Studies On Bioglogical Functions And Underlying Mechanism(s) Of BTG2 Gene In Breast Cancer

The BTG2 gene is one member of the BTG/TOB anti-proliferative gene family. A few studies have suggested that BTG2 may be a new candidate of tumor suppressor gene. The current study was designed to determine the impacts of BTG2 overexpression by stable transfection of pcDNA3-BTG2 expression vector on cell growth, migration, metastasis and radiosensitivity in vitro and in vivo in human breast cancer MDA-MB-231 and MCF-7 cells. The thesis consists of five sections: (1) Construction and establishment of cell lines with high expression of BTG2 by stable transfection of pcDNA3-BTG2 expression vector; (2) In vitro biological characteristics of BTG2 overexpression in breast cancer cells;(3) BTG2 association with ER-?and its regulation of ER-?transcriptional activity. (4) Biological characteristics of BTG2 overexpression in nude-mice transplanted tumor model; (5) BTG2-mediated radiosensitivity in breast cancer cells;Human breast cancer cell lines, MCF-7 and MDA-MB-231, with BTG2 overexpression were established by stable transfection with a mammalian expression vector pcDNA3 ecoding a full-length of BTG2 cDNA. Successful establishment and characterization of these BTG2 highly expressing cell lines were conducted by Western blot and RT-PCR assays, a significant higher of BTG2 expression in both MCF-7 and MDA-MB-231 cells with a stable transfection of pcDNA3-BTG2 expression vector. A series of experiments have been done to understand biological function and the underlying mechanism(s) of BTG2 overexpression in both MCF-7 and MDA-MB231 cells.In both of MCF-7 and MDA-MB-231 cell lines, increased expression of BTG2 did not cause any alteration of cell morphology, however, reduced cellular adhesion, migration and invasive ability, as evidence by in vitro scratch assay and“Boyden”chamber assay. BTG2 inhibition of invasion and migration associated with down-regulation of RHOC and PLAUR and up-regulation of SET, NME1, NME2 and RB1 genes, as obtained by SuperArray microarray analysis. BTG2 causes G1 arrest in MCF-7 cells and G2 arrest in MDA-MB-231 cells and up-regulation of BTG2 increase caused a G0/G1 arrest in MCF-7 cells and G2/M arrest in MDA-MB-231 cells, which were associated with a decrease of cyclin D1 and cyclin B1.As observed by GST pull-down and immunoprecipitation-Western blot assays, BTG1 associated with ER-?through a LXXLL motif located at 20LXXLL24 of the BTG2 protein sequence, which is required for the co-activators to bind to nuclear receptors. This interaction also needed the presence of E2. Furthermore, BTG1 significantly enhanced ER-?transcripitional activation driven by E2.There were 50% mice (3/6) to form tumors with the MDA-MB-231/BTG2 cells which were injected to nude mice, compared to those with MDA-MB-231 parental cells (5/6) or MDA-MB-231/Neo cells (6/6). Similarly, 2/6 in MCF-7/BTG2 cells compared to 5/6 for MCF-7 parental cells and 6/6 for MDA-MB-231/Neo cells. The tumor size of MCF-7 and MAD-MB-231 cells with high expression of BTG2 was significantly smaller with a slower growth compared to the control cells tumors it was found that local and far metastasis of tumors was significantly blocked in either MCF-7/BTG2 or MDA-MB-231/BTG2 tumors. As for experiments with a lung metastasis model by injection with cells from mouse tails, the number of metastatic colonies on the lung surfaces was significantly lower in BTG2 overexpressed cells than the controls.As observed by clonogenic survival assay, over expression of BTG2 gene can increases the radiosensitivity of both MCF-7 and MDA-MB-231 cell lines to radiotherapy, which was associated with an enhanced G0/G1 arrest and apoptosis induction caused by radiation and accompanied with a decreased expression of cyclin D1 protein and an increased expression of BAX protein. Additionally, overexpression of BTG2 caused a down- expression of DNA repair proteins Ku-70, FEN-1 and XRCC1.In conclusion, BTG1 not only functions as tumor suppressor in cell growth, invasion and migration in vitro models, but also act as perfect repressor in tumor growth and metastasis in animal models. Also BTG2 exhibits similar tumor suppression activities in both human breast cancer MCF-7 and MDA-MB-231. Although the exact mechanisms will need to further explore, multiple working pathways, involved in cell cycle progression, apoptosis and DNA damage repair and others, have been noted from mechanism studies here. Additionally, enforced expression increases radiosensitivity and BTG1 mediated transcriptional activation of ER-?will provide further evidence to support potential application of BTG2 as a novel marker and gene therapy in diagnosis, treatment and prognosis of breast cancer in near future...