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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. A 14-3-3γ knockdown clone also showed an override of both checkpoint pathways. These phenotypes were reversed upon expression of a shRNA resistant 14-3-3γ cDNA. Override of the G 2 DNA damage checkpoint pathway was accompanied by a decrease in the levels of inhibitory phosphorylation on cdc25C and cdk1. However, there was no difference in the γ-H2AX foci formation and levels of phospho-chk1 and phospho-chk2, suggesting that activation of the DNA damage checkpoint response and subsequent activation of the checkpoint kinases Chk1 and Chk2 was not perturbed. These results suggest that the override of checkpoint observed in 14-3-3γ knockdown cells is due to failure to inhibit cdc25C function.

The regulation of cell-cell adhesion is important for the processes of tissue formation and morphogenesis. Here we report that loss of 14-3-3γ leads to a decrease in cell-cell adhesion and a defect in the transport of plakoglobin (PG) and other desmosomal proteins to the cell border in HCT116 cells and in the mouse testis. 14-3-3γ binds to PG in a PKCμ dependent fashion resulting in microtubule dependent transport of PG to the border. Transport of PG to the border is dependent on the KIF5B/KLC1 complex. Knockdown of KIF5B in HCT116 cells or in the mouse testis, results in a phenotype similar to that observed with 14-3-3γ knockdown. Our results suggest that loss of 14-3-3γ leads to decreased desmosome formation and a decrease in cell-cell adhesion in vitro and in vivo in the mouse testis leading to defects in testis organization and spermatogenesis.

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T-type Ca2+ channels are a group of low voltage-gated calcium channels which seem to be crucial for embryonic or stem cell proliferation and differentiation; however, they could be also aberrantly expressed in several human tumors. Since calcium entry is required for smooth transition through the cell cycle, it has been hypothesized that the inhibition of T-type Ca2+ channels blocks cells in G0/G1 phase of cell cycle, while a release from inhibition leads to increased number of cells entering cell cycle and thus increased susceptibility to conventional antitumor therapy. In this study we investigated the effects of T-type channel inactivation, or downregulation, on cell cycle progression, cell death and survival, and resistance to radiotherapy (RT). The experiments were conducted using several cancer cell lines expressing T-type Ca2+ channels (glioblastoma, colon and prostate) and selective and structurally unrelated calcium channels antagonists, including Mibefradil, a drug that is currently undergoing clinical trial for its antitumor properties, and is planned to be tested as radiosensitizing agent in recurrent gliobastoma tumors. The effects of T-type Ca2+ channel inhibition on cell cycle progression, cell death and the expression/activation of cell cycle regulated proteins were assessed. Finally, the combination treatments including T-type channel antagonist and radiotherapy were assayed. We show that T-type Ca2+ channels are important for cell cycle progression and resistance to RT, and that their inhibition leads to enhanced cell death and reduced survival. Our study demonstrates that one of the earliest events evoked by T-type channel inhibition is a deregulation of pro-survival pathway PI3K/Akt/mTOR and activation of pro-apoptotic stress-activated p38MAPK pathway. The results support the idea of T-type Ca2+ channel as a novel molecular target for antitumor therapy and provide a rational and plausible biological mechanism responsible for the effects induced by T-type Ca2+ channel antagonists in cancer cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A239. Citation Format: Barbara Dziegielewska, Nicholas C.K. Valerie, Amol S. Hosing, James M. Larner, David L. Brautigan, Lloyd S. Gray, Jaroslaw Dziegielewski. T-type calcium channels as a novel molecular target for tumor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A239.