Potent androgen receptor inhibition unleashes oncogenic action of the KLF5 stem cell transcription factor in castration-resistant prostate cancer [ChIP-seq]

Advanced prostate cancer (PC) can be treated with endocrine therapies that inhibit transcriptional activity of the androgen receptor (AR). However, PC will ultimately evolve under the selection pressure of these therapies and progress to a lethal phenotype termed castration-resistant PC (CRPC). Recent studies suggest that de-differentiation away from an AR-driven luminal cell identity may be a key, early step. However, early therapy-induced mechanisms of prostate cancer cell de-differentiation are poorly understood. Here we show that the stem cell transcription factor Kruppel-like factor 5 (KLF5) is transcriptionally repressed by AR, and de-repressed by enzalutamide. KLF5 expression is high in a subset of CRPC tissues, but low in primary adenocarcinoma. KLF5 functioned as oncogene in CPRC cells, promoting cellular migration, anchorage-independent growth, expression of basal cell markers, and transcriptional signatures of CRPC lineage plasticity, but had modest effects on cell proliferation. Chromatin immuno-precipitation (ChIP)-sequencing, RNA-sequencing, and co-immunoprecipitation experiments established a physical interaction between KLF5 and AR, and integrative analysis revealed that KLF5 and AR drive opposing transcriptional programs. We identified ERBB2 as a point of transcriptional convergence that was activated by KLF5 and repressed by AR. Accordingly, KLF5 expression levels in CRPC tissues correlated with predicted sensitivity to the dual EGFR inhibitor lapatanib, and the ERBB2-specific inhibitor mubritinib blocked KLF5-driven cell migration. Collectively, these findings implicate KLF5 as an AR-suppressed oncogene that drives early steps in CRPC de-differentiation and disease progression. Overall design: KLF5 ChIP-seq in R1-AD1 prostate cancer cells cultured in medium supplemented with 10% charcoal-stripped serum (CSS) and stimulated 4h with 1 nM DHT (or 0.1% ethanol as vehicle control) as well as KLF5 ChIP-seq in AR gene-engineered R1-D567 prostate cancer cells cultured in medium supplemented with 10% CSS.

晚期前列腺癌（prostate cancer, PC）可采用抑制雄激素受体（androgen receptor, AR）转录活性的内分泌疗法进行治疗。然而，前列腺癌最终会在这些疗法的选择压力下演化，并进展为致死性的去势抵抗性前列腺癌（castration-resistant PC, CRPC）表型。近期研究表明，脱离AR驱动的腔上皮细胞身份的去分化过程，可能是疾病进展的关键早期事件。但目前对治疗诱导的前列腺癌细胞去分化的早期分子机制仍知之甚少。
本研究发现，干细胞转录因子Krüppel样因子5（Kruppel-like factor 5, KLF5）可被AR转录抑制，并可被恩扎卢胺（enzalutamide）解除抑制。KLF5在部分去势抵抗性前列腺癌组织中呈高表达，而在原发性腺癌中呈低表达。KLF5在去势抵抗性前列腺癌细胞中发挥癌基因功能，可促进细胞迁移、非锚定依赖性生长、基底细胞标志物表达以及去势抵抗性前列腺癌谱系可塑性的转录特征，但对细胞增殖的影响较为温和。染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）、RNA测序（RNA-seq）以及免疫共沉淀（co-immunoprecipitation, CoIP）实验证实，KLF5与AR之间存在物理相互作用；整合分析显示，KLF5与AR调控的转录程序完全相反。本研究鉴定出ERBB2是转录交汇靶点：KLF5可激活其转录，而AR则抑制其表达。据此，去势抵抗性前列腺癌组织中KLF5的表达水平与双重表皮生长因子受体（epidermal growth factor receptor, EGFR）抑制剂拉帕替尼（lapatanib）的预测敏感性呈正相关；而ERBB2特异性抑制剂莫布替尼（mubritinib）可阻断KLF5介导的细胞迁移。综上，本研究结果表明，KLF5是一种受AR抑制的癌基因，可驱动去势抵抗性前列腺癌去分化及疾病进展的早期进程。
**整体实验设计**：在添加10%活性炭吸附血清（charcoal-stripped serum, CSS）的培养基中培养R1-AD1前列腺癌细胞，分别用1 nM二氢睾酮（dihydrotestosterone, DHT）刺激4小时（以0.1%乙醇作为溶剂对照），并对其进行KLF5染色质免疫沉淀测序；同时在添加10%活性炭吸附血清的培养基中培养经AR基因工程改造的R1-D567前列腺癌细胞，同样进行KLF5染色质免疫沉淀测序。