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

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 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.

RESULTS: 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).

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.