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...

Lipocalin 2 is a siderophore‐binding protein that regulates iron homeostasis. Lipocalin 2 expression is elevated in multiple tumor types; however, the mechanisms that drive tumor progression upon Lipocalin 2 expression remain unclear. When Lipocalin 2 is over‐expressed, it leads to resistance to 5‐fluorouracil in colon cancer cell lines in vitro and in vivo by inhibiting ferroptosis. Lipocalin 2 inhibits ferroptosis by decreasing intracellular iron levels and stimulating the expression of glutathione peroxidase4 and a component of the cysteine glutamate antiporter, xCT. The increase in xCT levels is dependent on increased levels of ETS1 in Lipocalin 2 over‐expressing cells. Inhibiting Lipocalin 2 function with a monoclonal antibody leads to a decrease in chemo‐resistance and transformation in vitro, and a decrease in tumor progression and chemo‐resistance in xenograft mouse models. Lipocalin 2 and xCT levels exhibit a positive correlation in human tumor samples suggesting that the pathway we have identified in cell lines is operative in human tumor samples. These results indicate that Lipocalin 2 is a potential therapeutic target and that the monoclonal antibody described in our study can serve as the basis for a potential therapeutic in patients who do not respond to chemotherapy.

A decrease in the levels of the desmosomal plaque protein, plakophilin3 (PKP3), leads to a decrease in desmosome size and cell-cell adhesion. To test the hypothesis that PKP3 is required for desmosome formation, the recruitment of desmosomal components to the cell surface was studied in the PKP3 knockdown clones. The PKP3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared to vector controls, and did not form desmosomes in a calcium switch assay. Further analysis demonstrated that PKP3, plakoglobin (PG) and E-cadherin are present at the cell border at low concentrations of calcium. Loss of either PG or E-cadherin led to a decrease in the levels of PKP3 and other desmosomal proteins at the cell border. The results reported here are consistent with the model that PG and E-cadherin recruit PKP3 to the cell border to initiate desmosome formation.