Abstract
Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker β-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, β-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.
Highlights
Mutant p53 is deficient in tumor suppression and acquires additional activity, called gain of function
Mutant p53 Alters Normal MCF-10A Cell Polarity in Threedimensional Culture via epithelial-to-mesenchymal transitions (EMT)—Because the normal expression patterns for E-cadherin, -catenin, and laminin V in MCF-10A three-dimensional culture were altered by mutant p53, we examined whether the expression levels of these proteins are altered by mutant p53
We found that Snail-1 and Slug were increased by ectopic expression of G245S, R175H, R248W, and R273H regardless of p53 knockdown (p53-KD) (Fig. 7E, compare lane 1 with lanes 2–9, respectively)
Summary
Reagents—Growth factor-reduced Matrigel (BD Biosciences) was used for the three-dimensional culture. The resulting p53-KD cell lines were selected with puromycin, and p53-KD was confirmed by Western blot analysis. To generate stable p53-KD cell lines with mutant p53 overexpression, pBabe-U6-siRNA was co-transfected with pcDNA3-mutant p53 into MCF-10A cells. The resulting cell lines were selected with puromycin and G418 Both p53-KD and mutant p53 expression were confirmed by Western blot analysis. The medium for MCF-10A cells is composed of DMEM/ F12 supplemented with 5% donor horse serum, 20 ng/ml EGF, 10 g/ml insulin, 0.5 g/ml hydrocortisone, and 100 ng/ml cholera toxin. Confocal Microscopy—The three-dimensional structures in Matrigel were fixed in 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.5% Triton X-100 in PBS for 30 min at 4 °C.
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