ABSTRACT
EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was recently described as an antagonist of angiogenesis. Motivated by a strong dependence of tumor growth and metastasis on angiogenesis, we investigated the role of EFEMP1 in human breast cancer. We applied RNA microarray expression analysis and quantitative real-time PCR (QRT) in a total of 45 sporadic breast cancer tissues and found EFEMP1 down-regulation in 59% and 61% of the analyzed tissues, respectively. This down-regulation was confirmed on protein level. Immunohistochemistry in 211 breast cancer tissues resulted in reduced or even abolished EFEMP1 expression in 57-62.5% of the tumors. Bisulphite genomic sequencing in breast cancer cell lines and primary breast cancer tissues revealed promoter methylation as the major cause of this down-regulation. Furthermore, analysis of 203 clinically well characterized primary breast cancers displayed a significant correlation of reduced EFEMP1 protein expression with poor disease-free (p = 0.037) and overall survival (p = 0.032), particularly in those node-positive patients who received adjuvant anthracycline-based chemotherapy, but not in those treated by either cyclophosphamide-methotrexate-5-fluorouracil (CMF) or Tamoxifen. In summary, the presented data demonstrate for the first time the reduced EFEMP1 expression on RNA and protein level in a substantial number of sporadic breast carcinomas and its correlation with epigenetic alterations. Furthermore, these data point towards a possible predictive impact of EFEMP1 expression in primary breast cancer.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , DNA Methylation/genetics , Extracellular Matrix Proteins/biosynthesis , Neovascularization, Pathologic/genetics , Promoter Regions, Genetic/genetics , Adult , Aged , Aged, 80 and over , Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents, Hormonal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , DNA Mutational Analysis , Epigenesis, Genetic , Female , Gene Expression , Humans , Immunohistochemistry , Loss of Heterozygosity , Middle Aged , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Tissue Array AnalysisABSTRACT
The identification of novel disease-associated genes in gynaecological tumours has important implications for understanding the process of tumourigenesis and the development of novel treatment regimens. cDNA libraries from disease tissues may represent a valuable source to identify such genes. Recently, a bio-informatic procedure based on an 'electronic Northern' approach was established to screen expressed sequence tag (EST) libraries for genes differentially expressed in tumour and normal tissues, and identified 450 candidate genes differentially expressed in breast and ovarian cancer. In this report, the validation of an initial set of 40 candidate genes, which were selected due to their localization in chromosomal regions frequently altered in gynaecological tumours, is described. Differential expression of 29 of these genes, including three uncharacterized novel genes, was confirmed by applying cancer profiling arrays with 106 matched pairs of tumour/normal cDNAs and quantitative reverse transcription-polymerase chain reaction (RT-PCR) on 60 clinical specimens. The majority of these differentially expressed genes have not been described previously in the context of breast and ovarian cancer, and may constitute novel diagnostic markers for these tumour entities.
Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Ovarian Neoplasms/genetics , Carcinoma, Ductal, Breast/genetics , DNA, Complementary/genetics , DNA, Neoplasm/genetics , Expressed Sequence Tags , Female , Gene Expression Profiling , Genetic Markers , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Ovarian Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
Allelic imbalance constitutes a major mechanism of genetic aberrations in breast cancer and strongly indicates the involvement of tumor associated genes in the affected chromosomal regions. Preliminary results from our study indicated the existence of a tumor suppressor gene located on chromosomal arm 15q which may be involved in breast cancer progression.1 In the present study, 210 primary breast carcinomas, 30 metastases and 26 local recurrences from primary breast carcinomas have been analyzed with a panel of 18 highly polymorphic microsatellite markers spanning the chromosomal region 15q11-21.3. Allelic imbalance at 15q with at least 1 marker was seen in 36 of 56 (64.3%) metastases and recurrences, but only in 58 of 210 (27.6%) primary tumors (p<0.0001). We identified a subregion defined by microsatellite marker CYP19 (15q21.1) that showed significantly higher frequencies of allelic imbalance in metastases and recurrences (57.6%) when compared to primary carcinomas (8.9%; p<0.0001). Allelic imbalance at 15q was correlated with histopathologic parameters of the patients with primary breast carcinomas. We detected a significant association with established predictors of poor prognosis, i.e., negative estrogen receptor status (p=0.003), negative progesterone receptor status (p=0.028), high grade (p=0.014) and positive axillary lymph nodes (p=0.013). In summary, our data provide further evidence for a novel prognostic marker in breast carcinomas located in the chromosomal region 15q21.
