Erika Marton

MMP-2 and MMP-9 Expression in Breast Cancer-Derived Human Fibroblasts is Differentially Regulated by Stromal-Epithelial Interactions

Tissue remodeling is a key element in the local invasion and metastasis of malignant breast tumors. The degradation of extracellular matrix that is associated with this process is thought to be mediated by a number of Zn2+-dependent matrix metalloproteinases (MMPs). In most cases these enzymes are not produced by the malignant epithelium itself but by adjacent breast stroma, suggesting an important role for cell-cell interactions. We have analyzed Gelatinase A (MMP-2) and Gelatinase B (MMP-9) gene expression in a panel of six breast cancer cell lines and six primary cultures of stromal cells deriving from breast cancer biopsies. With one exception we did not detect MMP-2 or MMP-9 gene expression in any of the established tumor cell lines. Conversely, tumor stroma-derived fibroblasts expressed MMP-2 mRNA, although no MMP-9 mRNA was seen in RNase protection assays. When fibroblasts were cultured in the presence of media conditioned by MCF-7 tumor cells, MMP-2 enzyme production increased but MMP-9 activity remained undetectable. However, when fibroblasts and MCF-7 tumor cells were co-cultured together, MMP-9 was induced. These observations were confirmed by immunocytochemical analysis of co-cultures of MCF-7 and tumor-derived fibroblasts in which MMP-2 and MMP-9 protein expression was confined to stromal cells adjacent to MCF-7 tumor cells. No MMP-2 or MMP-9 staining was detected in monocultures of the two respective cell types. We conclude that MMP-2 expression is present in the stroma of malignant tumors and is increased by paracrine stimulation mediated by soluble factors. In contrast, MMP-9 expression tumor-derived fibroblasts requires direct contact with malignant tumor epithelium.

Expression of Sex Steroid Receptors and their Co-Factors in Normal and Malignant Breast Tissue: AIB1 is a Carcinoma-Specific Co-Activator

The differential expression pattern of estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β) and their co-activator/co-repressor proteins is thought to modulate estrogenic action and to be present already during the early stages of tumorigenesis. It has therefore been postulated that certain co-activator and co-repressor proteins contribute to the development of breast cancer. There are some reports providing information on gene amplification and mRNA over-expression of certain co-factors in breast cancer, but to date there is only limited knowledge about their respective protein expressions. The aim of this study was to examine the expression of four steroid receptor co-activators (steroid receptor co-activator 1 (SRC-1), transcription intermediary factor 2 (TIF 2), protein 300 kDa/CREB binding protein (p300/CBP), amplified in breast cancer 1 (AIB1)), and of the co-repressor nuclear receptor co-repressor (NCoR), in malignant breast tissues and in matching normal breast biopsies of the same individuals. Protein expression was analyzed by immunohistochemistry and was compared to prognostic parameters such as lymph node involvement, tumor grading and receptor status. All members of the co-regulatory protein family were detected in both, benign and matching malignant tissue samples, except for AIB1, which was found to be expressed exclusively in malignant epithelium. AIB1 was preferentially present in carcinomas with high tumor grade (r = 0.48, p = 0.014), and was co-expressed with p300/CBP (r = 0.54, p = 0.006). TIF 2 correlated significantly to nodal status (r = 0.46, p = 0.025). Furthermore, protein levels of ER-β, p300/CBP and AIB1 were higher in invasive ductal carcinomas than in normal mammary tissue. The tumoral ER-α protein expression was significantly correlated with that of PgR (r = 0.61, p = 0.001) and NCoR (r = 0.4, p = 0.043), whereas ER-β expression was associated with SRC-1 (r = 0.68, p ≤ 0.001), TIF 2 (r = 0.64, p = 0.001) and NCoR (r = 0.48, p = 0.014) protein levels in malignant specimens. In our hands, 20% of ER-β positive tumors did not express ER-α protein, thereby suggesting that a substantial fraction of ER-beta positive tumors is falsely considered to be ‘estrogen receptor negative’ if only ER-α specific antibodies are employed in the histological assessment of the ER status.

