Cancer Research Group, School of Life Sciences, University of Westminster, London W1W 6 UW, UK.
Tissue Architecture and Regeneration Research Group, School of Life Sciences, University of Westminster, London W1W 6 UW, UK.
Int J Mol Sci. 2021 Jan 30;22(3):1396. doi: 10.3390/ijms22031396.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.
胰腺导管腺癌 (PDAC) 是一种侵袭性最强的恶性肿瘤之一，其生存率有限。肽基精氨酸脱亚氨酶 (PAD) 的作用已在一系列癌症中得到研究，其作用涉及表观遗传调控（包括组蛋白修饰和 microRNA 调控）、癌症侵袭和细胞外囊泡 (EV) 释放。然而，目前对于 PDAC 中 PAD 的知识仍然有限。在本研究中，用泛 PAD 抑制剂 Cl-amidine 以及 PAD2、PAD3 和 PAD4 同工酶特异性抑制剂处理两种 PDAC 细胞系（Panc-1 和 MiaPaCa-2）。评估了对 EV 特征变化的影响，包括 EV microRNA 载物（miR-21、miR-126 和 miR-221）、与胰腺癌细胞进展和侵袭相关的细胞蛋白表达变化（膜突蛋白）、线粒体管家基因（抑制素、PHB）和基因调控（脱亚氨组蛋白 H3、citH3）。发现这两种胰腺癌细胞系主要表达 PAD2 和 PAD3，并且在 Panc-1 中的表达水平高于 MiaPaCa-2 细胞。PAD2 同工酶特异性抑制剂对降低 Panc-1 细胞侵袭能力的作用最强，同时膜突蛋白表达增加，在胰腺癌细胞中发现膜突蛋白表达降低与胰腺癌细胞侵袭性有关。PHB 水平也有一定程度的降低，但无显著意义，而组蛋白 H3 脱亚氨作用则各不相同。EV 特征对 PAD 抑制剂治疗有反应，PAD2 抑制剂的作用最强，其次是 PAD3 抑制剂，导致促癌性 EV microRNA 载物（miR-21、miR-221）减少，而抑癌性 microRNA 载物（miR-126）增加。虽然 PAD2 抑制剂、其次是 PAD3 抑制剂对降低癌细胞侵袭、升高膜突蛋白表达和调节 EV 特征的作用最强，但 PAD4 抑制剂作用微不足道，泛 PAD 抑制剂 Cl-amidine 也效果较差。与 MiaPaCa-2 细胞相比，PAD 抑制剂在 Panc-1 细胞中具有更强的调节作用，重要的是，Panc-1 细胞对 PAD3 抑制剂也有强烈反应，这与之前的观察结果一致，即 Panc-1 细胞显示出神经元/干细胞样特性。我们的研究结果报告了 PDAC 中 PAD 同工酶调节作用的新发现，强调了根据癌症类型和癌症亚型（包括 PDAC）进行 PAD 同工酶特异性治疗的作用。
