二甲双胍逆转乳腺癌HER2/ER信号交联所致耐药的作用及分子机制

HER2 and ER are two main therapeutic targets of breast cancer. The complex crosstalk between HER2 and ER signal pathways is the leading reason for endocrine resistance of HER2+/ER+ breast cancer. So, for this type of breast cancer, clarifying its endocrine resistance mechanism, searching for effective intervention methods and therapeutic targets is immediate areas of research focus. Our initial studies found that metformin, the traditional insulin sensitizer, could inhibit the proliferation of HER2+/ER+ breast cancer cells and reverse drug resistance, the possible mechanism was up-regulating miR-17/miR-101 and down-regulating molecules relevant to resistance. Bioinformatics and luciferase reporter gene analysis revealed that miR-17/miR-101 could directly regulate AIB1, which is not only the hub molecule associated with HER2/ER signal crosstalk, but also the key molecule related to drug resistance. We hypothesized that metformin might reverse drug resistance phenotype through up-regulating miR-17/miR-101 and down-regulating AIB1. Our study aims to probe the role of metformin in malignant phenotype and drug resistance of HER2+/ER+ breast cancer, to explore its possible mechanism from molecular, cellular, creatural and clinical fields. Our study could afford a new train of thought and theoretical basis for endocrine resistance of breast cancer.

HER2和ER是乳腺癌的两个主要治疗靶点。HER2和ER信号通路间的复杂交联是导致HER2+/ER+乳腺癌内分泌耐药的主要原因。因此，明确该型乳腺癌的耐药机制，寻找有效的干预手段增强内分泌治疗敏感性，是目前研究的热点。我们前期实验发现，传统胰岛素增敏剂二甲双胍可有效抑制HER2+/ER+乳腺癌细胞增殖、逆转耐药性，其可能机制是通过上调miR-17/miR-101来抑制耐药相关分子的表达。生物信息学和荧光素酶报告基因分析证实miR-17/miR-101可直接调控HER2、ER信号交联的枢纽分子AIB1，而AIB1正是介导内分泌耐药的关键分子。我们推测二甲双胍可能通过上调miR-17/miR-101抑制AIB1的表达，从而逆转耐药发生。本研究拟从细胞、动物、临床多个层次研究二甲双胍在HER2+/ER+乳腺癌恶性表型和耐药中的作用及机制，为研究内分泌耐药机制及探索新型治疗策略提供新的思路。

HER2和ER是乳腺癌的两个主要治疗靶点，HER2和ER信号通路间的复杂交联是导致HER2+/ER+乳腺癌内分泌耐药的主要原因。因此，明确该型乳腺癌的耐药机制，寻找有效的干预手段增强内分泌治疗敏感性，是目前研究的热点。我们分别构建了对他莫昔芬耐药的BT474细胞（BT474-TR）、HER2+的MCF-7细胞（MCF-7/HER2+）以及MCF-7/HER2+细胞他莫昔芬的耐药株（MCF-7/HER2+-TR）。我们的研究发现，二甲双胍可明显抑制BT474-TR和MCF-7/HER2+-TR的增殖，促进细胞凋亡并抑制迁移。二甲双胍可有效逆转HER+/ER+乳腺癌细胞对他莫昔芬的耐药，二甲双胍处理后的BT474-TR及MCF-7/HER2+-TR细胞对他莫昔芬的敏感性显著增强，进一步的裸鼠体内实验结果也验证了这一结果。前期，我们通过芯片分析得到了miR17及miR101这两个相关miRNA，通过软件预测到了AIB1基因可与其发生作用，通过荧光素酶报告基因实验验证了miR17/miR101有效作用于AIB1基因3’UTR，同时上调miR17及miR101能够上调AIB1的表达。从临床样本中同样发现了AIB1的表达与他莫昔芬的获益具有明显的相关性。进一步的研究提示他莫昔芬和二甲双胍可显著提高MCF-7及BT474细胞中miR101的表达，用软件预测了另外两个与之结合且与乳腺癌增殖密切相关的基因SOX2和NCOA6。对两种癌细胞进行双药处理，发现相较于单药作用，SOX2和NCOA6在RNA及蛋白水平均有明显降低。同时，在两个细胞中进行了SOX2过表达，进行了细胞增殖检测，结果符合预期。进而，对SOX2的3’UTR与miR-101结合位点的突变进行了荧光素酶检测，证实SOX2与miR-101有结合。进一步证实了SOX2分子可能是二甲双胍提高乳腺癌细胞对他莫昔芬敏感性的关键分子。该发现为下一步探索乳腺癌的内分泌治疗及耐药机制提供了新的思路。