Amplification of human int-2 in breast cancers and squamous carcinomas

Murine int-2 is one of the genetic loci implicated in the induction of mouse breast cancers by murine mammary tumor virus. An homologous gene has recently been identified in the human genome (Casey et al., 1986). The human int-2 locus was found to be amplified 7- to 25-fold in 4 of 46 infiltrating ductal breast cancers and 30- to 60-fold in 2 of 8 squamous carcinomas of the head and neck region, but not in other cancers. All of the involved tumors had metastasized to regional lymphatics at the time of analysis and five exhibited clinically aggressive behavior.     

Epigenetic silencing of maspin gene expression in human breast cancers

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer.     

Elastin gene expression in elastotic human breast cancers and epithelial cell lines

Elastosis is a prominent feature of the desmoplastic reaction in many invasive breast cancers. It is widely held that the elastic tissue is produced by fibroblastic cells of the breast stroma, but several studies have suggested that it derives from breast cancer epithelium. In studies directed to examining the mechanisms regulating desmoplasia in breast cancers, cell lines of human breast cancer derivation have been shown to synthesize immunoreactive tropoelastin in cell culture. Stromal fibroblasts, grown out from breast cancers, produced as much elastin as did nuchal ligament fibroblasts at similar passages. The human breast cancer cell lines, grown under similar conditions, produced elastin in culture at rates equivalent to 1.6-15% of those of the control fibroblastic cells. These included two estrogen receptor positive and one estrogen receptor negative cell types. Northern blot analysis of total RNA showed the presence, under high stringency conditions, of a 3.5-kilobase elastin mRNA band in both the fibroblastic cells and the cancer cell lines. In situ hybridization, with an elastin complementary RNA probe (prepared from a short segment of the translated region of human elastin mRNA), has been carried out on a selection of 21 invasive ductal breast cancers and 9 normal breast samples. It has been found that, while fibroblastic cells of the stroma and of the periductal region are responsible for elastin synthesis in most breast cancers, the malignant epithelium is a source of the elastin in the desmoplastic tissue of a significant proportion of such neoplasms. Vascular endothelium also expresses the elastin gene in some breast cancers. The elastotic elastin may have different cellular origins in different portions of a single ductal breast cancer. The results indicate that elastosis in breast cancers is very likely to be a complex process with multifactorial regulatory mechanisms. Subclassifying cancers according to the cellular source of the desmoplastic elastin, on the basis of in situ hybridization of elastin mRNA, may provide insights into the prognostic significance of elastosis in breast cancers.     

Identification and characterization of retinoblastoma gene mutations disturbing apoptosis in human breast cancers

Background  

The tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro - versus anti - apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer.     

Amplification in human breast cancer of a gene encoding a c-myc mRNA-binding protein

The coding region determinant-binding protein (CRD-BP) binds in vitro to c-myc mRNA and is thought to stabilize the mRNA and increase c-Myc protein abundance. The CRD-BP gene has 15 exons and 14 introns, is single-copy, and is located on chromosome 11 in mice and 17 in humans, close to HER-2/neu. The CRD-BP gene is moderately amplified in 12 of 40 human breast cancers; it is highly amplified in 2 others (14.4 and 20 copies). Despite their proximity, CRD-BP and HER-2/neu genes can be amplified independently. Amplification of a gene that might up-regulate c-Myc abundance could accelerate breast cancer.     

Mutations in the p53 gene in primary human breast cancers.

Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.     

Histoculture of human breast cancers

Breast cancers from 53 patients were explanted in culture, and 39 with two-year or more follow-up and documentable tissue forms of cancer in culture (histoculture) are the subject of this report. Clinicopathologic findings have been correlated with dynamic aspects of the cancers in short-term histocultures, including cell cohesiveness, yield and survival time of the cancer cells in culture, emigration of macrophages from the explants, and the occurrence of special interactions of lymphocytes with cancer cells. The outstanding finding was the association of special lymphocyte-cancer cell interactions with a more favorable prognosis; six of seven patients (86%) with infiltrating ductal cancer showing lymphocyte congregation, emperiopolesis, or other special activity were tumor-free survivors. Additional findings were: 1) patients having four or fewer lymph nodes positive for cancer had a survival rate of 50%, but this fell to 20% when emigrating macrophages were not present in the cancer cultures; 2) eight of the 12 patients (67%) having discoid histoculture survival time of less than one week were tumor-free, as opposed to three of eight patients (27%) where tumor survival in vitro was greater than one week. The reverse was true for nondiscoid, poorly cohesive colonies of cancer; 3) the combination of tumor-negative lymph nodes and low in vitro cancer yield was associated with the best patient survival rate, 64%. However, the combination of negative nodes and high in vitro yield was linked to the worst patient survival (20%). Although the overall study does not permit definitive conclusions, there is an undeniable potential for the use of histocultures in the evaluation of human cancers. Expanded studies are warranted, including larger numbers of tumors and cultures, and longer patient follow-up periods.     

Identification of Fat4 as a candidate tumor suppressor gene in breast cancers

Fat, a candidate tumor suppressor in Drosophila, is a component of Hippo signaling pathway involved in controlling organ size. We found that a ～3 Mbp deletion in mouse chromosome 3 caused tumorigenesis of a non‐tumorigenic mammary epithelial cell line. The expression of Fat4 gene, one member of the Fat family, in the deleted region was inactivated, which resulted from promoter methylation of another Fat4 allele following the deletion of one Fat4 allele. Re‐expression of Fat4 in Fat4‐deficient tumor cells suppressed the tumorigenecity whereas suppression of Fat4 expression in the non‐tumorigenic mammary epithelial cell line induced tumorigenesis. We also found that Fat4 expression was lost in a large fraction of human breast tumor cell lines and primary tumors. Loss of Fat4 expression in breast tumors was associated with human Fat4 promoter methylation. Together, these findings suggest that Fat4 is a strong candidate for a breast tumor suppressor gene. © 2008 Wiley‐Liss, Inc.     

