To Investigate The Key Genes Of Cancer Proliferation And The Mechanism Of Cancer Metastasis By Genome-wide Screening And Cell Fusion Method

Purpose:Uncontrolled cellular proliferation and metastasis are the key characteristics of malignant tumors; they are also the principle targets for cancer therapeutics. In this study, we aimed to establish a cellular screening system for the identification of key regulators for pancreatic cancer cell proliferation, and to investigate the role of tumor cell fusion in tumor metastasis.Pancreatic cancer is among the leading cancers that characterized with rapid tumor cell proliferation, early metastasis and high mortality. To some extent, cancer is a disease that cancer cells over-proliferate because of abnormal regulation. In the first part of this study, our goal is to develop a genome-wide screening platform for the identification of the genes controlling cancer cell proliferation.Malignant melanoma is a leading cause of mortality related to skin cancer, mainly due to the early distant metastasis. However, there is no effective treatment for malignant melanoma. Cell fusion is often observed during tumor progression and has been proposed to play a role in tumor metastasis. Tumor cell fusions have been seen in many cancers (such as breast cancer, prostate cancer and melanoma), and are associated with aneuploidy, tumor heterogeneity and poor prognosis. In the second party of this study, our goal is to generate melanoma tumor-tumor cell fusions and investigate their role in tumor metastasis and relavent gene expression profile.Methods:I Firstly we constructed the gene search vector TNO-PB by molecular clonning.obtained stable pancreatic cancer AsPC-1-2D2cell lines with tetracycline mediated gene expression regulation; Then we generated genome-wide random mutagenesis library; applied CFSE fluorescent probes to analyze the proliferation of pancreatic cancer cells; obtained several clones of pancreatic cancer cell with inhibited proliferation and further validated these clones with tetracycline mediated gene regulation. Ⅱ We established the phytohemagglutinin-polyethylene glycol (PHA-PEG) mediated cell fusion method and successfully obtained stable melanoma fusion cells following fluorescence activated cell sorting (FACS). DNA content and chromosome number of the fusion cells were analyzed with flow cytometry and karyotype analysis; Cell proliferation and tumorigenicity of the tumor fusion cells were analyzed, and experimental metastatic potential was determined in mouse; the gene and protein expression of the fusion cells were analyzed by microarray and western blot.Results:Ⅰ We successfully constructed a genome-wide screening system, and generated a mutagenesis library of AsPC-1pancreatic cancer cells containing about740,000mutated clones. We established the "FACS selection method" to select the cell polulations of low proliferation rate and the "clone screening method" to screen the clones of low proliferation rate. Pancreatic validated clones of low proliferation rate have been successfully screened by the above methods and checked to be low proliferation rate and regulated by DOX. Further study will found the key genes controlling cancer proliferation.Ⅱ The fusion efficiency was significantly enhanced by adding the PHA to agglutinate the cells together before adding fusion-promoting reagent PEG (about10times). Pure and Stable melanoma tumor-tumor cell fusion hybrids were successfully generated; the fusion tumor cells have been shown with near doubled cell size, DNA content, and chromosomes; while the cell proliferation and tumorigencity of the fusion tumor cells were decreased, their metastatic potential was increased; further analysis with microarray and western blot showed that PI3K-AKT pathway related to cell proliferation was significantly down-regulated, the expression of phospho-S6was significantly decreased. These results suggested that cell fusion reduced the melanoma-cell proliferation rate through the PI3K-AKT pathway. Bioinformatics analysis also showed that β-tubulin2expression was significantly enhanced (>10times), indicating β-tubulin2may play a role in melanoma metastasis.Conclusions:In summary, we successfully integrated a genome-wide mutagenesis technology with the CFSE cell proliferation tracking probe in human pancreatic cancer cells, obtained several clones of tumor cells with inhibited cell proliferation. The established screening system will provide novel strategy to identify key genes that controlling cancer cell proliferation. Using a highly efficient cell fusion method, we successfully obtained stable fusion melanoma cells, which demonstrated significantly decreased in cell growth and tumorigenicity, while dramatically increased in metastatic potential; further characterization indicated that the PI3K-AKT pathway and (3-tubulin2may play important roles in melanoma cell growth and metastasis.