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

Kengo Matsumura, Susumu Nakata, Keiko Taniguchi, Hiromi Ii, Eishi Ashihara, Susumu Kageyama, Akihiro Kawauchi, Tatsuhiro Yoshiki

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 p21(WAF1/CIP1) and p16(INK4A) were assessed by qRT-PCR and Western blotting. Effects of simultaneous double knockdown of p21(WAF1/CIP1) and p16(INK4A) 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 p21(WAF1/CIP1) and/or p16(INK4A) were upregulated in all cell lines tested. Simultaneous knockdown of p21(WAF1/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 p16(INK4A), which played a regulatory role in senescence induction, instead of p21(WAF1/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.