14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

Amitabha Mukhopadhyay,Arunabha Bose,Rahul Thorat,Anushree Gulvady,Tapas K Kundu,Renu Balyan,Lalit Sehgal,Amol S Hosing,Sorab N Dalal,Srikanta Basu,Lalit Borde,Parijat Senapati,Khyati Bhatt

doi:10.1038/srep26580

Abstract

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

Highlights

The centrosome is the major microtubule nucleating and organizing center in mammalian cells, consisting of two cylindrical centrioles, surrounded by multi-layered toroid of pericentriolar matrix (PCM)[1,2]
In addition to the loss of checkpoint control, an increase in mitotic index was observed in 14-3-3γ-knockdown cells (Supplementary Fig. S1a,b), a phenotype often associated with centrosome amplification[32,33]
Expression of an shRNA-resistant 14-3-3γconstruct resulted in a reversal of centrosome amplification in the 14-3-3γ-knockdown cells (Supplementary Fig. S1d–f), suggesting that centrosome amplification was solely due to loss of 14-3-3γ

Summary

Introduction

The centrosome is the major microtubule nucleating and organizing center in mammalian cells, consisting of two cylindrical centrioles, surrounded by multi-layered toroid of pericentriolar matrix (PCM)[1,2]. The expression of a 14-3-3-binding-deficient mutant of cdc25C (S216A) in 14-3-3γ-knockdown cells, at high passage, led to an extensive increase in spindle multi-polarity, a decrease in centrosome clustering, a decrease in cell survival and a reversal of tumor formation in nude mice. These results suggest that 14-3-3γ-mediated premature activation of the mitotic program during interphase results in an induction in spindle multi-polarity, a decrease in centrosome clustering and an inhibition of tumor formation

Methods

Results

Conclusion