The Regulation of p53 Activity in Breast Cancer

Institution: University of California, San Diego
Investigator(s): Yang Xu, Ph.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5JB-0056 Award: $51,393
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (1999)
Hereditary breast cancers account for about 5-10% of all breast cancers and a large percentage of them are early-onset breast cancers. Mutations of a number of tumor suppressor genes, including BRCA1, BRCA2, p53 and PTEN/MMAC1, are associated with the hereditary breast cancers. Recent studies have suggested that p53 mutations are necessary for tumor formation in BRCA-deficient tumors. In addition, p53, the most commonly mutated tumor suppressor in various human cancers, is also mutated at a high frequency in non-hereditary (sporadic) breast cancers. Therefore, understanding the regulation of p53 function in tumor suppression is essential to design new therapeutic strategies to treat this disease.

It appears that p53 can have two roles in tumor suppression, leading to either cell-cycle arrest or apoptosis (programmed cell death). Both p53-dependent functions appear to be involved in maintaining the integrity of the genome, such that excess DNA mutations leading to cancer will not accumulate. Normally p53 is present in an inactive form and at very low levels. But, with DNA damages (e.g., radiation or chemotherapy), p53 activity is induced. Our studies are aimed to employ genetic approaches to address the involvement of phosphorylation, which is the addition of phosphate groups to specific Ser/Thr sites in the protein structure, in p53 activation for both gene regulation and apoptosis functions.

We will employ new genetic approaches to introduce mutations of various p53 phosphorylation sites and use embryonic stem cells to study the effects of these changes. An important advantage of this assay is that the mutant p53 is expressed under the control of its own gene promoter. In addition, the mutant embryonic stem cells can differentiate into other primary cell types that we can use to address the question of apoptosis. In conclusion, these mutant embryonic stem cells will provide an ideal physiological model system to study the effects of these p53 mutations on the p53 responses to various DNA damages.

Our eventual goal is to screen breast cancer cells for not only the p53 mutations but also the defects in these signaling pathways involved in the p53 phosphorylation. But first, we need the results from this study to determine which phosphylation sites appear linked to events critical to cell cycle arrest and apoptosis. When these issues become resolved we will be in a better position to deal with problem of failed therapies for breast cancer.


Final Report (2000)
Note: This award was resigned after four months, so the PI could accept other funding.

Recent studies have suggested that Ser20 of p53 is phosphorylated following ionizing radiation and this phosphorylation might be involved in the stabilization of p53. To determine the importance of this phosphorylation event in the p53 response to DNA damage as proposed in the California BCRP grant, we have constructed the knock-in vector and introduced it into ES cells to mutate ser20 of the endogenous p53 into Ala in embryonic stem cells. We have generated homozygous mutant ES cells. We are in the process of deleting the PGK-neor gene from the mutant alleles so that the PGK-neor-deleted ES cells will express a p53 with the ser20 mutated to ala.

Ser15 is phosphorylated by ATM family kinases and might play a role in p53 responses to genotoxic stresses. We have introduced the ser15 to ala missense mutation into both alleles of the endogenous p53 gene of ES cells, therefore, these ES cells will express a p53 identical to the germline one except that ser15 is mutated to ala and thus it cannot be phosphorylated. We analyzed the p53 responses to DNA damage induced by ionizing radiation or W in these mutant ES cells and these studies provide compelling evidence that phosphorylation of p53 at serl5 is required for efficient p53 upregulation following DNA damage. While the generation of the mutant ES cells preceded the funding of CA BCRP grant, the analysis of the p53 responses to DNA damage was funded by CA BCRP grant.