FSIP1调控CD44-/CD24-三阴性乳腺癌细胞去分化为肿瘤干细胞的机制研究

Breast cancer stem cells (BCSCs) are one of the main reasons leading to the metastasis of triple negative breast cancer(TNBC), and the regulation of the characteristics of BCSCs remains an open question. We reported that FSIP1 regulates the oncogenesis and invasiveness in breast cancer cells by binding to the intracellular domain of HER2 directly, may become a potential target for breast cancer therapy (PNAS, 2017). Our preliminary studies revealed that the purified CD44-/CD24- differentiated MDA-MB-231 dedifferentiated into CD44+/CD24- stem-like cells in vitro. The further purified CD44+/CD24- BCSCs had similar stemness compared to the primarily purified BCSCs. Furthermore, the knockout of FSIP1 by Crispr/Cas9 enhanced the dedifferentiation of CD44-/CD24- MDA-MB-231 cells into CD44+/CD24- BCSCs, followed by YAP/TAZ highly expression. Accordingly, we hypothesize that FSIP1-mediated microtubule disorders activate YAP/TAZ, together with stem cell related genes over expression, promote the dedifferentiation of triple negative breast cancer CD44-/CD24- into CD44+/CD24- BCSCs. We will examine the characteristics of triple negative breast cancer CD44-/CD24- cells and investigate the nature and mechanisms underlying the dedifferentiation of triple negative breast cancer CD44-/CD24- cells into CD44+/CD24- BCSCs. Theoretically, our findings may provide new insights into regulation of the stemness of BCSC and potentially aid in design of new therapies for triple negative breast cancers.

乳腺癌干细胞（BCSCs）是三阴性乳腺癌复发的关键，其特性维持机制是肿瘤干细胞研究领域的瓶颈。申请人曾报道纤维鞘相互作用蛋白1（FSIP1）通过促进HER2蛋白胞内段络氨酸激酶的磷酸化调控乳腺癌的增殖，为乳腺癌治疗提供了新靶点（PNAS，2017）。预实验发现敲除FSIP1能促进三阴性乳腺癌中CD44-/CD24-细胞去分化为CD44+/CD24-BCSCs，伴随YAP/TAZ高表达。本研究拟首先检测三阴性乳腺癌中CD44-/CD24-细胞的肿瘤特性，其次比较初次纯化和去分化获得的BCSCs的干细胞特性差异，再次分析临床样本分离的原代细胞中FSIP1表达水平和CD44-/CD24-细胞去分化为BCSCs的相关性，最后探讨FSIP1是否通过Hippo/YAP/TAZ 通路调控CD44-/CD24-细胞去分化成BCSCs。本研究将初步阐明BCSCs特性维持机制，为三阴性乳腺癌的治疗提供新思路。

乳腺癌干细胞（BCSCs）是三阴性乳腺癌复发的关键，其特性维持机制是肿瘤干细胞研究领域的瓶颈。前期研究已经证明，敲除FSIP1能促进三阴性乳腺癌中CD44-/CD24-细胞去分化为CD44+/CD24- BCSCs。我们把临床获得的新鲜三阴性乳腺癌标本分离乳腺癌原代细胞，用免疫磁珠（MACS）原代细胞中纯化已分化的CD44-/CD24-乳腺癌细胞进行培养，将其与初次纯化的CD44+/CD24- BCSCs相比，发现去分化新获得的BCSCs的功能与CD44+/CD24- BCSCs功能一致。通过去分化过程中不同时间节点的细胞测序，我们发现RHBDL2、CRM1以及SOST均在去分化过程中发挥功能。动物体内实验发现三阴性乳腺癌CD44-/CD24-细胞亚群同样能够成瘤，将瘤体消化进行流式分析时，发现RHBDL2敲除后的CD44-/CD24-瘤体内CD44+/CD24- BCSCs细胞比例明显比对照组高。进一步通过大量临床患者数据发现CD44-/CD24-细胞频率较高的患者的DFS和OS明显短于CD44-/CD24-细胞频率较低的患者的DFS和OS。此外，TNBC患者无论化疗与否，CD44-/CD24-细胞频率较高的患者在肿瘤切除后4年发生进行性转移的风险都很高。通过免疫组化442例乳腺癌组织中SOST表达，进行了与乳腺癌骨转移的相关性分析，发现SOST表达上调与乳腺癌骨转移和乳腺癌患者生存率较差有关。SOST沉默显著降低了SCP2细胞的骨转移能力。SOST与STAT3相互作用以增强TGF-β/KRAS信号传导，增加肿瘤生长和骨转移。最后，我们针对CRM1基因设计可阻断BCSC的小分子药物，通过筛选发现LFS-1107可以选择性地消除CD44+CD24-富集的BCSC。给予LFS-1107可以强烈抑制小鼠异种移植模型中的肿瘤生长并根除残留肿瘤组织中的BCSC。此外，与一些批准的抗癌药物相比，LFS-1107可以显着消融TNBC的患者来源肿瘤类器官，且LFS-1107可以增强化疗药物的杀伤作用。综上，课题组发现维持肿瘤干细胞的新途径及内在机制，并筛选出靶向BSCS的小分子LFS-1107，有希望成为临床治疗剂，可能有助于TNBC疗法的设计。