Abstract
Mutation of the p53 gene is the most common genetic alteration in human malignances and associated clinically with tumor progression and metastasis. To determine the effect of mutant p53 on epithelial differentiation, we developed three-dimensional culture (3-D) of Madin-Darby canine kidney (MDCK) cells. We found that parental MDCK cells undergo a series of morphological changes and form polarized and growth-arrested cysts with hollow lumen, which resembles branching tubules in vitro. We also found that upon knockdown of endogenous wild-type p53 (p53-KD), MDCK cells still form normal cysts in 3-D culture, indicating that p53-KD alone is not sufficient to disrupt cysts formation. However, we found that ectopic expression of mutant R163H (human equivalent R175H) or R261H (human equivalent R273H) in MDCK cells leads to disruption of cyst polarity and formation of invasive aggregates, which is further compounded by knockdown of endogenous wild-type p53. Consistently, we found that expression of E-cadherin, β-catenin, and epithelial-to-mesenchymal transition (EMT) transcription factors (Snail-1, Slug and Twist) is altered by mutant p53, which is also compounded by knockdown of wild-type p53. Moreover, the expression level of c-Met, the hepatocyte growth factor receptor and a key regulator of kidney cell tubulogenesis, is enhanced by combined knockdown of endogenous wild-type p53 and ectopic expression of mutant R163H or R261H but not by each individually. Together, our data suggest that upon inactivating mutation of the p53 gene, mutant p53 acquires its gain of function by altering morphogenesis and promoting cell migration and invasion in part by upregulating EMT and c-Met.
Highlights
Wild-type p53, a tumor suppressor, plays an important role in cell-fate determination through the regulation of the cell cycle, programmed cell death, differentiation or senescence [1,2]
Consistent with that in human, p53 “hot-spot” mutations were found in canine TP53 gene, such as R163H and R261H [36]
We found that Madin-Darby canine kidney (MDCK) cells with R163H mutant exhibited spindleshaped morphology in 2-D culture, which represents the property of mesenchymal cells (Figure 1C)
Summary
Wild-type p53, a tumor suppressor, plays an important role in cell-fate determination through the regulation of the cell cycle, programmed cell death, differentiation or senescence [1,2]. Six p53 mutations are described as “hot-spot” because they are the most frequent mutations in human cancers [15]. These mutants are defective in binding to consensus wild-type p53 responsive element and defective in transactivation of wild-type p53 target genes [16]. Based on the effect on the core DNA-binding activity, these mutations can be classified into two main groups, conformational mutation and contact-site mutation. The conformational mutants, such as R175H and R249S, have an altered conformation of the core DNA-binding domain [17]. The contact-site mutants, such as R273H and R248W, have mutations at residues that directly contact target DNA [18]
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