FSIP1自分泌调控乳腺癌干细胞特性的作用和机制

Cancer stem cells (CSC) have the capacity of self-renewal and differentiation into multiple cancer cells. CSC have the characters of mensenchymal cells and express high levels of drug efflux transporter proteins, such as P-gp and BCRP, naturally resistant to many drugs. Accordingly, CSC are thought to be key of recurrence，relapse and drug resistance of cancer. However, little is known about the factors regulating the stemness of CSC. Our preliminary studies found that fibrous sheath interacting protein 1 (FSIP1), one of the cancer testis antigens, was selectively expressed in breast cancer cells and associated with metastasis and poor prognosis. FSIP1 was also expressed and secreted by breast cancer cell lines as well as freshly isolated breast CSC. Furthermore, FSIP1 silencing inhibited the proliferation, migration and invasion of breast cancer cells, reduced the stemness of breast CSC, and anti-FSIP1 sensitized breast cancer cells to several chemotherapeutic drugs. Accordingly, we hypothesize that autocrine FSIP1 regulates the proliferation, EMT, invasion and stemness of breast CSC and contributes to the recurrence, metastasis and drug resistance of breast CSC. We will examine the frequency of FSIP1+ CSC in breast cancer specimens and its association with clinical pathogenic characteristics of breast cancer; investigate its role and molecular mechanisms as well as the potential signaling that result in FSIP1 regulating proliferation, EMT, invasion, stemness and drug resistance of breast CSC; and identify the FSIP1 receptors on breast cancer cells. Our data will reveal novel molecular mechanisms underlying the action of FSIP1 in regulating the growth, metastasis and drug resistance and provide new targets for therapy design of intervention of breast cancer.

肿瘤干细胞是导致乳腺癌复发、转移和耐药的关键因素。前期工作发现纤维鞘蛋白FSIP1与乳腺癌转移及不良预后显著相关(Oncotarget,2015)，预实验表明乳腺癌和乳腺癌干细胞表达和分泌FSIP1，沉默FSIP1明显抑制乳腺癌细胞的增殖和迁移，且中和自分泌的FSIP1显著增强乳腺癌细胞系对化疗药物的敏感性，但FSIP1促进乳腺癌干细胞特性的机制尚不清楚。本研究拟在细胞水平上通过沉默或过表达FSIP1确认其对乳腺癌干细胞增殖、EMT和耐药性的调控；进而从分子水平确定介导FSIP1促进乳腺癌干细胞特性的信号通路，筛选并鉴定乳腺癌细胞表面FSIP1受体及下游信号传导蛋白；然后在动物水平上验证FSIP1表达变化对乳腺癌干细胞成瘤和转移能力的影响；最终在乳腺癌样本中检测FSIP1阳性乳腺癌干细胞比率与生物学行为关系。本研究将揭示FSIP1调控乳腺癌干细胞特性的分子机制，为乳腺癌新靶点筛选提供依据。

纤维鞘相互作用蛋白1(FSIP1)是一种与精子发生相关的睾丸抗原，在乳腺癌尤其是高表达人表皮生长因子受体2(HER2)的乳腺癌中大量表达，但对其在乳腺癌细胞生长和转移中的调控作用知之甚少。前期研究已经证明，FSIP1在乳腺癌中的表达与HER2阳性、复发和转移呈正相关，与生存期呈负相关。我们利用免疫共沉淀和微量热电泳法，发现FSIP1直接与HER2的胞内结构域结合。进一步研究证明shRNA诱导的SKBR3和MCF-7乳腺癌细胞FSIP1基因敲除抑制细胞增殖，刺激细胞凋亡，减弱上皮-间充质转化及侵袭、侵袭能力。将稳定转染sh-FSIP1的SKBR3细胞移植到nu/nu小鼠体内，与sh-NC移植相比，肿瘤体积减小，这与抑制增殖和促进凋亡一致。此外，使用shFSIP1基因敲减与野生型细胞进行高通量测序，KEGG通路结果显示FSIP1与细胞外基质蛋白途径有关，Western blot分析证实SNAI2蛋白表达减少。作为补充，使用相同的基因签名查询连接图谱得到已知抑制上皮-间充质转化的化学物质，包括雷帕霉素、17-N-烯丙氨基-17-去甲氧基格尔达那霉素和LY294002。这些化合物显示了sh-FSIP1对SKBR3细胞活力的影响。因此，抑制FSIP1能够限制乳腺癌细胞的致瘤性和侵袭性，并且考虑到它在包括正常乳腺在内的大多数其他组织中的缺失，可能成为乳腺癌治疗的潜在靶点。另外，我们还研究了FSIP1在三阴性乳腺癌(TNBC)进展和药物敏感性中的作用，通过CRISPR-Cas9基因敲除技术沉默FSIP1导致的FSIP1缺陷显著抑制了TNBC细胞的增殖和侵袭，并削弱了体内化疗诱导的生长抑制。计算模型预测FSIP1与ULK1结合，这是通过免疫共沉淀建立的。FSIP1缺乏促进了自噬，增强了腺苷酸活化蛋白激酶信号，降低了雷帕霉素的机制靶点和Wnt/β-catenin的活性。相反，抑制AMPK或抑制自噬可以恢复TNBC细胞对化疗药物的敏感性。我们的发现揭示了FSIP1的作用以及FSIP1在药物敏感性中潜在的作用机制，因此可能有助于TNBC疗法的设计。