Genetic alterations of the p14ARF -hdm2-p53 regulatory pathway in breast carcinoma.

TP53 is the most commonly mutated tumor suppressor gene in human cancers. The amplification and overexpression of HDM2 plays a role in tumorigenesis via inactivation of p53-dependent cell cycle arrest. p14ARF, an alternate transcript of the INK4A tumor suppressor locus, prevents hdm2-induced transcriptional silencing of p53 by binding hdm2. The role of this p14ARF-hdm2-p53 regulatory pathway in breast carcinoma is unknown. We hypothesized that p14ARF mutations and HDM2 gene amplification may be alternative mechanisms of p53 inactivation in breast cancer. Mutational analysis of TP53 (exons 5-9) and exon 1beta of pl4ARF was performed by PCR-SSCP and putative mutations were confirmed by sequencing. p14ARF mRNA expression was evaluated by RT-PCR and the presence of HDM2 gene amplification by differential PCR. Among the cell lines, 7/14 (50%) harbored TP53 mutations and 2/14 (14%) had a deletion ofp14ARF exon 1beta with no detectable p14ARF mRNA. None demonstrated HDM2 gene amplification. TP53 mutations were identified in 7/36 (19%) breast tumors and HDM2 amplification in 2/30 (7%) tumors. All the tumors contained an intact p14ARF exon 1beta with corresponding expression of the mRNA. Alterations in the various components of this regulatory pathway were identified in nine (64%) cell lines and 25% of the 36 breast cancers with TP53 mutation being the predominant aberration. Although p14ARF mutations and HDM2 gene amplification appear to be uncommon events in breast carcinoma, deregulation of this pathway may occur via alternative mechanisms in breast carcinogenesis.

Molecular analysis of the INK4A and INK4B gene loci in human breast cancer cell lines and primary carcinomas.

The INK4A and INK4B loci are located at 9p21 and have been implicated in the tumorigenesis of various human malignancies. The INK4A gene encodes two cell cycle regulators, p16(INK4A) and ARF, while INK4B encodes p15(INK4B). Previously, we have shown that the p16(INK4) tumor suppressor was not mutated or deleted in primary breast carcinomas. However, primary and metastatic breast carcinomas exhibited a relative hypomethylation of p16(INK4A), which is associated with expression, compared to normal breast tissue. The present study was conducted to determine if inactivation of p15(INK4B) and INK4A exon 1beta (ARF) are common events in breast carcinoma. Mutational analysis was performed by PCR-SSCP, and mRNA expression was evaluated by RT-PCR. Methylation-specific PCR was used to determine the methylation status of the p15(INK4B) promoter. Our results demonstrate that the p15(INK4B) gene was altered in 3 (21%) of the 14 breast cell lines; one had a silent mutation and two had homozygous deletion of the gene. None of the cell lines showed methylation of p15(INK4B). Two (14%) cell lines had homozygous deletion of INK4A exon 1beta. All normal and malignant breast tissue samples were wild-type and non-methylated for p15(INK4B) and wild-type for exon 1beta. Our results show that these structurally and functionally related genes are not invariably affected together, and the most frequently observed alteration at the INK4A and INK4B loci in breast carcinoma appears to be p16(INK4A) hypomethylation.

Expression of E2F-1 and E2F-4 is reduced in primary and metastatic breast carcinomas.

The E2F family of transcription factors can induce both cell proliferation and apoptosis. Whether they function as oncogenes or tumor suppressors appears to be tissue specific. Their role in breast carcinogenesis remains unclear. We found a decreased expression of E2F-1 and E2F-4 in 70% (7/10) of primary breast carcinomas and in all (10/10) metastatic nodal tissues when compared with the corresponding normal breast tissue. No tumor-specific mutation was detected, but polymorphisms were identified in E2F-1 exon 5 and in the polyserine tract of E2F-4. The presence of polymorphisms did not correlate with E2F expression. Among the 12 human breast cancer cell lines, one contained a missense mutation in E2F-1 exon 2. Five (42%) cell lines overexpressed E2F-1, while three (25%) expressed low levels of the protein. Our results suggest that not only are the E2Fs likely to function as tumor suppressors in breast cancer, but also that their down-regulation may be important in the development of metastases.

In microdissected ductal carcinoma in situ, HER-2/neu amplification, but not p53 mutation, is associated with high nuclear grade and comedo histology.

