P53 Silencing and Mammosphere Formation in Breast Cancer Cells Harbouring P53 Gain-of-Function Mutations Trigger Epithelial-Mesenchymal Transition

Background: Mutations in p53 gene are observed in ∼50% of all human cancers. In breast cancer alone, 12%-32% of luminal, 84% of basal-like and 75% of HER-2 expressing tumors have apparent p53 mutations. Tumor cells undergo epithelial-mesenchymal transition (EMT) to metastasise from primary sites to form secondary tumors at distant regions of the body. EMT is a complex biologic phenomenon which governs the transition of cancer cells with epithelial characteristics to mesenchymal traits, gaining new properties such as aggressiveness and invasiveness. Recent studies revealed that mutations in the p53 gene can give rise to alternate functional phenotypes leading to tumor initiation and progression. Aim: The aim of this study is to develop a robust human breast cancer cellular model to investigate p53 gain-of-function (GOF) mutations and EMT as well as evaluating the EMT phenotype associated with these mutations. Methods: Two breast cancer cell lines, namely MDA-MB-468 and HCC38 carrying the R273H and R273L missense mutations, respectively, were subjected to p53 knockdown using shRNA directed against p53 gene through lentiviral vector transduction. The transduced cells were then harvested for Western blotting to evaluate the protein expression of EMT markers which includes E-cadherin, SNAIL, ZEB1 and vimentin compared with the nontransduced control cells. Subsequently, both cell lines were subjected to mammosphere generation and redifferentiation to determine the basal expression of the EMT markers. Results: Silencing of p53 using siRNA in MDA-MB-468 and HCC38 downregulated E-cadherin expressions but upregulated vimentin levels. Furthermore, E-cadherin levels reduced significantly after conversion from adherent parental cells to mammospheres, but rebound upon redifferentiation. Conversely, vimentin was upregulated in mammospheres as compared with the parental and redifferentiated groups. Conclusion: p53 knockdown in breast cancer cells harboring R273H and R273L mutations favor vimentin expression but not E-cadherin, suggesting that p53-GOF mutants are involved in EMT and the development of metastatic tumors. The mammosphere model accurately recapitulates cell plasticity between epithelial and mesenchymal states, as evidenced by the expression of mesenchymal cell markers in the mammospheres, and epithelial cell markers in adherent and redifferentiated cells.