| Breast cancer is the most common type of cancer in women worldwide. The incidence of breast cancer has been rising slowly; about 1 million women are diagnosed with breast cancer every year in the world. In china especially, with rapidly rising incidence in recent years, breast cancer is the malignant tumor of the female morbidity and mortality, seriously affecting the lives and health of women. The majorities of deaths from breast cancer are not due to the primary tumor itself, but are the result of metastasis to other organs in the body. At the time of clinical diagnosis, approximately 10% have occurred in patients with metastatic. In case of tumor metastasis, prognosis is poor; 5-year survival rate is only 20%. Study of breast cancer metastasis related signaling pathway is important for the management and prediction of breast cancer progression.As a breast cancer suppressor gene, the Fork head transcription factor Foxp3 has got more and more attention in recent years. Studies have shown that Foxp3 expression in normal breast epithelial cells, but in breast cancer, the expression of Foxp3 is low or missing. It is becoming clear that this failure of normal FOXP3 expression can result in deregulation of the expression of a range of oncogenes which have been implicated in the development and metastasis of cancer. Importantly, mice that are heterozygous for Foxp3 mutationsspontaneously develop mammary carcinomas at a high frequency. It is now known that Foxp3 can play an important role in controlling oncogenic factors in epithelial cells by controlling the expression of a number of genes implicated in cancer, such as HER-2/Erb B2,SKP2. Because many important cancer-related signaling pathways are regulated by FOXP3, it may be a potential therapeutic target for human malignancies.Although a large number of studies have shown that Foxp3 inhibits the proliferation of breast cancer, however, there are rarely evidence between Foxp3 expression and breast cancer metastasis. The results from Overbeck-Zu have show that the low expression of Foxp3 can promote breast cancer cells to chemokine-driven shift, which suggest Foxp3 can inhibits the metastasis of breast cancer, however, the mechanism is not clear, and there still many issues to be explored. First, is there any relationship between Foxp3 and breast cancer metastasis in clinical samples. Second, which kinds of metastasis associated genes can be regulated by Foxp3 in breast cancer cells? Third, what are the mechanisms by which Foxp3 regulate the expression these gene expressions? The last, can the metastasis of breast cancer cells in animal models be inhibited by reactivating or reintroducing wild type Foxp3 to cancer cells which lack normal expression of this protein?The answers to these questions will clarify the mechanisms of Foxp3 inhibits breast cancer metastasis and provide a new function of Foxp3 in breast cancer. To this end, these following research works have been done. ① To confirm the relationship between failure expression of Foxp3 and breast cancer metastasis, we first analysis the expression of Foxp3 in 200 cases of breast cancer in clinical samples, and census the differential expression levels in normal breast tissue, breast cancer, distant metastases of breast tissue by tissue microarray technique. ② In order to find out which kinds of metastasis associated genes can be regulated by Foxp3 in breast cancer cells, we also analysis the relationship between the levels of Foxp3 and the cell adhesion molecule CD44 in breast cancer specimen and human breast cancer cell lines by immunohistochemistry, real-time PCR, western blot. ③ We next ask whether Foxp3 is a determining regulator of CD44 expression in breast cancer cells by elevate Foxp3 expression in SKBr-3、MDA-MB-231; To determine whether CD44 expression will increase when decrease Foxp3 expression, weknock down Foxp3 expression in MCF-7、T47D using three si RNA oligonucleotides designed to target Foxp3 transcripts specifically.④ we then ask whether the expression of CD44 would fluctuate concordantly with changes in activated Foxp3. Quantification of this association of CD44 expression with the presence of Foxp3 was demonstrated by isolating the highest and lowest 10% of CD44-expressing viable MDA-MB-231 cells by FACS sorting and co-staining with CD44 and Foxp3 antibody. ⑤ To elucidate the mechanisms of Foxp3 regulate CD44 expression, we performer conventional CHIP, EMSA and dual luciferase reporter gene analyses.⑥ To investigate whether increased Foxp3 expression influence breast cancer cells transfer capacity, We carried out the transwell and cell adhesion experiments. ⑦ To determine whether elevated Foxp3 expression would have a similar effect on the breast cancer cells metastasis in vivo, we evaluated mammary glands of transgenic mice the occurrence of pulmonary metastasis using animal imaging technology.We have obtained the following results through these experiments. ① From the results of tissue microarray technology, we found that, compared with normal breast tissue and breast cancer tissue without metastasis, Foxp3 expression in breast cancer tissue has occurred in shift significantly decreased, which shows that there is a close relationship between Foxp3 and metastasis of breast cancer.② The level of expression of CD44 and Foxp3 were negatively correlated in breast cancer tissues, suggests a close relationship between them and Foxp3 may be a regulator of CD44 gene.③ After forced a transient elevation in the expression of Foxp3,reduced in both CD44 transcript levels were obtained in both cases; Conversely, when we knock down Foxp3 expression in breast cancer cells by si RNA, the result was a increased in both transcript and protein levels of CD44.④ using FACS sorting, we observed a >2-fold difference in Foxp3 levels between the subsets expressing the highest and lowest levels of CD44. ⑤ The results of cell immunofluorescence technique have showed the distribution of Foxp3 and CD44 proteins in the cells.⑥ Further, transient elevation in the expression of Foxp3 lead to a 50% decrease in CD44 promoter activity.⑦ Ch IP analyses provide direct evidence of the ability of Foxp3 to bind to the promoters of both CD44 genes in two human breast cancercell lines.⑧ The result of EMSA confirmed the interaction between Foxp3 and CD44 promoter.⑨ The transgenic mice had lower level of metastasis as compared to wild type cells in vivo.In the study, we confirmed Foxp3 can inhibit breast cancer metastasis by regulate the expression of CD44 first time. These results complement Foxp3 as a breast cancer suppressor, at same time, we also elucidate the molecular mechanism of CD44 expression in breast cancer. This report provide a new idea for inhibiting breast cancer metastasis and provide a new target and method for the clinical treatment of breast cancer. |
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