CyclinD1-CDK4/6磷酸化XBP1S介导其入核参与ER+乳腺癌他莫昔芬耐药的机制研究

Tamoxifen (TAM) is the first choice in endocrine therapies for ER+ breast cancer, but 30% of patients will develop drug resistance. Our previous study has found out that IRE1-XBP1 pathway of endoplasmic reticulum stress is involved in tamoxifen resistance of breast cancer. TAM activated IRE1 and induced the overexpression and nuclear translocation of XBP1S, and the specific inhibitor of IRE1 can reverse tamoxifen resistance. But the manner of XBP1S’ nuclear translocation and its downstream effect are still unclear. We have analyzed the XBP1S’ transcriptional genes in breast cancer by Chip-sequence, and this project plan to explore the effect of Cyclin D1 transcribed by XBP1S on tamoxifen resistance and the related molecular mechanism: (1) to test the expression of Cyclin D1 and its related factors in tamoxifen resistance breast cancer cells, nude mice models and clinical breast cancer tissues; (2) to check whether XBP1S can be phosphorylated by Cyclin D1-CDK4/6 on S346, and improve XBP1S’ nuclear translocation and activate the positive feedback cycle of XBP1S-Cyclin D1; (3) to interfere the XBP1S-Cyclin D1 cycle or to inhibit IRE1 and phosphorylation of XBP1S at the same time, and to check their synergistic effects on reverse tamoxifen resistance, and aim to find out the new target for improving endocrine therapies’ efficacy in breast cancer.

他莫西芬（TAM）是ER+乳腺癌内分泌治疗首选，但30%患者耐药，我们前期发现IRE1-XBP1通路参与TAM耐药，TAM激活IRE1导致XBP1S的过度表达及入核，抑制IRE1可逆转耐药，但XBP1S入核及下游机制尚待研究。本项目拟采用Chip-seq分析乳腺癌中XBP1S基因转录谱，深入探讨Cyclin D1在IRE1-XBP1导致TAM耐药中的作用及机理：在细胞-裸鼠-临床标本中检测Cyclin D1及相关分子的表达，分析其与细胞周期活化的关系；验证Cyclin D1-CDK4/6可否磷酸化XBP1S的S346位点，促进其入核，从而形成“XBP1S-Cyclin D1正反馈调控网络”，导致细胞周期激活及肿瘤耐药、复发；最终检测干扰XBP1S-Cyclin D1循环以及“TAM-IRE1抑制-XBP1S磷酸化抑制”三联给药对TAM耐药的逆转，为提高乳腺癌内分泌治疗疗效提供新治疗靶点。

本项目研究要点是分析乳腺癌中XBP1s基因转录谱，深入探讨Cyclin D1在IRE1-XBP1导致它莫昔芬耐药中的作用及机理。我们明确了XBP1的在乳腺癌它莫昔芬耐药中与临床病理特征的显著相关性。但XBP1s与Cyclin D1之间的相互作用未能得到验证。通过对正常非耐药细胞及它莫昔芬耐药细胞进行转录组测序， 并于通过与大数据生信的结合分析，我们发现SRC\Sirt1及CXCR4等多个或可在乳腺癌它莫昔芬耐药机制中发挥重要作用的基因，但尚未进行深入研究。我们的进一步研究发现，SRC和SIRT1在它莫昔芬耐药的乳腺癌细胞中均上调，并与它莫昔芬治疗的乳腺癌预后不良有关。SRC通过上调SIRT1 的表达促进它莫昔芬的耐药性。此外，它莫昔芬治疗的乳腺癌患者中， CXCR4的高表达与预后不良相关， 且CXCR4在它莫昔芬耐药的乳腺癌细胞中明显上调。CXCR4的特异性拮抗剂AMD3100可以通过抑制AKT磷酸化来部分逆转乳腺癌细胞对它莫昔芬的耐药性。