Depletion of γ-glutamylcyclotransferase inhibits breast cancer cell growth via cellular senescence induction mediated by CDK inhibitor upregulation.

Chromosome 7 open reading frame 24 (C7orf24) was originally identified as a highly expressed protein in various types of cancer, and later shown to be a γ-glutamylcyclotransferase (GGCT). GGCT depletion in cancer cells has anti-proliferative effects in vitro and in vivo, and it is therefore considered a promising candidate as a therapeutic target. However, the cellular events induced by GGCT depletion remain unclear. GGCT was depleted by siRNA in MCF7, MDA-MB-231, PC3, A172, Hela, and LNCaP cells. Induction of cellular senescence was evaluated with senescence-associated β-galactosidase (SA-β-Gal) staining. Expression levels of p21WAF1/CIP1 and p16INK4A were assessed by qRT-PCR and Western blotting. Effects of simultaneous double knockdown of p21WAF1/CIP1 and p16INK4A together with GGCT on cell cycle regulation and cell growth was measured by flow cytometry, and trypan blue dye exclusion test. We found that GGCT knockdown induces significant cellular senescence in various cancer cells. Cyclin dependent kinase inhibitor p21WAF1/CIP1 and/or p16INK4A were upregulated in all cell lines tested. Simultaneous knockdown of p21WAF1/CIP1 recovered the cell cycle arrest, attenuated cellular senescence induction, and rescued the subsequent growth inhibition in GGCT-silenced MCF7 breast cancer cells. In contrast, in GGCT silenced MDA-MB-231 breast cancer cells, GGCT depletion upregulated p16INK4A, which played a regulatory role in senescence induction, instead of p21WAF1/CIP1. Our findings demonstrate that induction of cellular senescence mediated by the upregulation of cyclin-dependent kinase inhibitors is a major event underlying the anti-proliferative effect of GGCT depletion in breast cancer cells, highlighting the potential of GGCT blockade as a therapeutic strategy to induce cellular senescence. The online version of this article (doi:10.1186/s12885-016-2779-y) contains supplementary material, which is available to authorized users.

7号染色体开放阅读框24（C7orf24）最初被鉴定为在多种癌症中高表达的一种蛋白质，后来被证明是一种γ -谷氨酰环转移酶（GGCT）。癌细胞中GGCT的缺失在体外和体内都具有抗增殖作用，因此它被认为是一个有希望的治疗靶点。然而，由GGCT缺失诱导的细胞事件仍不清楚。 通过小干扰RNA（siRNA）在MCF7、MDA - MB - 231、PC3、A172、Hela和LNCaP细胞中使GGCT缺失。用衰老相关的β -半乳糖苷酶（SA - β - Gal）染色评估细胞衰老的诱导情况。通过实时定量聚合酶链反应（qRT - PCR）和蛋白质印迹法（Western blotting）评估p21WAF1/CIP1和p16INK4A的表达水平。通过流式细胞术和台盼蓝拒染试验测量同时双敲低p21WAF1/CIP1和p16INK4A以及GGCT对细胞周期调控和细胞生长的影响。 我们发现GGCT敲低在多种癌细胞中诱导显著的细胞衰老。细胞周期蛋白依赖性激酶抑制剂p21WAF1/CIP1和/或p16INK4A在所有测试的细胞系中均上调。在MCF7乳腺癌细胞中，同时敲低p21WAF1/CIP1恢复了细胞周期停滞，减弱了细胞衰老诱导，并挽救了随后因GGCT沉默而导致的生长抑制。相反，在MDA - MB - 231乳腺癌细胞中，GGCT缺失上调了p16INK4A，它在衰老诱导中起调节作用，而非p21WAF1/CIP1。 我们的研究结果表明，由细胞周期蛋白依赖性激酶抑制剂上调介导的细胞衰老诱导是乳腺癌细胞中GGCT缺失抗增殖作用背后的一个主要事件，突显了GGCT阻断作为一种诱导细胞衰老的治疗策略的潜力。 本文的在线版本（doi:10.1186/s12885 - 2016 - 2779 - y）包含补充材料，可供授权用户使用。