MUTATIONS IN P53 ARE FREQUENT IN THE PRENEOPLASTIC STAGE OF MOUSE MAMMARY-TUMOR DEVELOPMENT

Preneoplastic lesions in the mammary gland represent a population of c ells at increased risk of progression to tumors. Because p53 is the mo st commonly mutated gene in human breast cancer, we sought to determin e whether mutations in p53 were present in preneoplastic lesions or we re acquired during progression to overt tumors. In the mouse mammary g land, hyperplastic alveolar nodules (HAN) are the most common preneopl astic lesion. Analysis of the TM series of transplantable murine HAN o utgrowths and tumors allowed the status of p53 to be determined at dis tinct stages of mammary tumorigenesis. Alterations in the p53 gene or in the pattern of p53 protein expression were observed in all five HAN outgrowth lines examined. Altered expression of p53 protein was detec ted in 3 of 5 TM HAN outgrowth lines as determined by immunohistochemi stry. Overexpression of nuclear p53 was detected in only a fraction of the cells (10-50%) in TM3 and TM4 HAN outgrowths, whereas in tumors t hat arose from TM4 HAN outgrowths, the proportion of cells overexpress ing p53 protein approached approximately 100%. Despite overexpression of p53 in TM3 HAN outgrowths, no tumors have developed in this line. T he TM9 outgrowth line exhibited a different pattern of p53 expression by immunohistochemistry: p53 protein was overexpressed in the cytoplas m of virtually all cells in the HAN outgrowths but was localized to th e nuclei of TM9 tumor cells. Direct sequencing of p53 transcripts from tumors and cell lines revealed various genetic changes: point mutatio ns in exons 4 and 5 (TM2H, nonsense; TM4, missense); a deletion in exo n 5 (TM4); and an insertion in exon 7 (TM3). Although p53 protein was overexpressed in TM9 tumors, it was shown to be wild-type both by immu noprecipitation and direct sequencing of the entire coding region of t he cDNA. TM4 cells were homogeneous with respect to mutant p53 genotyp e and uniformly expressed p53 by immunohistochemical staining in vitro , but transplantation of TM4 cells to fat pads of BALB/c hosts resulte d in HAN outgrowths in situ in which <50% of the cells expressed the m utant p53 at detectable levels. In summary, mutation of the p53 gene a nd overexpression of p53 protein can occur in preneoplastic mammary ep ithelial cells, and those mutations are maintained in tumors that aris e from the HAN. It appears that mutation of p53 conferred a biological ly relevant growth advantage to cells in vivo. Overexpression of p53 i n one outgrowth line in the absence of mutations in the coding sequenc e suggests the presence of cellular factors that can enhance accumulat ion of wild-type p53 protein. Conversely, expression of mutant p53 was decreased when cells were grown in situ, implicating the presence of cellular factors that can suppress p53 expression in vivo. These obser vations demonstrate that the p53 pathway may be a common target for mu tation in murine mammary tumorigenesis.