Title: Gain-of-Function Effects of Mutant p53 Explored Using a Three-Dimensional Culture Model of Breast Cancer
Description: p53 is the most frequent target for mutation in human tumors and mutation at this locus is a common and early event in breast carcinogenesis. Breast tumors with mutated p53 often contain abundant levels of this mutant protein, which has been postulated to actively contribute to tumorigenesis by acquiring pro-oncogenic ("gain-of-function") properties. To elucidate how mutant p53 might contribute to mammary carcinogenesis, we employed a three-dimensional (3D) culture model of breast cancer. When placed in a laminin-rich extracellular matrix, non-malignant mammary epithelial cells form structures highly reminiscent for many aspects of acinar structures found in vivo. On the other hand, breast cancer cells, when placed in the same environment, form highly disorganized and sometimes invasive structures. Modulation of critical oncogenic signaling pathways has been shown to phenotypically revert breast cancer cells to a more acinar-like morphology. We examined the role of mutant p53 in this context by generating stable, regulatable p53 shRNA derivatives of mammary carcinoma cell lines to deplete endogenous mutant p53.

We demonstrated that, depending on the cellular context, mutant p53 depletion is sufficient to significantly reduce invasion or in some cases actually induce a phenotypic reversion to more acinar-like structures in breast cancer cells grown in 3D culture. Additionally, using stable overexpression of a panel of tumor-derived p53 mutants in non-malignant mammary epithelial cells, we found that mutant p53 is sufficient to disrupt normal acinar morphogenesis. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates revealed that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on mammary tissue architecture. We then showed that mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed mevalonate pathway genes in human breast tumors and elevated expression of the mevalonate pathway correlates with a poor prognosis in breast cancer.

We also queried a number of pathways/proteins that had previously been implicated in breast cancer and shown to be sufficient to bring about a phenotypic reversion in 3D culture to search for additional mechanisms by which mutant p53 might contribute to mammary carcinogenesis. Using this approach, we identified integrin β4 as a novel target of mutant p53 in breast cancer cells and demonstrated that stable knockdown of integrin β4 is sufficient to dramatically reduce invasive processes in breast cancer cells grown in 3D culture. We also show that mutant p53 associates with the promoter of ITGB4, the gene encoding integrin β4. Finally, we demonstrated that inhibition of NF-κB, a downstream mediator of integrin β 4 signaling, can mimic the phenotypic effects of mutant p53 depletion. These findings contribute to our understanding of breast carcinogenesis and may offer novel prognostic indicators and therapeutic targets for tumors bearing mutations in p53.