Gene Knock out Mice Model for Studying the Molecular Mechanisms of Early Onset Breast Tumor Microenvironment Regulation.(2/3)

A、Specific Aim: Early-onset breast tumors have been detected in more Taiwanese patients than in patients from Western countries for more than 10 years. This study will focus on the tumor microenvironment, which is involved in the formation of breast cancer at a young age. B、Preliminary results: 1. From breast tumor tissues: Hyaluronan synthase (HLS) was detected at lower levels in young (<35 y, n=76) compared old (>70 y, n=65) patients in a total cohort of 436 cases (*p<0.001). 2. From HLS knock/out mice: Three genes (HLS-1, -2, -3) were targeted to establish K/O mice. The cancer cells (e0771) derived from the mouse breast tumors were transplanted into C57BL/6J mice, and the results showed that HLS-2 and -3 K/O provide an environment that facilitates tumor formation. 3. From HLS-overexpressing breast cancer cells: The overexpression of HLS-2 and -3 proteins induced human breast cancer apoptosis. In contrast, apoptotic effects were not observed in the absence of HLS genes in mouse embryonic fibroblasts (MEFs). 4. From in vivo antitumor tests: The I P. injection of hyaluronan inhibited tumor cell proliferation in SCID mice with transplantation of human breast cancer (MDA-MB-231) tumor cells. 5. A previous study (Nature, 18:346-349,2013) demonstrated that high-molecular-weight hyaluronan mediates cancer resistance in a naked mole-rat model. C、Hypothesis: HLS was detected at a low level in the microenvironment of young age-specific breast tumor tissues. The hyaluronan, which was synthesized by HLS in the matrix of tumor tissues, was not able to against tumor cell proliferation, invasion, or migration, thus promoting young age (<35 y)-specific breast cancer formation in Taiwanese people. D、Study design: Year 1 proposal: Studies of the mechanisms of HLS deficiency involved in early-onset breast cancer formation Aim-1、Studies examining the expression level of HLS and its correlation with the occurrence of breast cancer at a young age. 參 Statistical analysis (H-score and real-time PCR) of the HLS level in the tumor tissues of young vs. old patients. Aim-2、Knockout of the HLS gene to test its biological functions in the breast tumor microenvironment. • Establishment of HLS gene K/O mice: mouse breast cancer (e0771) cells will be transplanted into HLS-1, -2, and -3 gene K/O mice, and the tumor growth will be observed. • Establishment of HLS gene K/O human breast cancer cells to observe the effects of HLS K/O on the growth of human breast tumor cells. • Establishment of HLS gene K/O MEFs: MEF cells will be co-cultured with human breast cancer cells to investigate the antitumor effects of hyaluronan synthesized by HLS in the co-cultured medium. Year 2 Proposal ： Study of the mechanisms of HLS-induced cell death in human breast cancer cells Aim-1、Over-expression of the HLS genes induced antitumor effects in human breast cancer cells. • Establishment of HLS-overexpressing breast cancer cells using the Tet-on technique. 參 Live cell model of the effects of cell growth cycle arrest in HLS-overexpressing breast cancer cells. • Observation of a live cell model of tubulin disorganization-induced apoptosis in HLS-overexpressing breast cancer cells. Aim-2、Investigation of HLS expression in cancer-associated fibroblasts (CAFs) isolated from young breast cancer patients. • Establishment of CAFs specific to young breast cancer patients: • Identification of HLS high (or low)-expressing CAFs to investigate the antitumor role of HLS in the tumor microenvironment. • Co-culture of CAFs and breast cancer cells specific to young patients to observe the anti-tumor and anti-metastasis effects of hyaluronan synthesized by HLS. Year 3 proposal ： Study of intracellular HLS regulation for the prevention of young age-specific breast cancer formation. Aim-1 、 Study of the mechanisms underlying HLS deficiency involved in carcinogenesis formation. • HLS-overexpressing cancer cell lines (established in year 2) will be used to demonstrate that HLS deficiency enhances malignancy in breast cancer formation. • In vivo animal model to demonstrate that K/O of HLS in human breast cancer cells enhances tumor growth and metastasis. Aim-2、Study of the regulation of HLS expression in human breast cancer cells induced by the effects of the tumor microenvironment. • The effects of negative regulatory mechanisms of the tumor microenvironment (such as smoking and hormones) on HLS expression in human breast cancer cells will be examined. • The effects of positive regulatory mechanisms of the tumor microenvironment (such as hypoxia and growth factor deficiency) on HLS expression in human breast cancer cells will be examined. Aim-3、Study of HLS expression as a molecular target for the prevention of young age-specific breast cancer formation. • Young age-specific CAF cells will be used to demonstrate the antitumor effects of hyaluronan, which is synthesized by HLS. • Natural compounds that upregulate HLS expression in young age-specific CAFs will be screened. • HLS K/O human cancer cells will be used as a cell model to screen for antitumor drugs.