γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells.

Abstract

Background/aim: γ-Glutamylcyclotransferase (GGCT) depletion suppresses breast cancer cell proliferation by inducing cellular senescence. However, the underlying molecular mechanisms have not been fully elucidated. Therefore, the objective of this study was to elucidate the mechanisms by which GGCT depletion suppresses cancer cell proliferation.

Materials and methods: Human breast cancer MCF-7 cells were transfected with GGCT-specific or control siRNAs. Transcriptomic profiling by RNA sequencing identified differentially expressed genes (q<0.01, |log₂ fold change|>1), and Gene Ontology and KEGG analyses characterized affected pathways. Key genes and functional effects on the TGF-β2/SMAD3 axis, cell-cycle progression, and senescence were validated by qRT-PCR, western blotting, and SA-β-Gal assays.

Results: Comprehensive gene expression analysis revealed that depletion of GGCT increases the expression levels of the cell cycle arrest factors CDKN1A (p21^Cip1) and CDKN2B (p15^INK4b), accompanied by elevated transforming growth factor-β2 (TGFB2) expression. Blocking this pathway through the simultaneous knockdown of TGFB2 was found to significantly restore the growth-inhibitory effect mediated by cellular senescence induced by GGCT depletion. This finding demonstrated that these phenotypes depend on the TGF-β2 pathway. Furthermore, we identified SMAD3 as a TGF-β2 downstream factor essential for the increase in p21^Cip1 and p15^INK4b and the growth-inhibitory effect induced by GGCT depletion.

Conclusion: Activation of the TGF-β2/SMAD3 pathway is a mechanism by which cellular senescence is induced through GGCT depletion, suggesting that GGCT inhibition represents a promising therapeutic strategy for the treatment of breast cancer.