School of Science and Technology, Centre for Health, Ageing and Understanding of Disease, Nottingham Trent University, Nottingham, NG11 8NS, UK.
Department of Biological and Biomedical Sciences, Science Centre, School of Health, Science and Wellbeing, Staffordshire University, Leek Road, Stoke-on-Trent, ST4 2DF, UK.
Cell Death Dis. 2023 May 9;14(5):317. doi: 10.1038/s41419-023-05818-9.
Androgen independency is associated with poor prostate cancer (PCa) survival. Here we report that silencing of transglutaminase-2 (TG2) expression by CRISPR-Cas9 is associated with upregulation of androgen receptor (AR) transcription in PCa cell lines. Knockout of TG2 reversed the migratory potential and anchorage independency of PC3 and DU145 cells and revealed a reduced level of mucin-1 (MUC1) RNA transcript through unbiased multi-omics profiling, which was restored by selective add-back of the truncated TG2 isoform (TGM2_v2). Silencing of AR resulted into increased MUC1 in TG2KO PC3 cells showing that TG2 affects transcriptional regulation of MUC1 via repressing AR expression. Treatment of PC3 WT cell line with TG2 inhibitor ZDON led to a significant increase in AR expression and decrease in MUC1. ZDON also blocked the formation of MUC1-multimers labelled with TG amine-donor substrates in reducing conditions, revealing for the first time a role for TG2, which we show to be externalised via extracellular vesicles, in MUC1 stabilisation via calcium-dependent transamidation. A specific antibody towards TGM2_v2 revealed its restricted nuclear location compared to the canonical long form of TG2 (TGM2_v1), which is predominantly cytosolic, suggesting that this form contributes to the previously suggested TG2-mediated NF-κB activation and AR transcriptional repression. As TGM2_v2 transcription was increased in biopsies of early-stage prostate adenocarcinoma (PRAD) patients compared to subjects presenting inflammatory prostatitis, and total TG2 protein expression significantly increased in PRAD versus normal tissue, the role of TG2 and its truncated form as a prostate malignancy marker is suggested. In conclusion, this investigation has provided the first unbiased discovery of a novel pathway mediated by TG2 via MUC1, which is shown to contribute to androgen insensitivity and malignancy of PCa cells and be upregulated in PCa biopsies, with potential relevance to cancer immune evasion.
雄激素非依赖性与前列腺癌（PCa）不良预后相关。本研究报道，CRISPR-Cas9 敲低转谷氨酰胺酶-2（TG2）表达与 PCa 细胞系中雄激素受体（AR）转录上调有关。TG2 敲除逆转了 PC3 和 DU145 细胞的迁移潜能和锚定独立性，并通过无偏多组学分析揭示了粘蛋白-1（MUC1）RNA 转录本水平降低，通过选择性添加截断 TG2 同工型（TGM2_v2）得到恢复。AR 沉默导致 TG2KO PC3 细胞中 MUC1 增加，表明 TG2 通过抑制 AR 表达影响 MUC1 的转录调控。用 TG2 抑制剂 ZDON 处理 PC3 WT 细胞系导致 AR 表达增加和 MUC1 减少。ZDON 还阻止了在还原条件下用 TG 胺供体底物标记的 MUC1 多聚体的形成，首次揭示了 TG2 的作用，我们发现它通过细胞外囊泡外化，通过钙依赖性转酰胺作用稳定 MUC1。针对 TGM2_v2 的特异性抗体显示其与主要位于细胞质中的经典长型 TG2（TGM2_v1）相比，其核定位受限，这表明该形式有助于先前提出的 TG2 介导的 NF-κB 激活和 AR 转录抑制。由于与患有炎症性前列腺炎的受试者相比，早期前列腺腺癌（PRAD）患者活检中 TGM2_v2 转录增加，并且与正常组织相比，PRAD 中总 TG2 蛋白表达显著增加，因此提示 TG2 及其截断形式作为前列腺癌恶性标志物的作用。总之，本研究首次提供了 TG2 通过 MUC1 介导的新型途径的无偏发现，该途径被证明有助于 PCa 细胞的雄激素不敏感性和恶性程度，并在 PCa 活检中上调，可能与癌症免疫逃避有关。
