Contribution of Different MCF10A-TP53 Mutants to Epithelial-Mesenchymal Transition and Invasiveness

High-throughput sequencing has confirmed a striking prevalence of somatic TP53 mutations in breast cancer (~35% overall), frequently associated with high-grade tumors and worse clinical outcomes. This prevalence and its role in cell cycle regulation bring significance to studying TP53 mutations. Murine and cell line models have shown that TP53 mutations lead to gain of function phenotypes, such as enhanced invasion, tumor growth and metastases. These functions cannot be considered as exclusively negative effects that lead to a null phenotype.  Unique TP53 mutations might have distinct functional effects in the hallmarks of cancer. This study focused on two assays: the transition from epithelial to mesenchymal state (EMT) and invasion. MCF10A stable cells were generated expressing the ten most frequent breast cancer mutations located in the DNA binding domain of TP53 via transduction.  These mutant cells were treated with tumor growth factor beta (TGF-β), which is known for its dual role in inducing a more mesenchymal phenotype and apoptosis. Protein expression and immunofluorescent staining showed that some TP53 mutants exhibited more mesenchymal state compared to wt-p53 even in the absence of TGF-β and mutants could be classified into 3 groups based on invasiveness: highly invasive -G245S, R248Q , R248W, moderately invasive -R248W, Y220C, Y234C, R273C ,and H179R and the less invasive -R175H and Y163C. However all mutants were more mesenchymal  in  presence of TGF-β as demonstrated by protein expression of mesenchymal markers such as SLUG and immunofluorescent staining which correlated with the invasiveness of the cells. Interestingly, all mutants in presence of TGF-β displayed disrupted epithelial markers (β-catenin and E-cadherin) by immunofluorescent staining despite the detection of the expression of the protein. This mesenchymal state of the mutants is comparable to null-p53 in the absence of TGF-β, a cell type known for its increased mesenchymal phenotype. The p53-null cells includes a mixture of epithelial and mesenchymal cells with spheroidal shape in the invasion assays. This data suggests that different TP53 mutations result in distinct cellular programs whose role is involved in breast carcinogenesis. This is seen by the p53^G245S that consistently in all the methods used to analyze EMT had a more mesenchymal phenotype compared to all the other mutants.