Comparison of gene expression data from human and mouse breast cancers: identification of a conserved breast tumor gene set

The aim of our work was to establish a database for breast cancer gene expression data in order to compare human and mouse breast cancer. We identified human and mouse homologues genes and compared the expression profile of 24 human breast tumors with 6 WAP-SVT/t breast tumors (WAP-SVT/t animals, line 8). Our studies confirmed the heterogeneity in gene expression of human as well as mouse breast cancer cells. However, 63 genes were found to be differentially expressed (upregulated: 40; downregulated: 23 genes) in at least 75% of the breast tumors of both species. To differentiate between early and late events in tumor formation, we compared the 63 differentially expressed genes with a mouse data set obtained from hyperplastic mammary glands. This revealed that the majority of the early deregulated genes are cell proliferation specific. These early changes seem to be necessary although not sufficient for breast cancer formation. Late alterations concern mainly genes belonging to the category of cell communication and metabolism. Interestingly, most of the 63 conserved genes are commonly associated with tumorigenesis.     

EGFR gene and protein expression in breast cancers.

AIMS: To measure the EGFR gene amplification and protein expression, and to compare the EGFR expression with HER2 expression similarly measured. METHODS: EGFR gene amplification was assayed by fluorescence in situ hybridization and its protein expression was evaluated by immunohistochemistry on tissue microarray of 165 consecutive invasive breast cancers. RESULTS: EGFR protein overexpression was observed in 20.6% of the 165 studied cases, but gene amplification was observed in 7.9% only. EGFR protein was expressed in 33.3% of HER2-amplified tumors, and in only 16.3% of HER2 non-amplified tumors. EGFR expression significantly increased in HER2 gene-amplified and protein-expressing tumors as well. On survival analysis, EGFR expression was a significant prognostic factor for the disease-free and overall survival of the patients. CONCLUSIONS: EGFR protein expression was independent of EGFR gene amplification status, whereas it was intimately associated with HER2 amplification and overexpression in breast cancer.     

Microsatellite instability in sporadic human breast cancers

Human breast-cancer specimens from 100 patients were analyzed for microsatellite instability (referred to as replication error; RER) at 12 genomic loci on 7 chromosomes, and results were correlated with clinicopathologic characteristics. In 42 of 100 breast-cancer patients, we investigated whether RER was associated with the amplification of oncogenes and/or suppression of tumor-suppressor genes. Of the 100 patients, 8 (8%) were RER-positive at one or more chromosomal loci. The majority of RER-positive patients had early-stage disease with ER-positive tumors, suggesting that RER occurs early in breast tumorigenesis. However, no significant correlation was observed between RER and oncogenes or tumor-suppressor genes. Thus, the mechanism of RER in sporadic human breast cancer may be independent of the multi-step carcinogenesis caused by the alterations of oncogenes and tumor-suppressor genes. © 1996 Wiley-Liss, Inc.     

Targeting the endothelin system: novel therapeutic options in gynecological,urological and breast cancers

The endothelin system comprises the three peptide hormones endothelin (ET)-1, -2, -3, their G protein-coupled receptors, endothelin-A-receptor (ET_AR) and endothelin-B-receptor (ET_BR), and the enzymes of endothelin biosynthesis and degradation. In the past two decades, an impressive amount of data has been accumulated investigating the role of the endothelin system in a variety of malignancies. In many cancers, ET-1/ET_AR interaction induces proliferation, angiogenesis, antiapoptosis and resistance to chemotherapy. Data indicate a pivotal role of the endothelin system in tumorigenesis, local progression and metastasis. Subsequently, novel drugs have been designed inhibiting ET-1 biosynthesis or ET_AR interaction. A wide range of preclinical data is available on the role of ET_AR antagonists in gynecological, urological and breast cancers providing evidence for their antiangiogenic, proapoptotic and growth inhibitory effects. Of particular interest is the anti-invasive and antimetastatic efficacy of ET_AR antagonists and synergism when co-administered with established cancer therapies. Data indicate a future role of ET_AR antagonists in oncologic therapies.     

Targeting the endothelin system: novel therapeutic options in gynecological, urological and breast cancers

The endothelin system comprises the three peptide hormones endothelin (ET)-1, -2, -3, their G protein-coupled receptors, endothelin-A-receptor (ET(A)R) and endothelin-B-receptor (ET(B)R), and the enzymes of endothelin biosynthesis and degradation. In the past two decades, an impressive amount of data has been accumulated investigating the role of the endothelin system in a variety of malignancies. In many cancers, ET-1/ET(A)R interaction induces proliferation, angiogenesis, antiapoptosis and resistance to chemotherapy. Data indicate a pivotal role of the endothelin system in tumorigenesis, local progression and metastasis. Subsequently, novel drugs have been designed inhibiting ET-1 biosynthesis or ET(A)R interaction. A wide range of preclinical data is available on the role of ET(A)R antagonists in gynecological, urological and breast cancers providing evidence for their antiangiogenic, proapoptotic and growth inhibitory effects. Of particular interest is the anti-invasive and antimetastatic efficacy of ET(A)R antagonists and synergism when co-administered with established cancer therapies. Data indicate a future role of ET(A)R antagonists in oncologic therapies.