Expression And Mechanistic Studies Of C-Abl Regulating The Malignant Behavior Of Epithelial Ovarian Cancer

The cervical carcinoma，endometrial cancer and ovarian malignantcancer are the most common gynecological malignancies, the ovarianmalignant cancer is the third most common gynecologic cancer, but themortality rate of the ovarian malignant cancer exceeds that for all othergynecologic malignancies combined, nearly85%～90%of ovarianmalignancies are classified as epithelial ovarian carcinomas. The followingare some reasons that may lead to the high mortality of ovarian cancer ingynecologic cancer:1) in the human the paired ovarian lie within in thepelvic cavity, and the pelvic examination is notoriously inefficient atdetecting presymptomatic early ovarian cancer;2) most patients with theearly of EOC experience have no or few symptoms, and more people havenot concern themselves,75%of EOC patients are diagnosed at anadvanced and metastatic stage when they appeared the first examination;3)ovarian cancer usually spreads via cellular shedding into the peritonealcavity followed by implantation on the peritoneal surface, and it is easy torecurrent after surgery;4) EOC patients with platinum or taxol-resistant. According to the reviews, EOC patients with a5-year survival rate of about40%. In the world whole, there are more than204,000new cases of ovariancancer，125,000deaths from the disease each year. Despite extensiveclinical and basic research efforts have been undertaken, thecellular/molecular mechanisms of EOC metastasis are still nascent. Thus,identification of novel molecular markers and the cellular/molecularmechanisms of EOC could be helpful for predicting metastasis andindividualized therapy.Cell migration is the first and essential step for cancer cell metastasis.The ability of invasive cancer cells to migrate through the extracellularmatrix (ECM) and enter the blood or lymphatic system depends onalterations in the actin cytoskeleton. Reorganization of actin filaments istightly regulated by a variety of proteins including the Rho family smallGTPases, cofilin/ADF, Arp2/3complex and WASp family protein. Ourprevious studies have shown that up-regulated WAVE1expression iscorrelated with an unfavorable outcome and is an independent prognosticfactor for EOC. Various research studies have demonstrated thatAbl-mediated phosphorylation of WAVE1is critical for cancer cellmigration, invasion and metastasis. However, there is little informationregarding whether c-Abl affects the development or progression of EOC.As a non-receptor tyrosine kinase, c-Abl is implicated in the regulation ofcell proliferation, actin reorganization, cell migration and apoptosis. Several groups have found that c-Abl not only plays an important role inactin reorganization and invadopdia formation, but also increases solidtumors invasiveness and is associated with a poor clinical outcome. c-Ablprotein was overexpressed and constitutively activated in highly invasivebreast cancer cells, which demonstrated that c-Abl plays a critical role inbreast cancer progression and invasion. Down-regulation of c-Abl has beenfound to be related to decreased invasion of melanoma cells, and resultssuggest that it might be used as a molecular target for preventing cellmetastasis. A recent study has shown that c-Abl is involved in the cisplatinresistance of esophageal adenocarcinoma. Imatinib, a small moleculetyrosine kinase inhibitor, has been shown to specifically inhibit thepathognomonic BCR-ABL tyrosine kinase in chronic myeloic leukemia(CML), acute lymphoblastic leukemia, and gastrointestinal stromal tumour(GIST). Furthermore, imatinib was able to inhibit tyrosine kinases likec-Abl, c-kit and PDGFR (PDGFR-α、PDGFR-β). Some clinical trials havedemonstrated that imatinib has limited activity as monotherapy in recurrentor platinum-and taxane-resistant ovarian cancer. Some studies havedemonstrated that imatinib may be useful for the treatment of ovariancarcinoma as an addition to conventional chemotherapy. Despite these andother findings, study on the role of c-Abl in epithelial ovarian cancerinvasion and metastasis remains deficiency. In the present study, we aimedto determine the expression and mechanism of c-Abl in malignant behaviors in EOC by the following four parts.PART ONETHE EXPRESSION AND CLINICAL SIGNIFICATION OF C-ABLIN EPITHELIAL OVARIAN CANCERObjectivesThe aims of this study are to determine the expression of c-Abl andinvestigate a possible relationship between c-Abl and prognosis in EOC.Methods1. c-Abl protein level was evaluated in137EOC specimens byimmunohistochemical staining, and the association of c-Abl proteinexpression with clinicopathogic features in137cases of invasive ovariancancer was investigated.2. c-Abl protein level was evaluated in32EOC specimens by Westernblot analysis, and the association of c-Abl protein expression withclinicopathogic features in32cases of invasive ovarian cancer wasinvestigated.3. Survival analysis was performed to assess the correlation betweenc-Abl expression and survival.4. Expression of c-Abl in ovarian cancer cell lines was measured byWestern blot analysis and immunofluorescence. Results1. Immunohistochemical staining revealed that c-Abl was overexpres-sed in EOC compared with samples from a non-invasive ovarian tumor andnormal ovaries (P<0.05). Furthermore, expression of c-Abl wassignificantly associated with advanced FIGO stage, poor grade, serumCa-125and residual tumor size (P<0.05).2. Western blot analysis revealed that the protein expression of c-Ablin EOC tissues was higher compared with the borderline, benign andnormal ovary tissues (1.704±0.382,0.346±0.068,0.334±0.066,0.317±0.062, respectively; all P<0.05). No statistical significance was foundamong the borderline tumors, benign tumors and normal ovaries (allP>0.05).3. Survival analysis showed that patients with high c-Abl staining hada significantly worse overall and disease-free survival compared to patientswith low c-Abl staining (P<0.05). By multivariate analysis, high c-Ablexpression, poor grade, advanced tumor stage and residual tumor sizeremained independent prognostic factors of poor overall survival.4. The protein level of c-Abl in four cell lines by Western blot analysis(1.044±0.138,0.905±0.096,0.943±0.170and0.855±0.750,respectively). Immunofluorescence was also used to detect the expressionof c-Abl in the SKOV3and3AO cell lines. ConclusionsOur present study finds that c-Abl overexpression is associated withan unfavorable outcome. c-Abl may be a crucial predictor for EOCmetastasis. PART TWOCONSTRUCTION OF SHRNA LENTIVIRAL VECTORTARGETING C-ABL GENE AND SELECTION ACONSTRUCTED-STABLY TRANSFECTED OVARIAN CANCERCELL LINEObjectivesThe aims of this study are to construct shRNA lentiviral vectortargeting c-Abl gene and select a construct-stably transfected the humanovarian carcinoma cell line for next step.Methods1. Design and synthesis of four c-Abl specific shRNA sequences and anegative control sequence. DNA sequencing and double restrictiondigestion were performed to verify the recombinant plasmid.2. The recombinant plasmid and construction of lentiviral vectors wereco-transfected into the293T cells using pMagic7.1to collect and purify. Establishing constructed-stably transfected human ovarian carcinoma celllines using the manufacturer’s protocol.3. Verified the best inhibited effect of c-Abl in the stably transfectedhuman ovarian carcinoma cell lines using western blot and real-time PCR.Results1. The DNA sequencing and double restriction digestion showed thatwe successfully designed and synthesized four c-Abl specific shRNAsequences.2. Using the manufacturer’s protocol, we have harvested stablytransfected human ovarian carcinoma cell lines by puromycin.3. The protein and mRNA levels of c-Abl were down-regulated inSKOV3by Western blot and RT-PCR. The SKOV3-Ri2was with the bestinhibitory effects of c-Abl.ConclusionsIn this study we have successfully designed and synthesized fourspecific shRNA sequences. Using lentivirus vector, we have harvestedSKOV3-Ri2with the best inhibitory effect of c-Abl. PART THREEC-ABL GENE SILENCING WITH RNA INTERFERENCE IMPACTON THE MALIGNANT BEHAVIOR OF SKOV3ObjectivesThe aims of this study are to examine the effect of c-Abl genesilencing using RNAi impact on the migration, proliferation, adhesion andinvasion of SKOV3.Methods1. Transwell asssys were carried out to analyze the cell migration andinvasion of SKOV3before and after inhibition of c-Abl.2. Cell adhesion asssy was performed to test the cell adhesion ofSKOV3before and after inhibition of c-Abl.3. MTT asssy was performed to examine the cell proliferation ofSKOV3before and after inhibition of c-Abl.4. Soft agar assay was performed to determine the cell anchorage-independent growth of SKOV3before and after inhibition of c-Abl.5. In nude mice mode we have measured the the tumor formation ofSKOV3before and after inhibition of c-Abl.Results1. c-Abl silencing reduces cell migration and invasion:Transwell asssys were carried out to analyze the effects of c-Ablsilencing on ovarian cancer cell invasion and migration. In SKOV3-Ri2, a noticeable decline in the number of migratory cells compared with thecontrol (P<0.05), and decrease in the number of invading cells comparedwith the controls.The SKOV3-Ri2showed a significant reduces in cell adhesioncompared with the controls.2. c-Abl silencing inhibits cell proliferation and colony formation:The SKOV3-Ri2presented a significant growth-inhibiting effect byMTT assay.The SKOV3-Ri2showed the number of colonies formed、the size ofthe colonies and the growth rate was significantly lower than the control bysoft agar assay.3. c-Abl silencing inhibits tumor formation in nude mice.The size of the xenografts and the growth rate of SKOV3-Ri2weresignificantly smaller and slower than the control.ConclusionsOur present study demonstrated the down-regulation of c-Abl reducedcell invasion, cell migration, cell adhesion, colony formation and cellproliferation in vitro. PART FOURTHE MOLECULAR MECHANISM OF C-ABL REGULATING THEMALIGNANT BEHAVIOR OF HUMAN EPITHELIAL OVARIANCANCERObjectivesThe aims of this study are to determine the molecular mechanism ofc-Abl how regulating invasive、proliferative malignant behaviors ofovarian cancer.Methods1. Using the HE staining to demonstrate the morphological changes ofxenografts.2. Using immunohistochemical staining and Western blot analysis toexam the levels of MMP-2、MMP-9、VEGF、cyclin D1、E-cadherin inxenografts.3. Using Western blot analysis to detect proliferation-related signalingpathways. The protein level of ERK1/2, p-ERK1/2, AKT, P38MAPK, p-AKT and p-P38MAPK were tested in SKOV3-Ri2and SKOV3-NC.Results1. HE staining demonstrated the morphology of SKOV3-Ri2changed.2. In SKOV3-Ri2, the expression of c-Abl、MMP-9、MMP-2、cyclinD1、 VEGF were significantly reduced, while the protein level ofE-cadherin was increased by immunohistochemical staining and Western blot analysis, compared with controls.3. In SKOV3-Ri2，the total protein levels of AKT, p-AKT, andp-P38MAPK were down-regulated by Western blot. In contrast, there areno changes have observed in total ERK1/2, p-ERK1/2and P38MAPKlevels.ConclusionsOur study demonstrated the effect of c-Abl in regulating the malignantbehavior of EOC might via PI3K/AKT and P38MAPK pathway.