The Role of 14-3-3 Gamma in Promoting Genomic Instability

Abstract

The 14-3-3 family members are a group of highly conserved scaffolding proteins that are present in all eukaryotes. Despite having limited endogenous activity, the 14-3-3s bind to numerous client proteins and directly modulate their activities by a variety of mechanisms. In mammals, there are seven isoforms (beta, gamma, epsilon, eta, sigma, theta, and zeta), and between them over 200 known binding partners, which allow them to function in nearly every aspect of cellular biology. The 14-3-3 family has long been studied in regards to cancer, as aberrant changes in their expression patterns have been associated with numerous human cancers. It is, therefore, a shared goal, among many, to elucidate the role of 14-3-3 proteins in tumorigenesis. For the last decade, our laboratory has been keenly focused on the tumor-promoting roles of 14-3-3 proteins in the context of lung cancer, as their expression patterns are highly dysregulated in this cancer setting. Herein, we expand on this area of research and demonstrate that four of the seven 14-3-3 isoforms are significantly elevated in both adenocarcinomas and squamous cell carcinomas of the lung. We then investigated the consequences of these isoforms being increased in malignant lung tissues and showed that when upregulated, 14-3-3 sigma, gamma, and zeta correlate with poorer prognosis in patients with lung adenocarcinoma (LUAD). Interestingly, these associations with survival were not observed in patients with lung squamous cell carcinoma (LUSC), suggesting that the upregulation of these 14-3-3 isoforms may influence patient survival and serve as suitable prognostic biomarkers. To explore the cellular consequences of 14-3-3 upregulation, our laboratory previously overexpressed 14-3-3 gamma, the isoform that demonstrated the strongest prognostic capacity in the LUAD-TCGA dataset, in human lung adenocarcinoma cells. Overexpression of this isoform caused a fraction of cells to become polyploid, meaning they contained more than two sets of chromosomes. Amassing data have demonstrated that polyploid cells are uniquely resistant to chemo- and radiotherapy, making polyploid cells integral components in driving the ongoing evolution of the patient disease and recurrence. We, therefore, became interested in illuminating the molecular mechanism(s) driving 14-3-3 gamma-induced polyploidy, the net effect these cells had on genomic integrity, and whether this phenomenon occurred in vivo. By utilizing the fluorescence ubiquitin cell cycle indicator (FUCCI) system, we were able to show that overexpression of 14-3-3 gamma resulted in inhibition of mitotic entry, forcing some cells to bypass mitosis entirely, thereby facilitating the polyploid phenotype. In pursuit of investigating whether these polyploid cells could re-enter the cell cycle and undergo cell division, we developed a widely-applicable, nontoxic procedure for measuring DNA content in live cells by fluorescence microscopy. This capacity allowed a cell’s temporal location within the cell cycle and its DNA ploidy to be coupled with a variety of imaging directed analyses. By tracking these polyploid cells over time in combination with the nuclear-reporter H2B-GFP, we were able to show that these polyploid cells are capable of entering mitosis, and when they do, they experience a prolonged and error-prone cell division. Collectively, these data demonstrated that the overexpression of 14-3-3 gamma resulted in a genetically unstable polyploid intermediate with the capacity to undergo mitosis and plausibly facilitate the transition into an aneuploid cell state. Equipped with this knowledge, we examined whether this phenomenon occurred in vivo. To do this, we turned back to the TCGA and confirmed that polyploid tumors had significantly elevated expression of 14-3-3 gamma compared to diploid tumors. This data may explain one avenue as to why elevated expression of 14-3-3 gamma correlates with more reduced survival in patients with lung adenocarcinomas. Taken together, our studies suggest that 14-3-3 gamma may play a role in tumorigenesis by inducing polyploidy, which may set the stage for further changes that lead to neoplastic progression.