不同亚细胞定位的FOXP3在乳腺癌转移中的作用及其分子机制探讨

Breast cancer is the common female cancer and although many advanced treatments have resulted from improving clinical instruments and methods, metastasis still leads to cancer mortality and poor prognosis. Forkhead Box Protein 3 (FOXP3) was demonstrated to be an X-linked tumor suppressor gene and inhibited mammary carcinogenesis and tumor cell proliferation by acting as a transcriptional repressor of breast cancer oncogenes such as SKP2 (S-phase kinase protein 2) and HER2 (human epidermal growth factor receptor 2). However, the research on biological functions and significance of FOXP3 in breast cancer metastasis presently remained unclear because of conflicting clinical results and were limited to clinical epidemiological studies without related research on molecular mechanism. Some reports showed that there were somatic FOXP3 mutations in cancerous tissues and some of these mutations affected the nuclear localisation,which might potentially limit the function of FOXP3. We found that only reduced FOXP3 nuclear expression was significantly associated with tumor clinical stages and lymph node metastasis in breast?cancer samples and that over-expression wild-type FOXP3, restricted to the nucleus, in breast cancer cells could inhibit cell migration in vitro. We also found that wild-type FOXP3 might be a transcriptional repressor of CD44 through RNA-seq analysis and that FOXP3 expression substantially repressed activity of the CD44 gene promoter. The adhesion molecule CD44, as cell surface receptor for hyaluronic acid (HA), had a long-standing association with breast cancer cell invasion and metastasis. Based on the results of preliminary experiment and literature research, we planed to illuminate the following questions in this project. First, the effect of FOXP3 in cytoplasmic or nuclear localisation on breast cancer metastasis was identified through epidemiological studies of large-scale breast cancer samples, cytological research, and animal models. Second, it would be proved that FOXP3 could inhibit breast cancer metastasis through down-regulating CD44 expression. At last, the mechanism of down-regulation of FOXP3 in breast cancer tissues was preliminary explored. The study highlights the biological functions and molecular mechanism of FOXP3 in different subcellular localization in breast cancer metastasis and provides a potential target for clinic control of breast cancer metastasis in the future.

乳腺癌是女性最常见的恶性肿瘤，转移是其主要致死原因。研究发现转录因子FOXP3表达于正常乳腺上皮细胞，在乳腺癌组织中表达降低或缺失；并且FOXP3是X-染色体连锁的乳腺癌抑制基因，抑制乳腺癌的发生和增殖。但关于FOXP3表达与乳腺癌转移关系的报道仅停留在对临床样本统计学分析层面，且结果相互矛盾，这可能与其在肿瘤细胞中的亚细胞定位多样性相关。本课题组前期研究发现，乳腺癌标本中FOXP3的细胞核阳性表达率与肿瘤淋巴结转移呈负相关；过表达野生型FOXP3能抑制乳腺癌细胞的迁移，转录组学分析FOXP3能抑制粘附分子CD44的表达。基于此，本课题拟首先通过系统的临床分析，体外细胞行为学，体内动物实验明确不同亚细胞定位的FOXP3在乳腺癌转移中的作用；然后深入阐明FOXP3通过抑制CD44来抑制乳腺癌转移的作用及分子机制；最后，初步探讨乳腺癌中FOXP3下调的机制。该研究为乳腺癌转移的防治提供新思路。

乳腺癌是女性最常见的恶性肿瘤，转移是其主要致死原因。研究发现转录因子FOXP3 表达于正常乳腺上皮细胞，在乳腺癌组织中表达降低或缺失；并且 FOXP3 是 X-染色体连锁的乳腺癌抑癌基因，抑制乳腺癌的发生和增殖。但关于 FOXP3 表达与乳腺癌转移关系的报道仅停留在对临床样本统计学分析层面，且结果相互矛盾。本课题通过系统的临床分析发现，乳腺癌中FOXP3表达水平下降并且亚细胞定位发生了变化，只有细胞核内FOXP3表达水平与乳腺癌的进展，转移成负相关；体内动物实验结果显示FOXP3能够显著抑制乳腺癌细胞的肺转移能力；体外Transwell和细胞粘附实验结果说明，FOXP3能够显著抑制乳腺癌细胞的侵袭和转移能力；以上结果证实FOXP确实具有抑制乳腺癌转移的作用。. 本课题进一步通过转录组学测序筛选FOXP3调控的下游靶分子，结果显示细胞黏附分子CD44可能是FOXP3参与乳腺癌转移调控的重要靶分子；通过分子生物学实验、细胞学实验和临床组织标本证实FOXP3通过抑制CD44的启动子活性抑制CD44的表达从而抑制乳腺癌的转移。本课题还探讨了部分乳腺癌细胞核中的FOXP3失去抑癌功能的原因，通过分子生物学实验，细胞行为学实验以及临床样本分析等实验技术发现乳腺癌中细胞核内的Gal-1与FOXP3相互作用导致野生型FOXP3失去抑癌功能。 . 本课题通过研究对不同亚细胞定位的 FOXP3 在乳腺癌侵袭转移中的作用及其分子机制，明确定位于细胞核的有功能的 FOXP3 是乳腺癌转移的抑制基因， 进一步扩展了 FOXP3 分子在乳腺癌中的作用；证实 FOXP3 通过调控CD44 的启动子活性抑制 CD44 的表达，从而抑制乳腺癌的转移; 初步证实乳腺癌中细胞核内的Gal-1与FOXP3相互作用导致FOXP3失去抑癌功能。该研究丰富了乳腺癌的转移机制，也为乳腺癌的临床诊断治疗提供了潜在的靶点。