C-Cbl 逆转了人乳腺癌细胞中 HER2 介导的他莫昔芬耐药性。
C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells.
机构信息
Department of Medical Oncology, the First Hospital of China Medical University, Shenyang, 110001, Liaoning, China.
Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning Province, the First Hospital of China Medical University, NO. 155, North Nanjing Street, Heping District, Shenyang, 110001, Liaoning, China.
出版信息
BMC Cancer. 2018 May 2;18(1):507. doi: 10.1186/s12885-018-4387-5.
BACKGROUND
Tamoxifen is a frontline therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women. However, many patients develop resistance to tamoxifen, and the mechanism underlying tamoxifen resistance is not well understood. Here we examined whether ER-c-Src-HER2 complex formation is involved in tamoxifen resistance.
METHODS
MTT and colony formation assays were used to measure cell viability and proliferation. Western blot was used to detect protein expression and protein complex formations were detected by immunoprecipitation and immunofluorescence. SiRNA was used to examine the function of HER2 in of BT474 cells. An in vivo xenograft animal model was established to examine the role of c-Cbl in tumor growth.
RESULTS
MTT and colony formation assay showed that BT474 cells are resistant to tamoxifen and T47D cells are sensitive to tamoxifen. Immunoprecipitation experiments revealed ER-c-Src-HER2 complex formation in BT474 cells but not in T47D cells. However, ER-c-Src-HER2 complex formation was detected after overexpressing HER2 in T47D cells and these cells were more resistant to tamoxifen. HER2 knockdown by siRNA in BT474 cells reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was also disrupted and tamoxifen resistance was reversed in BT474 cells by the c-Src inhibitor PP2 and HER2 antibody trastuzumab. Nystatin, a lipid raft inhibitor, reduced ER-c-Src-HER2 complex formation and partially reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was disrupted by overexpression of c-Cbl but not by the c-Cbl ubiquitin ligase mutant. In addition, c-Cbl could reverse tamoxifen resistance in BT474 cells, but the ubiquitin ligase mutant had no effect. The effect of c-Cbl was validated in BT474 tumor-bearing nude mice in vivo. Immunofluorescence also revealed ER-c-Src-HER2 complex formation was reduced in tumor tissues of nude mice with c-Cbl overexpression.
CONCLUSIONS
Our results suggested that c-Cbl can reverse tamoxifen resistance in HER2-overexpressing breast cancer cells by inhibiting the formation of the ER-c-Src-HER2 complex.
背景
他莫昔芬是绝经前妇女雌激素受体（ER）阳性乳腺癌的一线治疗药物。然而，许多患者对他莫昔芬产生耐药性，其耐药机制尚不清楚。在这里，我们研究了 ER-c-Src-HER2 复合物的形成是否与他莫昔芬耐药有关。
方法
MTT 和集落形成实验用于测量细胞活力和增殖。Western blot 用于检测蛋白表达，免疫沉淀用于检测蛋白复合物的形成，免疫荧光用于检测蛋白复合物的形成。siRNA 用于研究 HER2 在 BT474 细胞中的功能。建立体内异种移植动物模型研究 c-Cbl 在肿瘤生长中的作用。
结果
MTT 和集落形成实验表明，BT474 细胞对他莫昔芬耐药，而 T47D 细胞对他莫昔芬敏感。免疫沉淀实验显示，BT474 细胞中存在 ER-c-Src-HER2 复合物，但 T47D 细胞中不存在。然而，在 T47D 细胞中过表达 HER2 后，检测到 ER-c-Src-HER2 复合物形成，这些细胞对他莫昔芬的耐药性增强。BT474 细胞中 HER2 的 siRNA 敲低减少了 ER-c-Src-HER2 复合物的形成，逆转了他莫昔芬耐药性。BT474 细胞中 c-Src 抑制剂 PP2 和 HER2 抗体曲妥珠单抗也破坏了 ER-c-Src-HER2 复合物的形成，并逆转了他莫昔芬耐药性。质膜窖抑制剂制霉菌素减少了 ER-c-Src-HER2 复合物的形成，并部分逆转了他莫昔芬耐药性。c-Cbl 的过表达破坏了 ER-c-Src-HER2 复合物，但 c-Cbl 的泛素连接酶突变体没有作用。此外，c-Cbl 可以逆转 BT474 细胞中的他莫昔芬耐药性，但泛素连接酶突变体没有效果。c-Cbl 的作用在体内 BT474 荷瘤裸鼠中得到了验证。免疫荧光也显示，裸鼠肿瘤组织中 c-Cbl 过表达后，ER-c-Src-HER2 复合物的形成减少。
结论
我们的结果表明，c-Cbl 通过抑制 ER-c-Src-HER2 复合物的形成，可以逆转 HER2 过表达的乳腺癌细胞对他莫昔芬的耐药性。
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