Genetic basis for p 53 overexpression in human breast cancer ( protein expression / gene mutation / alielic deletion )

Overexpression of an activated form of the p53 protein may be involved in neoplastic transformation. We found widespread overexpression of p53 by immunohistochemical staining in 11 (22%) of 49 primary invasive human breast cancers. Northern blot analysis showed that this overexpression was not due to an increase in the steady-state level of p53 mRNA. The p53 gene was directly sequenced in 7 of these tumors with elevated levels of the protein and, in each case, a mutation that altered the coding sequence for p53 was found in a highly conserved region of the gene. Whereas 4 of these tumors contained only a mutant p53 allele, the other 3 tumors exhibited coding sequences from both a mutant and a wild-type allele. p53 mutations have previously been correlated with allelic loss of part of chromosome 17p that contains the p53 locus. Examination of all 49 breast tumors revealed a 61% frequency of deletion at or near the p53 locus. However, the presence of allelic deletion did not correlate with overexpression of the protein. Six tumors that were deleted but did not express high levels of the protein were sequenced and all retained a wild-type p53 allele. In this series of human breast cancers, overexpression of the p53 protein, not allelic loss on chromosome 17p, was always associated with mutation of the p53 gene. The nuclear phosphoprotein p53 is expressed in normal cells and appears to be involved in the regulation of cellular proliferation (1, 2). The level of p53 protein is regulated through the cell cycle by two mechanisms: (i) p53 RNA transcription increases by 10to 20-fold late in the G1 phase (3, 4) and (ii) normal p53 protein is rapidly eliminated by virtue of its short half-life (6-30 min) (5-7). These two mechanisms serve to give the cell a short pulse of protein that is then quickly degraded. Levels of the protein, therefore, are normally low, even in actively dividing cells. Mutant p53 genes can cooperate with the ras oncogene to transform primary rodent fibroblasts (8, 9). In general, mutant forms of the p53 protein that are transforming have several properties that distinguish them from wild type. They fail to bind to the simian virus 40 tumor antigen (10) but do form stable complexes with hsc70 (11), a constitutively expressed member of the heat shock family. Perhaps as a result of this complex with hsc70, mutant p53 proteins have an increased half-life that, in part, gives rise to high steadystate levels of the protein in cells transformed with a mutant p53 gene (8). Altered p53 genes with proven in vitro transforming activity have been isolated from several sources including carcinogen-treated rodent cells (12) and primary human colorectal cancers (13). In addition, a variety of point and deletion mutations have been described for the p53 gene in primary tumors and cell lines derived from tumors (14). Although most ofthese mutations have not been tested for transforming activity, they almost invariably alter amino acid residues that have been highly conserved during evolution (15), suggesting that they are oncogenic mutations. Several lines of evidence suggest that p53 may act as a recessive oncogene or tumor suppressor, much like the retinoblastoma gene. (i) Mutations that activate the oncogenic potential of p53 have been found over a large region of the protein, suggesting that these mutations serve to eliminate a normal function ofthe protein (8, 14). (ii) The wild-type gene is capable of suppressing transformation of rodent cells by mutant p53 plus ras (16, 17). (iii) Mutant p53 protein is capable of forming oligomeric complexes with wild-type p53 (12, 18). This binding may inactivate the function of the normal protein and could explain how mutant p53 is able to transform cells in the presence of wild-type protein. (iv) The human p53 gene is located on the short arm of chromosome 17 in a region very frequently deleted in many different human tumors (19). Allelic loss on 17p is often accompanied by a mutation in the remaining copy of the p53 gene, thus eliminating all wild-type p53 in those cells. Nigro et al. (14) have proposed that mutation occurs first during tumor progression and promotes cell growth by a dominant negative effect on the wild-type protein. These mutations are then followed by deletion of the wild-type gene, leading to a further loss of growth control. The extremely high frequency (60-70%) of allelic deletion on 17p in many types of cancer would, therefore, predict an equally high frequency of p53 mutation, as appears to be true in colorectal cancer (14). We have examined primary human breast cancer to assess the generality of this hypothesis. Greater than 60% of breast cancers have been shown to be allelically deleted for sequences on the distal end of 17p (20, 21). In addition, a separate study has found that 45% ofbreast cancers overexpress the p53 protein (22). Since p53 mutations that are oncogenic also increase the stability of the protein, tumors that have elevated steady-state levels of the protein likely harbor activating mutations in the gene. Therefore, it appeared that p53 activation in breast cancer would follow the same pattern as colorectal cancer. We report, however, that deletion on 17p is not a good predictor of mutation but that widespread overexpression of the p53 protein does indicate the presence of a mutation in breast