BACKGROUND: HER-2/neu and p53 are two molecular markers that have been the focus of investigation in patients with invasive breast carcinoma. However, most of the published data have relied on immunohistochemical detection of the proteins as a surrogate marker of the underlying genetic alterations, a detection method that often gives variable results due to technical factors. In addition, there are limited data documenting HER-2/neu amplification and p53 mutations in the various histologic subtypes of ductal carcinoma in situ (DCIS). The authors evaluated a series of microdissected, pure DCIS lesions comprising a spectrum of morphologic subtypes (comedo, micropapillary, papillary, cribriform, and solid) and their corresponding normal breast tissue for genetic aberrations in HER-2/neu and p53. METHODS: HER-2/neu amplification was determined by differential polymerase chain reaction, and p53 mutations were identified by single-strand conformation polymorphism analysis. RESULTS: HER-2/neu amplification was identified in 12 of 30 DCIS samples (40%), and p53 mutations were identified in 6 of 30 DCIS samples (20%). The genetic alterations were not present in any of the normal breast tissue samples. HER-2/neu amplification occurred predominantly in the comedo subtype (69% vs. 18% of the noncomedo subtype; P = 0.008) and in lesions of high nuclear grade (63% vs. 14% of low grade; P = 0.01). There was no difference in the frequency of p53 mutations among the subtypes or between low grade and high grade lesions. No correlation between the presence of the two genetic alterations was observed. CONCLUSIONS: The presence of HER-2/neu amplification, but not p53 mutations, correlates with histologic subtype and nuclear grade. The relatively frequent occurrence of HER-2/neu amplification and p53 mutations in DCIS tissue and their absence in normal breast tissue suggest that these genetic aberrations are important early in breast duct carcinogenesis.

Hypomethylation and increased gene expression of p16INK4a in primary and metastatic breast carcinoma as compared to normal breast tissue.

Controversy continues to surround the role of p16INK4a in cell cycle control and carcinogenesis. Mutations, deletions and changes in methylation patterns of p16INK4a have been proposed as mechanisms leading to abnormal expression of the gene. We show here that primary and metastatic breast carcinomas demonstrate hypomethylation of p16INK4a which is associated with expression of p16INK4a mRNA, as compared to normal breast tissue which demonstrates a relative hypermethylation of p16INK4a associated with the absence of p16INK4a expression. These data suggest that methylation and lack of expression of p16INK4a is not a central mechanism in the development of breast carcinoma, but rather that the gene is functioning and expressed in breast carcinoma more frequently than in normal breast tissue. The role of p16INK4a is much more complex than has been previously hypothesized.

Absence of p16 gene (CDKN2) deletions in microdissected primary breast carcinoma specimens.

BACKGROUND: The p16 gene (CDKN2), a tumor suppressor gene located on chromosome 9p21, has been demonstrated to be mutated or deleted with high frequency in a variety of tumor cell lines, including breast. While previous studies have not demonstrated CDKN2 mutations in primary breast carcinomas, it is possible that gene deletion in neoplastic DNA was marked by the presence of contaminating normal stromal DNA in breast carcinoma specimens. METHODS: We investigated the incidence of homozygous deletion of CDKN2 by analyzing 20 microdissected pure populations of primary breast carcinoma cells. Using polymerase chain reaction (PCR) techniques, the entire coding region and intervening introns of CDKN2 were amplified. The PCR products were resolved by agarose gel electrophoresis and single-strand conformation polymorphism (SSCP) analysis. RESULTS: We detected no deletions or mutations of the p16 gene. CONCLUSIONS: CDKN2 is not deleted with high frequency in primary breast carcinomas, and the p16 gene does not play a role in breast carcinogenesis via this mechanism.

Microsatellite instability in breast cancer.

BACKGROUND: Microsatellites are short repetitive nucleotide sequences that, through mutation, can undergo either expansion or contraction. This novel mutational mechanism known as microsatellite instability may play a role in carcinogenesis. We investigated the incidence of microsatellite instability in a series of primary breast carcinoma surgical specimens. METHODS: Using polymerase chain reaction techniques followed by polyacrylamide/urea gel electrophoresis, we analyzed 46 pairs of normal and primary breast tumor samples at seven different microsatellite loci, five of which were located on chromosome 17. RESULTS: Thirteen of our 46 tumors (28.2%) demonstrated microsatellite instability. Five tumors (10.8%) were unstable at two or more loci, and of those, four (8.7%) were unstable at different loci on different chromosomes. An additional five tumors demonstrated loss of heterozygosity alone when compared with their normal counterparts. CONCLUSIONS: These findings indicate that microsatellite instability is present in primary breast cancer populations and, although the mechanism of action has yet to be elucidated, may play a role in breast carcinogenesis.