Plakophilin3 is a desmosomal plaque protein whose levels are reduced in poorly differentiated tumors of the oropharyngeal cavity and in invasive colon carcinomas. To test the hypothesis that plakophilin3 loss stimulates neoplastic progression, plakophilin3 expression was inhibited by DNA vector driven RNA interference in 3 epithelial cell lines, HCT116, HaCaT and fetal buccal mucosa. The plakophilin3-knockdown clones showed a decrease in cell-cell adhesion as assessed in a hanging drop assay, which was accompanied by an increase in cell migration. The HCT116 plakophilin3-knockdown clones showed a decrease in desmosome size as revealed by electron microscopy. These altered desmosomal properties were accompanied by colony formation in soft agar and growth to high density in culture. The HCT116-derived clones showed accelerated tumor formation in nude mice and increased metastasis to the lung, a phenotype consistent with the increased migration observed in vitro and is consistent with data from human tumors that suggests that plakophililn3 is lost in invasive and metastatic tumors. These data indicate that plakophilin3 loss leads to a decrease in cell-cell adhesion leading to the stimulation of neoplastic progression and metastasis. Plakophilins are desmosmal plaque proteins, which belong to the p120ctn subfamily of Armadillo repeat containing proteins (reviewed in Refs. 4 and 5). Unlike plakophilins 1 and 2, plakophilin3 is ubiquitously present in a wide range of epithelial cells and tissues with the exception of hepatocytes 6,7 and forms a complex with a number of desmosomal proteins. Plakophilin3 binds to the desmosomal cadherins desmoglein 1-3 and desmocollins 1 and 3, cytokeratin 18 and other desmosomal plaque proteins such as desmoplakin and plakoglobin.8 Therefore, plakophilin3 has been postulated to play a crucial role in the function of desmosomes and maintenance of the desmosomal structure.8 Immunofluorescence analysis followed by confocal microscopy has shown that in addition to being present at desmosomal plaques in epithelial tissues and in cell lines of epithelial origin, 6-8 plakophilin3 is also found in cytoplasmic stress granules in complex with RNA-binding proteins. 9 Furthermore, in epithelial cell lines, a speckled nuclear pattern of staining was also detected with antibodies to plakophilin3.6,7 Although plakophilin3 knockout mice are viable, they display severe defects in desmosome assembly in the basal membrane of the epidermis. The epidermis of the knockout mice show hyperplasia, and the mice are extremely susceptible to skin infections and inflammation. 10 These results suggest that in addition to regulating desmosome function and organization, plakophilin3 may play a role in integrating extra cellular signals with events occurring inside the cell.A number of reports have suggested that alterations in desmosome structure or composition could lead to neoplastic progression (reviewed in Ref. 11). To determine if plakophilin3 is required for desmosomal assembly and plays a role in inhibiting ep...

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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.

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