14-3-3sigma gene silencing during melanoma progression and its role in cell cycle control and cellular senescence

The family of 14-3-3 proteins plays an important role in cancer biology by interfering with intracellular signalling pathways and cell cycle checkpoints. The 14-3-3sigma isoform acts as a tumor suppressor and is often inactivated during tumor development.

Here, we demonstrate enhanced CpG methylation of the 14-3-3sigma gene in lymph node and cutaneous melanoma metastases compared with primary tumors, associated with dramatically reduced mRNA expression. In line with this, treatment of different metastatic melanoma cell lines with 5-aza-2'-deoxycytidine (5-Aza-CdR), a potent inhibitor of cytosine methylation, significantly induces 14-3-3sigma protein expression. Additional treatment with histone deacetylase inhibitor 4-phenylbutyric acid (Pba) further enhances 14-3-3sigma expression. Induction of 14-3-3sigma expression by 5-Aza-CdR/Pba treatment leads to almost complete inhibition of cell proliferation, with cells predominantly arrested in G2-M. The antiproliferative effect of 5-Aza-CdR/Pba was reversed in 14-3-3sigma knockdown cells. Similarly, melanoma cell lines stably overexpressing 14-3-3sigma show dramatically reduced cell proliferation rates. Moreover, synchronous 14-3-3sigma stably overexpressing cells do not progress through cell cycle, but display a permanent increase in the population of 4n DNA containing cells. Interestingly, overexpression of 14-3-3sigma induces senescence of melanoma cells and is involved in melanoma cell senescence under genotoxic stress. Finally, 14-3-3sigma knockdown supports migratory capacity of melanoma cells in vitro, while 14-3-3sigma overexpression has opposing effects.

Taken together, the present report indicates that epigenetic silencing of 14-3-3sigma might contribute to tumor progression in malignant melanoma via loss of cell cycle control, impaired cellular senescence program and support of migratory capacity.