IQGAP3 Promotes Cancer Proliferation And Metastasis In High Grade Serous Ovarian Cancer

Objective:To investigate the role of IQ motif containing GTPase activating protein 3(IQGAP3)in proliferation and metastatic capacities of high grade serous ovarian cancer.Background:Ovarian cancer is the deadliest gynecologic cancer,affecting women worldwide.Due to the lack of proper screening methods most of the cases are diagnosed at late stages when the survival rates are dismal.The incidence of new ovarian cancer cases has been falling on average by 2.3%each year in the past decade.Despite this,the overall survival rate has not shown any significant rise in the recent years.The current 5-year survival rate for all stages of ovarian cancer cases in the US is around 47%.However,almost 60%of the new cases are diagnosed at advanced stages and in those cases,the 5-year survival rate is only around 29%.Additionally,there exists a high rate of recurrence even after aggressive multimodality treatment,which further worsens the prognosis.Thus,it is deemed a silent killer and is the deadliest of all the malignancies in women.IQGAP3(IQ motif containing GTPase Activating Protein 3)is a member of Rho family GTPases,which has crucial role in the development and progression of several types of cancers.Three members of the IQGAP family have been described in humans.All three members are equipped with 4 IQ motifs and a Ras GAP-related domains.The GAP related domain of IQGAPs mediates its binding to the Rho family of GTPases.One of the members of the Rho family of GTPases,CDC42 has been shown to serve critical roles in cell proliferation,survival,adhesion and migration,and is correlated with a less favorable prognosis in various types of cancer.IQGAP3 is hypothesized to be involved in the proliferation of epithelial cells.IQGAP3 is a novel member of the IQGAP family,which was discovered in 2007.IQGAP3 is located on chromosome 1 at 1q21.3 loci and has been reported to act as an oncogene in several types of cancer.It is a transmembrane protein,and has been speculated to be a potential therapeutic target.IQGAP3 has not been explored in the pathogenesis and progression of ovarian cancer till date.In this research we investigate the aberrant expression of IQGAP3 in ovarian cancer along with the mechanism involved in various oncogenic processes.This research has been divided into following parts:(1)The expression profile of IQGAP3 in ovarian cancer and its correlation to the clinicopathological parameters;(2)The effect of IQGAP3 in the biological behaviors of ovarian cancer;(3)The study of the mechanism involved in the effect of IQGAP3 in ovarian cancer.Part 1 The expression profile of IQGAP3 in ovarian cancer and its correlation to the clinicopathological parametersObjective:IQGAP3 has been found to have an elevated expression in several cancers including pancreatic,gastric,renal,breast,hepatocellular carcinoma etc.Data available in public database TCGA can be analyzed to compare the expression of IQGAP3 in ovarian cancer and normal tissue,which shows significantly elevated levels in cancer specimen.Similarly,analyzing the public dataset also indicate that overexpression of IQGAP3 correlates with a poorer prognosis of the patients.In this study we investigate the expression profile of IQGAP3 at mRNA and protein levels in the high grade serous ovarian cancer(HGSOC)samples and normal fallopian tubal samples.We also explore the correlation of the differential expression of IQGAP3 with the clinicopathological parameters to access the clinical significance of IQGAP3 in ovarian cancer and to identify the role of IQGAP3 in progression and aggressiveness of ovarian cancer.Method:We analyzed the differential expression level of IQGAP3 in ovarian cancer and normal fallopian tubal samples.Real-time PCR was used to analyze the expression of IQGAP3 mRNA levels,western blot and immunohistochemistry were employed to further study the protein expression levels of IQGAP3 between the cancer and the control samples.Then using the standard statistical methods,we determined the clinicopathological significance of the differential expression of IQGAP3 in ovarian cancer cases.We also used The cancer genome atlas(TCGA)database to analyze the samples and study the clinicopathological correlation.Result:1.TCGA data showed a significantly higher expression of IQGAP3 in HGSOC compared to the normal tissue(p<0.05)2.RT-PCR analysis showed a significantly higher expression of mRNA in HGSOC samples compared to the normal fallopian tube samples.(p<0.05)3.Western blot analysis also corroborated the finding of PCR showing higher expression of IQGAP3 in the HGSOC samples compared to the fallopian tube samples.(p<0.05)4.Immunohistochemical staining showed IQGAP3 was overexpressed in the HGSOC tissue samples compared to the fallopian tube samples.5.There was statistically significant correlation between high expression of IQGAP3 and several clinicopathological parameters(CA125,recurrence,peritoneal metastasis)6.Kaplan Meier survival analysis showed a significant difference in overall survival(OS)and progression free survival(PFS)among the TCGA cases with high and low expression of IQGAP3.Conclusion:IQGAP3 is significantly overexpressed in ovarian cancer compared with the normal fallopian tubal specimen.The elevated expression of IQGAP3 is correlated with several clinicopathological parameters and also indicates a poor prognosis in the patientsPart 2 The effect of IQGAP3 in the biological behaviors of ovarian cancerObjective:It has been demonstrated that IQGAP3 actively participates in various pathogenic processes of several cancers.It has been reported to play a role in crucial processes like proliferation,migration,invasion,apoptosis etc.In this study we have studied the functional role of IQGAP3 in the biological behavior of ovarian cancer.The study explored the role of IQGAP3 overexpression in