Supporting data for “Androgen receptor modulates chemotherapy response in breast cancer”

Breast cancer ranks as the second most diagnosed cancer globally and the most common among women. Chemotherapies, encompassing both systemic and targeted approaches, are critical for managing the disease, yet drug resistance often compromises treatment efficacy. The androgen receptor (AR), a widely expressed hormonal receptor in breast cancer, has an unclear role in disease progression. Emerging evidence suggests AR may contribute to drug resistance in various cancers, including breast cancer; however, the underlying mechanisms remain underexplored.<br>This study investigated the role of AR in chemotherapy response using two AR-positive breast cancer cell lines. We assessed responses to multiple chemotherapeutic agents—palbociclib, docetaxel, doxorubicin, paclitaxel, and cisplatin. <i>In vitro </i>and <i>in vivo</i> results revealed that AR activation reduced cell sensitivity to palbociclib and doxorubicin, indicating that AR modulated responses to these drugs. For palbociclib, a CDK4/6 inhibitor, AR activation induced RB1 polyubiquitination and degradation, diminishing RB1 levels—a key downstream target of CDK4/6—and thus impairing palbociclib efficacy. Chromatin immunoprecipitation (ChIP) sequencing identified 1,209 AR-regulated genes, with gene ontology (GO) analysis highlighting significant enrichment of ubiquitin-related genes. qPCR validation pinpointed TRIM37, an AR-regulated E3 ligase, as a mediator of RB1 degradation. TRIM37 overexpression decreased palbociclib sensitivity, while its knockdown enhanced sensitivity and attenuated the effect of AR on RB1 levels and drug response, establishing TRIM37 as a novel E3 ligase that bound RB1 via its MATH and C-terminal domains.<br>For doxorubicin, a DNA-damaging agent, AR activation mitigated induced DNA damage, suggesting a role in DNA repair. GO analysis of AR-regulated genes from ChIP sequencing implicated DNA damage-binding genes in AR-mediated doxorubicin response. qPCR validation identified CRY2 as an AR-regulated gene reducing DNA damage. CRY2 overexpression lowered doxorubicin sensitivity, whereas its knockdown increased sensitivity and diminished the influence of AR on drug response. Mechanistically, CRY2 enhanced non-homologous end joining (NHEJ) repair—rather than homologous recombination (HR), by binding to NHEJ proteins (DNA-PKcs, Artemis, Ku80, Lig4, and XLF). Interactions of CRY2 to DNA-PKcs, Artemis, Ku80, and Lig4 strengthened post-doxorubicin treatment. Inhibition of NHEJ with the DNA-PKcs inhibitor KU-57788 reversed the protective effect of CRY2, confirming AR modulates doxorubicin response via CRY2-driven NHEJ activation.<br>In conclusion, this study elucidates AR-mediated resistance mechanisms to palbociclib and doxorubicin in breast cancer through TRIM37 and CRY2, respectively. These findings highlight AR as a potential therapeutic target and underscore the need for further clinical validation to optimize treatment strategies for AR-positive breast cancer patients.These are the supporting data for the PhD thesis of “Androgen receptor modulates chemotherapy response in breast cancer”. The data included western blot, luciferase assay, cell viability assay, qPCR, ChIP qPCR, cell cycle assay, CoIP assay, Ubiquitination assay, IF staining, animal study, DNA repair assay including HR and NHEJ activities, and TUNEL assay. The procedures of the assays were described in the thesis.

乳腺癌是全球范围内确诊率第二高的癌症，也是女性群体中最常见的恶性肿瘤。包含系统性治疗与靶向治疗在内的化疗手段是该病临床管理的关键，但耐药性往往会削弱治疗效果。雄激素受体（androgen receptor, AR）是乳腺癌中广泛表达的激素受体，但其在疾病进展中的功能尚不明确。越来越多的研究证据表明，AR可能参与包括乳腺癌在内的多种癌症的耐药过程，但其背后的分子机制仍有待深入探索。<br>本研究采用两株AR阳性乳腺癌细胞系，探究了AR在化疗应答中的调控作用。我们检测了细胞对多种化疗药物的响应，包括帕博西尼（palbociclib）、多西他赛（docetaxel）、阿霉素（doxorubicin）、紫杉醇（paclitaxel）和顺铂（cisplatin）。体外（in vitro）与体内（in vivo）实验结果显示，AR激活会降低细胞对帕博西尼与阿霉素的敏感性，表明AR可调控细胞对这两种药物的应答。作为CDK4/6抑制剂的帕博西尼，其作用机制中，AR激活可诱导RB1发生多泛素化并降解，降低RB1蛋白水平——而RB1是CDK4/6的关键下游靶标——进而削弱帕博西尼的治疗效果。染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）测序共鉴定出1209个受AR调控的基因，基因本体（gene ontology, GO）分析显示，泛素相关基因存在显著富集。定量聚合酶链反应（quantitative PCR, qPCR）验证结果显示，受AR调控的E3泛素连接酶TRIM37介导了RB1的降解过程。过表达TRIM37会降低细胞对帕博西尼的敏感性，而敲低TRIM37则可增强敏感性，并削弱AR对RB1蛋白水平与药物应答的调控作用，证实TRIM37是一种新型E3泛素连接酶，可通过其MATH结构域与C端结构域结合RB1。<br>作为DNA损伤剂的阿霉素，其处理后，AR激活可减轻药物诱导的DNA损伤，提示AR可能参与DNA修复过程。对ChIP测序得到的AR调控基因进行GO分析发现，DNA损伤结合基因参与了AR介导的阿霉素应答过程。qPCR验证显示，CRY2是受AR调控的基因，可降低细胞的DNA损伤水平。过表达CRY2会降低细胞对阿霉素的敏感性，而敲低CRY2则可增强敏感性，并削弱AR对药物应答的调控作用。从分子机制来看，CRY2可通过结合非同源末端连接（non-homologous end joining, NHEJ）相关蛋白（DNA-PKcs、Artemis、Ku80、Lig4及XLF），增强NHEJ修复通路活性，而非同源重组（homologous recombination, HR）通路。阿霉素处理后，CRY2与DNA-PKcs、Artemis、Ku80及Lig4的相互作用会增强。使用DNA-PKcs抑制剂KU-57788抑制NHEJ通路后，CRY2的保护作用被逆转，证实AR可通过CRY2介导的NHEJ激活调控阿霉素的药物应答。<br>综上，本研究分别阐明了AR通过TRIM37与CRY2介导乳腺癌细胞对帕博西尼与阿霉素的耐药机制。上述研究结果表明，AR可作为潜在的治疗靶点，同时也强调需要开展进一步的临床验证，以优化AR阳性乳腺癌患者的治疗方案。本数据集为《雄激素受体调控乳腺癌化疗应答》博士学位论文的配套支持数据，涵盖蛋白质免疫印迹（western blot）、荧光素酶报告基因实验、细胞活力检测、qPCR、ChIP qPCR、细胞周期检测、免疫共沉淀（co-immunoprecipitation, CoIP）实验、泛素化检测实验、免疫荧光染色、动物实验、包含HR与NHEJ活性检测的DNA修复实验，以及TUNEL实验。各项实验的操作流程已在论文中详述。