken. Identification of novelmolecular markers and the cellular/molecular mechanisms of EOC couldimprove the prediction of metastasis, novel therapeutic methods and furtherunderstanding of EOC progression.The initial and essential step of cancer cell metastasis is cell migration.Invasive cancer cells must use their remodeled actin cytoskeleton tomigrate through the extracellular matrix (ECM) and enter the blood or lymphatic system. Reorganization of actin filaments is tightly regulated bya variety of proteins which are essential for cell movement. Our previouslystudy using the innovative reverse capture antibody microarray to identifyautoantibody biomarkers in the plasma samples of EOC patients suggesteda significantly elevated expression of Wiskott–Aldrich syndrome proteinfamily verprolin-homologous protein1(WAVE1) in EOC plasma comparedwithhealthy controls. WAVE1, which belongs to Wiskott–Aldrichsyndrome protein (WASP) family protein,has been proposed as acting as anenhancer. Various researchhas found that WAVE1is critical for cancer cellmigration, invasion and metastasis. Down-regμlation of WAVE1has beenfound to be related to decreased invasion of prostate cancer cells, andresμlts sμggest that it might be used as a molecμlar target for preventingcell metastasis. WAVE1protein was overexpressed in malignant melanomacells, which showedhigher Rac activity than non-invasive andnon-metastatic cells. A recent studyhas shown that WAVE1is a novelregulator of apoptosis that is involved in the multidrug resistance ofleukemia cells.however, no studyhas assessed the expression of WAVE1inpatients with EOC or investigated the role of WAVE1in ovarian cancer cellinvasion and metastasis. In the present study, we aimed to examine thefunction and mechanism of WAVE1in malignant behaviors in EOC by thefollowing four parts. PART ONETHE EXPRESSION AND CLINICAL SIGNIFICATION OFWAVE1IN EPITHELIAL OVARIAN CANCERObjectives: This study aims to determine the effect of WAVE1expression and investigate a possible relationship between WAVE1andprognosis in EOC.Methods:1. WAVE1protein level was measured in223EOC specimens byimmunohistochemical staining, and the association of WAVE1proteinexpression with clinicopathological characteristics in223cases of invasiveovarian carcinomas was investigated.2. WAVE1protein level was measured in46EOC specimens byWestern blot, and the association of WAVE1protein expression withclinicopathological characteristics in46cases of invasive ovariancarcinomas was investigated.3. Survival analysis was performed to assess the correlation betweenWAVE1expression and survival.4. Expression of WAVE1in ovarian cancer cell lines was evaluated byWestern blot analysis and immunofluorescence.Results:1.Immunohistochemical staining showed that WAVE1was over- expressed in EOC compared with samples from a non-invasive ovariantumor and normal ovaries (P<0.05). The expression of WAVE1wassignificantly associated with advanced FIGO stage, poorgrade, serumCa-125and residual tumor size (P<0.05).2.Western blot analysis showed that WAVE1was overexpressed inEOC compared with samples from a non-invasive ovarian tumor andnormal ovaries (0.930±0.188,0.586±0.098,0.542±0.103, and0.441±0.097,respectively)(P<0.05). The expression of WAVE1was significantlyassociated with advanced FIGO stage, poorgrade, serum Ca-125andresidual tumor size (P<0.05).3.Survival analysis showed that patients with low WAVE1staininghada significantly better survival compared to patients withhigh WAVE1staining (P<0.05). In multivariate analysis, WAVE1overexpression,advanced stage and suboptimal surgical debulking were independentprognostic factors of poor survival.4.By Western blot analysis, WAVE1expression was detected in fourovarian cancer cell lines (1.218±0.112,1.111±0.084,1.057±0.148, and0.968±0.055, respectively). Immunofluorescence was performed todemonstrate WAVE1expression in SKOV3and3AO cell lines.Conclusions: Our present study finds that WAVE1overexpression isassociated with an unfavorable prognosis. WAVE1is an independentprognostic factor for EOC, which suggests that it is a novel and crucial predictor for EOC metastasis. PART TWOCONSTRUCTION OF SHRNA LENTIVIRAL VECTORTARGETING WAVE1GENE AND SELECTION A STABLYTRANSFECTED EPITHELIAL OVARIAN CANCER CELLLINEObjectives: This study aims to construct short hairpin RNA (shRNA)lentiviral vector targeting WAVE1gene and select a stably transfectedepithelial ovarian cancer cell line for the following studies.Methods:1.Designed and synthesized two WAVE1specific shRNA sequencesand one negative control sequence. Double restriction digestion and DNAsequencing were used to identified the recombinant plasmid.2.The correct recombinant plasmid and lentiviral vector wereco-transfected into293T cells by Lipofectamine TM2000to collect andpurify lentivirus virus particles. Establishing stably transfected epithelialovarian cancer cell lines by the manufacturer’s protocol.3.Identified the best inhibited effect of WAVE1in the stablytransfected epithelial ovarian cancer cell