the development,proliferation,migration and invasion of ovarian cancer in vitro as well as in vivo.We further investigated the role of IQGAP3 in apoptotic potential of ovarian cancer cell lines.Furthermore,we studied the functional role of IQGAP3 in chemosensitivity to PARPi agent such as olaparib.Methods:We first demonstrated the expression level of IQGAP3 in the ovarian cancer cell lines(A2780,HEY,SKOV3,OVCAR3)and compared the expression profile with that of the fallopian tubal cell line FTE187 and normal ovarian epithelium cell line HOSEPIC using western blotting.We then used two siRNAs to knockdown the expression of IQGAP3 then proceeded to study its effect in various biological behaviors of the ovarian cancer cells.MTT assay and clonogenic assays were performed to assess the effect on the proliferative capacity of the cells in vitro.Transwell assay was used to investigate the role of IQGAP3 knockdown on migration and invasion in vitro.We also used a lentiviral vector to stably transfect the cell with IQGAP3 shRNA and these stably transfected cells were used in tumor forming assay and metastasis assay in vivo.The tumorigenesis and metastasis in nude mice were further studied through bioluminescence imaging technique.We also demonstrated the alterations in the expression of relevant markers due to the knockdown of IQGAP3 using western blot tenchnique.Furthermore,Annexin V-FITC/PI dual staining and flowcytometry was used to study the effect of IQGAP3 in the apoptotic potential of the cells in vitro.We used MTT assay to show that knockdown of IQGAP3 heightens the chemosensitivity of ovarian cancer cells to wards PARPi agents like olaparib.Results:1.The results of immunoblotting showed a significant decline in IQGAP3 expression level after transient as well as stable transfection in the respective cells.2,MTT assay showed a significant decrease in the proliferative potential of ovarian cancer cell lines after transient transfection with two siRNAs3.Clonogenic assay also showed the decrease in the number of clones in the cells transfected with siRNA compared to the corresponding controls.4.Trasnwell assay showed a lower migration and invasion in the cell lines transfected with siRNAs compared to the respective controls.5,In vivo tumorigenesis assay showed a decreased capacity of tumor formation in nude BALB-c mice compared to the control group.The mice inoculated with stably transfected cells showed a lower number of metastatic foci compared to the control group.Bioluminescence study also elaborated these findings.6.Flowcytometry showed a higher apoptotic potential in the cell lines after knockdown ofIQGAP3.7.Chemosensitivity with olaparib was increased when IQGAP3 was knockout using two siRNAs,which was assessed using MTT assay.8.Western blot showed alteration in several EMT-related proteins associated with migration and invasion following the knockdown of IQGAP3.There was an increased expression of epithelial markers E-CAD,while decreased expression of mesenchymal markers N-CAD,ZEB-1,Vimentin,Snail.The protein markers associated with apoptosis also showed alteration through western blot.There was raised expression of pro-apoptotic markers Bax,Caspase 3,Caspase 9 and decresed expression of anti-apoptotic protein marker Bcl-2.Conclusion:Overexpression of IQGAP3 significantly affects the proliferation,migration,invasion of ovarian cancer in vitro and in vivo.there was also a significant effect of elevated expression of IQGAP3 in the apoptotic potential and chemosensitivity towards PARPi agent olaparib.Part 3 The study of the mechanism involved in the effect of IQGAP3 in ovarian cancer.Objective:The mechanism behind the functional role of IQGAP3 has been studied in several cancers.There have been researches focusing on several signaling pathways which could be implicated in the working of IQGAP3.It has been reported that IQGAP3 exerts its pathogenic roles in lung cancer through EGFR-ERK pathway.It was also reported that IQGAP3 is the effector of CDC42 and IQGAP3 might function through regulation of CDC42.In this study,we study the mechanism,by which IQGAP3 exerts its effect on different aspects of HGSOC.The study also explored the role of CDC42 in the regulation of IQGAP3.We also delved into PI3K/AKT/mTOR signaling pathway as the underlying mechanism by which IQGAP3 exerted its effect of proliferation and migration in HGSOC.Method:To determine whether IQGAP3 was associated with CDC42 in ovarian cancer,the protein expression levels of CDC42 in ovarian cancer cells were assessed by knocking down IQGAP3 expression.We assessed the expression of CDC42 after knockdown of IQGAP3.Then,the effects of CDC42 on the cancer cells were assessed.We carried out the knockdown of CDC42 through transient transfection then determined the changes in the cellular functions like proliferation,migration,invasion and apoptosis.We used MTT assay for determining the proliferation,transwell assay with and without Matrigel was used to assess the migration and invasion potential while apoptotic potential was determined by flow cytometry.We also ran western blot to determine the changes in the protein expression level of PI3K/AKT/mTOR after knockdown of IQGAP3.Results:1.Knockdown of IQGAP3 simultaneously decreased the expression of CDC42.2.Knockdown of CDC42 expression,resulted in a significant decrease in the proliferative potential of HEY cells.3.There was also a decline in migration and invasion of cells after knockdown of CDC42.4.Apoptotic potential was found to be enhanced following CDC42 knockdown.5.Western blot analysis showed a decrease in the protein levels of PI3K,p-AKT,p-mTOR in the cells after the knockdown of IQGAP3.Conclusion:IQGAP3 is closely correlated to CDC42 and might exert its pathogenic functions through the regulation of CDC42.There is an important role of PI3K/AKT/mTOR signaling pathway in the biological functions of IQGAP3 in ovarian cancer.