Plakophilin3 downregulation leads to a decrease in cell adhesion and promotes metastasis

Abstract

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...

Structural and Functional Diversity of Desmosomes

“…In agreement with the murine data, analysis of samples isolated from patients with mutant plakoglobin or PKP1 revealed absent or severely compromised epidermal desmosomes, incapable of completing a connection to intermediate fi laments (Pigors et al, 2011;McGrath et al, 1997). Junctions between epithelial cells expressing PKP3 shRNA, likewise, failed to maintain strong intercellular adhesion when challenged mechanically in vitro (Kundu et al, 2008). Ruptured cardiac walls in PKP2-null mice speak to the importance of plakophilins in maintaining cellular adhesion in vivo (Grossmann et al, 2004).…”

14-3-3 gamma is required to enforce both the incomplete S phase and G2 DNA damage checkpoints

“…HCT116 cells were obtained from the lab of Dr. Bert Vogelstein 35 and cultured as described. 36 HCT116 cells were transfected with pTU6II(a) based or pEGFPf based constructs by calcium phosphate precipitation method. 37 To determine the efficiency of shRNA constructs, HCT116 cells were co-transfected with 2 μg of pEGFPf based shRNA constructs and 0.5 μg of constructs expressing HA-tagged 14-3-3γ followed by Western blots with antibody to the HA epitope.…”

Desmosome assembly, homeostasis, and desmosomal disease

“…Reduced expression of desmosomal cadherins, which may be responsible for the weakening of cell-cell adhesion observed in cancer cells, was observed in different tumors, such as skin, 151 head and neck, 152 lung, 153 breast, 154 and a variety of other epithelial malignancies, 155 including gastric 156 and colon 157 cancers. In one example, knockdown of Pkp3 in colon cancer cells promoted anchorage-independent growth and tumor growth in immunocompromised mice.…”