Identification of MUC1-C Dependence in Drug-Resistant Advanced Prostate Cancer Uncovers a New Target for Antibody-Drug Conjugate Therapy

Androgen receptor positive prostate cancer (PC), castration resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) represent a spectrum of malignancies that invariably become resistant to treatment with targeted and cytotoxic agents. There is no known common pathway responsible for these pleotropic mechanisms of resistance. The MUC1 gene is aberrantly expressed in CRPC and NEPC in association with poor clinical outcomes. The present results demonstrate that the oncogenic MUC1-C protein is necessary for resistance of (i) PC cells to enzalutamide (ENZ), and (ii) CRPC and NEPC cells to docetaxel (DTX). We show that MUC1-C-mediated ENZ and DTX resistance is conferred by upregulation of aerobic glycolysis and suppression of reactive oxygen species (ROS) necessary for self-renewal capacity. Common dependence of these drug-resistant phenotypes on MUC1-C for the cancer stem cell (CSC) state thus identified a potential new target for their treatment. cIn this context, we further demonstrate that targeting MUC1-C with an antibody-drug conjugate (ADC) is highly effective in suppressing (i) self-renewal of drug-resistant CRPC and NEPC CSCs and (ii) growth of t-NEPC tumor xenografts derived from drug-resistant cells and a patient with refractory disease. These findings reveal a shared MUC1-C-dependent pathway in drug-resistant CRPC and NEPC progression and identify MUC1-C as a target for their treatment with an ADC. Overall design: RNA Seq in LNCaP-ER, DU-145-DR, and H660 Cell lines with MUC1-C shRNA silencing. We conpared DMSO and DOX treatment cells for differential gene expression analysis. MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma, St. Louis, MO, USA) was inserted into the pLKO-tet-puro vector (Plasmid #21915; Addgene, Cambridge, MA, USA) . Vector-transduced cells were selected for growth in 1–2 µg/ml puromycin. Cells were treated with 0.1% DMSO as the vehicle control or 500 ng/ml doxycycline (DOX; Millipore Sigma).

雄激素受体阳性前列腺癌（androgen receptor positive prostate cancer, PC）、去势抵抗性前列腺癌（castration resistant prostate cancer, CRPC）与神经内分泌前列腺癌（neuroendocrine prostate cancer, NEPC）构成一类恶性肿瘤谱，此类肿瘤最终均会对靶向治疗及细胞毒性药物产生耐药性。目前尚未明确介导此类多效性耐药机制的共同通路。MUC1基因在CRPC与NEPC中存在异常表达，且与不良临床结局密切相关。本研究结果证实，致癌性MUC1-C蛋白是两类细胞产生耐药性的必要条件：（i）前列腺癌细胞对恩扎卢胺（enzalutamide, ENZ）的耐药；（ii）CRPC与NEPC细胞对多西他赛（docetaxel, DTX）的耐药。本研究表明，MUC1-C介导的ENZ与DTX耐药，是通过上调有氧糖酵解、抑制癌症干细胞（cancer stem cell, CSC）自我更新所需的活性氧（reactive oxygen species, ROS）实现的。上述耐药表型均依赖于MUC1-C以维持癌症干细胞状态，这一发现为这类肿瘤的治疗提供了潜在新靶点。

在此研究背景下，本团队进一步证实，采用抗体药物偶联物（antibody-drug conjugate, ADC）靶向MUC1-C，可高效抑制以下过程：（i）耐药性CRPC与NEPC干细胞的自我更新；（ii）源自耐药细胞及难治性患者的治疗相关神经内分泌前列腺癌（t-NEPC）肿瘤异种移植物的生长。本研究结果揭示了耐药性CRPC与NEPC进展中一条共有的MUC1-C依赖性通路，并证实ADC靶向MUC1-C可用于这类肿瘤的治疗。

总体实验设计：对转染MUC1-C短发夹RNA（shRNA）的LNCaP-ER、DU-145-DR及H660细胞系进行RNA测序。我们比较了二甲基亚砜（DMSO）处理组与多西环素（doxycycline, DOX）处理组的细胞，以开展差异基因表达分析。MUC1 shRNA（MISSION shRNA TRCN0000122938；Sigma，美国密苏里州圣路易斯市）被插入至pLKO-tet-嘌呤霉素载体（质粒#21915；Addgene，美国马萨诸塞州剑桥市）中。转染载体的细胞通过在1~2 μg/ml嘌呤霉素培养基中筛选获得。细胞分别以0.1% DMSO作为溶剂对照，或500 ng/ml多西环素（DOX；Millipore Sigma）进行处理。