title: Depletion of γ-glutamylcyclotransferase inhibits breast cancer cell growth via cellular senescence induction mediated by CDK inhibitor upregulation.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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/CIP1and p16INK4Awere assessed by qRT-PCR and Western blotting. Effects of simultaneous double knockdown of p21WAF1/CIP1and p16INK4Atogether with GGCT on cell cycle regulation and cell growth was measured by flow cytometry, and trypan blue dye exclusion test.
  
  
  RESULTS
  We found that GGCT knockdown induces significant cellular senescence in various cancer cells. Cyclin dependent kinase inhibitor p21WAF1/CIP1and/or p16INK4Awere upregulated in all cell lines tested. Simultaneous knockdown of p21WAF1/CIP1recovered 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Clinical Oncology, Kyoto Pharmaceutical University, Misasagi-Nakauchicho 5, Yamashinaku, Kyoto, 607-8414, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Matsumura
authors.affiliation: Department of Clinical Oncology, Kyoto Pharmaceutical University, Misasagi-Nakauchicho 5, Yamashinaku, Kyoto, 607-8414, Japan. snakata@mb.kyoto-phu.ac.jp.
authors.forename: Susumu
authors.initials: S
authors.lastname: Nakata
authors.affiliation: Department of Clinical Oncology, Kyoto Pharmaceutical University, Misasagi-Nakauchicho 5, Yamashinaku, Kyoto, 607-8414, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Taniguchi
authors.affiliation: Department of Clinical Oncology, Kyoto Pharmaceutical University, Misasagi-Nakauchicho 5, Yamashinaku, Kyoto, 607-8414, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ii
authors.affiliation: Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Misasagi-Nakauchicho 5, Yamashinaku, Kyoto, 607-8414, Japan.
authors.forename: Eishi
authors.initials: E
authors.lastname: Ashihara
authors.affiliation: Department of Urology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, 520-2192, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Kageyama
authors.affiliation: Department of Urology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, 520-2192, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kawauchi
authors.affiliation: Department of Clinical Oncology, Kyoto Pharmaceutical University, Misasagi-Nakauchicho 5, Yamashinaku, Kyoto, 607-8414, Japan.|Department of Urology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, 520-2192, Japan.
authors.forename: Tatsuhiro
authors.initials: T
authors.lastname: Yoshiki
keywords: Cell cycle arrest; Cellular senescence; p16INK4A; p21WAF1/CIP1; γ-glutamylcyclotransferase