lines by western blot and real time PCRResults:1.The double restriction digestion and DNA sequencing showed wesuccessfully designed and synthesized two WAVE1specific shRNAsequences, with a titer of2.2×108U/ml.2.According to the manufacturer’s protocol, we established stablytransfected ovarian cancer cell lines by puromycin.3.The western blot and Real time PCR showed the protein and mRNAlevels of WAVE1were down-regulated in stably transfected ovarian cancercell lines. The SKOV3-Ri1cell line was with the more inhibited effect ofWAVE1.Conclusions: Our present study successfully designed andsynthesized two WAVE1specific shRNA sequences. By lentivirus vector,weharvested the stably transfected epithelial ovarian cancer cell lineSKOV3-Ri1with the more inhibited effect of WAVE1. PART THREEWAVE1GENE SILENCING VIA RNA INTERFERENCEIMPACT ON THE MALIGNANT BEHAVIOR OFEPITHELIAL OVARIAN CANCER CELLSObjectives: This study aims to investigate the effect of WAVE1genesilencing via RNA interference impact on the proliferation, migration,invasion, and adhesion in SKOV3cells.Methods:1.The morphplogic changes of SKOV3cells before and afterinhibition of WAVE1were detected by confocal immunofluorescencemicroscopy.2.The cell migration and invasion of SKOV3cells before and afterinhibition of WAVE1were analyzed by transwell assay.3.The cell adhesion of SKOV3cells before and after inhibition ofWAVE1was tested by cell adhesion assay.4.The cell proliferation of SKOV3cells before and after inhibition ofWAVE1was investigated by MTT assay.5.The cell anchorage-independentgrowth of SKOV3cells before andafter inhibition of WAVE1was examined by soft agar assay.6.The tumor formation of SKOV3cells before and after inhibition ofWAVE1was measured in nude mice.Results:1.WAVE1silencing induces morphologic changes:The morphology of the SKOV3-Ri1, SKOV3-NC and SKOV3were examined using actin staining. The SKOV3-Ri1was unpolarized, missingtheir pseudopodia, presented reduced cellular protrusions, and wasreshaped compared with control cells.2.WAVE1silencing decreases cell migration and invasion:Transwell assays were carried out to determine the effects of WAVE1silencing on ovarian cancer cell migration and invasion. In SKOV3-Ri1, asignificant reduction in the number of migratory cells was observedcompared with the control cells (P<0.05), and there was also a significantdecrease in the number of invading cells compared with the control cells.The SKOV3-Ri1presented a significant decrease in cell adhesioncompared with the control cells (P<0.05).3.WAVE1silencing inhibits colony formation and cell proliferation:Using the MTT assay, we found that SKOV3-Ri1presented asignificantgrowth-inhibiting effect on day2(P<0.05).Using a soft agar assay, we found that the average number of coloniesformed by SKOV3-Ri1was significantly lower than the number formed bycontrol cells, and the size of the colonies was reduced (P<0.05).4.WAVE1silencing inhibits tumor formation in xenografts in nudemiceWe found that not only thegrowth rate but also the size of thexenografts of SKOV3-Ri1were significantly slower and smaller,respectively, compared with control cells. Conclusions: Our present study showed the down-regulation ofWAVE1had a significant effect on cell morphological changes. WAVE1silencing decreased cell migration, cell invasion, cell adhesion, colonyformation and cell proliferation in vitro. In addition, we also found thatdown-regulation of WAVE1inhibited tumor malignant behaviors in vivo. PART FOURTHE UNDERLYING MECHANISMS OF WAVE1REGULATING THE MALIGNANT BEHAVIOR OFEPITHELIAL OVARIAN CANCERObjectives: This study aims to investigate the underlying mechanismsof WAVE1regulating proliferative and invasive malignant behaviors ofepithelial ovarian cancer.Methods:1.HE staining was used to analyze the morphological changes ofxenografts which formed by SKOV3-Ri1and SKOV3-NC.2.Immunohistochemical staining and western blot analysis were usedto detect the levels of cyclin D1, VEGF, MMP-2, MMP-9, and E-cadherinin xenografts which formed by SKOV3-Ri1and SKOV3-NC.3.Western blot analysis were used to examine proliferation-related signaling pathways that regulate cancer cell tumorigenesis and metastasis.The protein level of AKT, p-AKT, p38MAPK, p-p38MAPK, ERK1/2, andp-ERK1/2were tested in SKOV3-Ri1cells and SKOV3-NC.Results:1.HE staining showed the morphology of SKOV3-Ri1changed.2.Immunohistochemical staining and western blot analysis showed thedown-regulation of WAVE1, MMP-2, MMP-9, VEGF, and cyclin D1expression, and the up-regulation of E-cadherin expression, were observedin xenografts formed by SKOV3-Ri1, compared with controls.3.Western blot analysis indicated that total AKT, phospho-AKT, andphospho-p38protein levels were down-regulated in SKOV3-Ri1. Incontrast, no WAVE1silencing-induced changes were observed in totalERK1/2, phospho-ERK1/2, and p38levels.Conclusions: Our present study showed WAVE1regulated themalignant behavior of epithelial ovarian cancer might through theactivation of the PI3K/AKT and p38MAPK signaling pathways.