Insulin-like growth factor (IGF)-I and IGF-II Serum concentrations in patients with benign and Malignant breast lesions: Free IGF-II is correlated with breast cancer size

Purpose: Insulin-like growth factors (IGFs) are potent mitogens for breast cancer cells in vitro, and elevated IGF-I serum levels are a risk factor for breast malignancies. This study evaluated IGF-I and IGF-II serum levels in healthy women and in patients with benign and malignant breast lesions and correlated them with tumor size. Experimental Design: Serum levels of the total and unbound fractions of IGF-I and IGF-II were analyzed in 65 patients with benign and malignant breast lesions and in 38 women without breast disease. ELISAs were used to detect serum IGF levels, with (total IGF) or without (free IGF) prior acid-ethanol extraction. Results: Total IGF-I serum concentrations were lower in healthy women than in breast cancer patients (P < 0.001) or patients with benign breast lesions (P = 0.010), but no differences were observed in free IGF-I levels. Conversely, healthy women had higher serum levels of free IGF-II than women with breast lesions (P = 0.003), and the free/total IGF-II ratio was significantly reduced in patients with breast disease (P = 0.001). Although IGF-I or IGF-II serum concentrations of breast cancer patients were similar to those of patients with benign lesions, the size of a malignant tumor was correlated to the ratio free/total IGF-II (P = 0.002). Conclusions: Malignant breast tumors cannot be distinguished from benign breast lesions by systemic IGF serum levels. However, women with breast lesions have decreased IGF-II concentrations, and free IGF-II levels are clearly correlated to the size of a breast cancer, indicating an involvement in tumor growth.

Stem cell marker expression in human trisomy 21 amniotic fluid cells and trophoblasts.

Down Syndrome is the most frequent genetic cause of mental retardation. Deregulation of specific differentiation processes is a major cause for the neuropathological cell features typical for this syndrome. The molecular mechanisms leading to Down Syndrome are likely to be operative from the very earliest time of embryonic/fetal development. We therefore analysed human amniotic fluid cell samples and cytotrophoblastic cells from placental biopsies, both with normal karyotypes and with trisomy 21, for the mRNA expression of stem cell marker genes. Here we describe for the first time that these human primary cell sources contain cells that express telomerase reverse transcriptase, leukemia inhibitory factor receptor, and bone morphogenetic protein receptor II. A specific difference between aneuploid and normal cells could not be detected. These data provide evidence that human amniotic fluid and cytotrophoblastic cell cultures might provide a new source for research on primary cell systems expressing these stem cell markers. In addition, it is suggested that early deregulation of the expression of these genes in the here analysed cell sources does not contribute to the molecular development of Down Syndrome.

Brca1 and differentiation

Breast cancer is one of the most frequent malignancies affecting women. The human breast cancer gene 1 (BRCA1) gene is mutated in a distinct proportion of hereditary breast and ovarian cancers. Tumourigenesis in individuals with germline BRCA1 mutations requires somatic inactivation of the remaining wild-type allelle. Although, this evidence supports a role for BRCA1 as a tumour suppressor, the mechanisms through which its loss leads to tumourigenesis remain to be determined. Neither the expression pattern nor the described functions of human BRCA1 and murine breast cancer gene 1 (Brca1) can explain the specific association of mutations in this gene with the development of breast and ovarian cancer. Investigation of the role of Brca1 in normal cell differentiation processes might provide the basis to understand the tissue-restricted properties.

Cell cycle and cell size regulation in Down syndrome cells.

Although the neuropathological features typical for Down Syndrome obviously result from deregulation of both, cell cycle control and differentiation processes, so far research focused on the latter. Considering the known similarities between the neuropathology of Down Syndrome and Alzheimers disease and the knowledge, that in Alzheimers disease neuronal degeneration is associated with the activation of mitogenic signals and cell cycle activation, it is tempting to investigate the consequences of an additional chromosome 21 on mammalian cell cycle regulation. We analysed the distribution of cells in different cell cycle phases on the flowcytometer and the cell size of human amniotic fluid cells with normal karyotypes and with trisomy 21. We could not detect any significant differences suggesting that the presence of an additional copy of the about 225 genes on human chromosome 21 does not trigger cell cycle effects in amniotic fluid cells. These data provide new insights into the cell biology of trisomy 21 cells.