Screening And Study Of Metastasis Suppressor Genes And Small Molecule Inhibitors In Ovarian Cancer

Ovarian malignancy is one of the most common causes of death in female gynecological oncology.Nearly 220,000 women are diagnosed with epithelial ovarian cancer（EOC）every year worldwide,and about 140,000 patients succumbing to this disease annually,it is the leading cause of cancer-related death in women and also the gynecological malignancy with the highest mortality rate^[1].The MUC16/CA125glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer.Targeting MUC16/CA125 might be an effective strategy for the specific treatment of ovarian cancer.In the current stage of cancer research,CRISPR screening have been extensively applied and enables high-throughput interrogation of gene functions in diverse tumor biologies,such as tumor growth,metastasis,synthetic lethal interactions,therapeutic resistance immunotherapy response and so on.By targeting coding and non-coding regions throughout the genome,the biological detection of target genes can be carried out conveniently and efficiently.Meanwhile,because of the high mortality rate of EOC,development of precision therapeutics for EOC is an area of critical need.Tumor immunology has promoted the development of ovarian cancer therapy in recent years,in particular,enzymes have served as direct targets for ground-breaking new medicines.However,due to its characteristics of insidious onset,high malignancy and strong metastasis,effective early screening methods,treatment and prognostic indicators are still not currently available in clinical practice.which leads to the poor survival rate and prognostic of patients with ovarian cancer.From the transcription level.Here,we aimed to use an independent screening system to screen out relevant genes that inhibit ovarian cancer metastasis and identify MUC16/CA125-specific small molecules and targets for development in the treatment of ovarian cancer.In this study,we successfully established an orthotopic implantation model of ovarian cancer using CRISPR/Cas9 human genome-wide knockout library and selected the human ovarian cancer low-metastatic cell line SKOV3.We aimed to remove the genes related to inhibition of ovarian cancer implantation metastasis by in vivo screening to induce ovarian tumorigenesis and metastasis,and thus screened the genes that inhibit ovarian cancer metastasis.Meanwhile,Utilizing a separate approach,we performed high-content small-molecule screening of 6 different libraries of 356,982compounds for MUC16/CA125 selective agents using isogenic dual florescence ovarian cancer cell lines and identified lead candidates that showed preferential cytotoxicity in MUC16+cells.we also performed a synthetic lethal kinase screen using a human kinome RNAi library and identified potential targets preferentially targetable in MUC16+cells.The result shows that among the 6722 significant candidate genes associated with metastasis selected from the CRISPR/Cas9 library,ITK（Interleukin-2-inducible T-cell kinase）which is a cytotoxic lymphocytes-related gene acting as a key factor to predict clinical outcomes of patients with ovarian cancer independently and powerfully in TCGA and GEO datasets.Previous studies have shown that in development of inhibitors,tumorigenesis and inflammatory process,ITK plays an significant role.Integrated analysis also showed that dysregulated molecular processes including predominantly oncogenic signaling pathways in low-ITK group but immune related pathways in high-ITK group.Furtherly,we discovered that ITK was positive correlation with CD244 and SOCS1（cancer suppressor gene）but negative correlation with SMARCD1（oncogene）between low and high-ITK group.Additionally,deconvolution of the cellular composition of all samples with MCP-counter and CIBERSORT methods validated the immune related analysis and discovered the closely correlation between ITK and cytotoxic lymphocytes.In the meantime,our screen of the human kinome identified integrin-linked kinase（ILK）and glucokinase（GCK）as potential targets.When ILK and GCK were silenced,they preferentially inhibited invasion of MUC16+cells in vitro and in vivo and we show that this is due to decreased activation of MAP kinase,and non-receptor tyrosine kinases.Through dual fluorescent high-content screen,the anti-tumor effect of flavopiridol which is a cyclin-dependent kinase（CDK）inhibitor was determined in vitro and in vivo.In summary,we successfully screen the relevant genes that inhibit ovarian cancer metastasis using a mature and well-established screening system,we also present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development,we provide a promising way for ovarian cancer diagnosis and treatment in the clinical practice.