Yolande Berthois

Effect of stromal and epithelial cells derived from normal and tumorous breast tissue on the proliferation of human breast cancer cell lines in co-culture.

Stromal and epithelial components surrounding neoplastic cells are believed to be important in tumor regulation. We have studied the effects of stromal and epithelial cells on the proliferation of a variety of breast‐cancer epithelial cell lines. Co‐culture experiments were performed in which the 2 cell types were separated by a microporous membrane. Under these conditions, fibroblasts from normal breast tissues inhibited the proliferation of MCF‐7 cells, but not that of immortalized normal S2T2 cells. In contrast, fibroblasts from cancerous breast tissues did not influence the proliferation of the 2 cell lines tested. Conditioned media (CM) of breast fibroblasts derived from normal tissues were not able to affect MCF‐7 cell growth, suggesting complex paracrine interactions between both cell types. Normal breast epithelial cells (NBEC) have also been tested for their ability to regulate the proliferation of breast‐cancer epithelial cell lines. Co‐culture experiments demonstrated that NBEC inhibited a variety of breast‐cancer cell lines. CM from NBEC induced similar results and the inhibitory effect appeared to be specific for epithelial cells from tumorous breast. Moreover, CM from NBEC and normal fibroblasts were shown to contain more TGFβ1 and amphiregulin than those of MCF‐7 cells. We conclude that both the tissue origin and the target tumor cells phenotype will determine the extent of proliferative response. More important, the tumor‐cell growth inhibition induced by fibroblasts and epithelial cells of normal breast tissue may constitute a tumor‐growth‐regulatory mechanism. Int. J. Cancer, 71:42–48, 1997.

Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line

The epidermal growth factor (EGF) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antigen of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggressive transformed cells. Dual staining of EGFR and AR was observed in 50 – 60% of NS2T2A1 cells, while 30 – 40% cells expressed AR only. To explore the potential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientation was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to <5%). The anchorage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4 – 6.8% relative to the control cell line transfected with the vector alone. The clones expressing AR antisense RNA showed a reversion of the malignant phenotype when injected in nude mice, since a significant reduction of tumor intake was observed coincident with a significant tumor mass reduction (>96%). Moreover, intra-tumoral vascularization decreased significantly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). These in vitro and in vivo data reveal the oncogenic nature of AR in transformed breast epithelial cells and imply a role for AR in tumor angiogenesis.

Role of epidermal-growth-factor receptor in tumor progression in transformed human mammary epithelial cells.

The presence of epidermal‐growth‐factor receptors (EGFR) and of its ligands (TGFα and amphiregulin) in breast‐cancer tissues suggests that they play a paracrine/autocrine role in tumor growth or progression. This hypothesis was tested on 3 cell lines, S2T2, NS2T2A and NS2T2A1. These epithelial cells are derived from a normal human breast‐epithelial‐cell culture transformed by SV40‐T Ag, are of the same clonal origin, have respectively increasing levels of EGFR, TGFα, amphiregulin and of thymidine‐kinase activity associated with increasing tumorigenic potential in nude mice (tumor intake and tumor volume). The monoclonal antibody MAb 425, which blocks ligands interaction with EGFR, reduced by more than 90% anchorage‐independent growth of the most tumorigenic cells, NS2T2A1. Another anti‐EGFR MAb, 528, reduced to 25% of controls the mean tumor mass after NS2T2A1 grafting in mice. Anti‐sense RNA expression of EGFR in these cells confirmed the importance of this receptor in tumor progression, since it reduced significantly the tumor volume and tumor weight of NS2T2A1 cells to 16% of those in mock‐transfected control cells. Int. J. Cancer 78:112–119, 1998.© 1998 Wiley‐Liss, Inc.

Clinical relevance of amphiregulin and VEGF in primary breast cancers

The characterization of novel prognostic markers in breast cancer is necessary to improve the identification of high‐risk populations. In our study, the prognostic significance of VEGF and amphiregulin (AR) was investigated and compared to conventional prognostic factors in primary breast cancers. The analysis was performed using enzyme‐linked immuno‐assay in a series of 193 patients, and univariate and multivariate analysis were performed in the overall population as well as in pre‐ and post‐menopausal patients subdivided in node‐negative (N−) and node‐positive (N+) subsets. AR (median, 44.8 pg/mg protein) appeared strongly correlated with progesterone receptors (PgR) (p = 0.0018) in the premenopausal N+ population, and with uPA (p= 0.020) and VEGF (p= 0.0053) in the postmenopausal/N+ patients. Despite these attractive data, AR expression was not significant for recurrence or survival outcome. Data revealed strong correlation between VEGF and uPA, and PAI‐1, in the N+ population. Moreover, patients with high VEGF levels displayed poor outcome, with an increased risk for N+ subset. These data were confirmed by multivariate analysis that presented histologic grade (HR, 10.55, p = 0.001) and VEGF (HR, 3.89, p = 0.03) as the prominent prognostic markers for overall survival for the N+ population. Furthermore, infiltrating ductal carcinomas (IDC) were shown to express higher levels of both uPA (p < 0.0001) and VEGF (p = 0.002) than intralobular carcinomas. This retrospective study reinforces the pejorative biological role of VEGF in the progression of breast tumors. Our data also suggest that VEGF and uPA might play particular role in the biology and progression of IDC.

POSITIVE REGULATION OF NORMAL AND TUMORAL MAMMARY EPITHELIAL CELL PROLIFERATION BY FIBROBLASTS IN COCULTURE

SummaryIn the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.

Adrenomedullin promotes cell cycle transit and up-regulates cyclin D1 protein level in human glioblastoma cells through the activation of c-Jun/JNK/AP-1 signal transduction pathway.

Adrenomedullin is a secreted peptide hormone with multiple functions. Although a number of reports have indicated that adrenomedullin may be involved in tumor progression, its mechanism of action remains obscure. In this study, we have analysed the signal transduction pathway activated by adrenomedullin in human glioma cells. Our results revealed that adrenomedullin induced the phosphorylation of both c-Jun and JNK in glioblastoma cells. Silencing JNK expression with siRNA reversed the phosphorylation of c-Jun induced by adrenomedullin, indicating that JNK is responsible of c-Jun activation. In addition, electrophoretic mobility-shift assays showed that the increase in phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays and co-immunoprecipitation demonstrated that c-Jun and JunD are part of the AP-1 complex, indicating that activated c-Jun is dimerized with JunD in response to adrenomedullin. Furthermore, adrenomedullin was shown to promote cell transit beyond cell cycle phases with a concomittant increase in cyclin D1 protein level, suggesting that adrenomedullin effects cell proliferation through up-regulation of cyclin D1. The inhibition of JNK activation or the suppression of c-Jun or JunD expression with siRNA impaired the effects of adrenomedullin on cell proliferation and on cyclin D1. Taken together, these data demonstrate that activation of cJun/JNK pathway is involved in the growth regulatory activity of adrenomedullin in glioblastoma cells.

Distribution of estrogen receptor heterogeneity in growing MCF‐7 cells measured by quantitative microscopy

The existence of interactive subpopulations is a biological feature that can modulate the proliferation of tumor cells. The hormone-responsive breast cancer cell line MCF-7 has been described as heterogeneous in terms of density. In this study we describe a quantitative image analysis methodology that we developed for the in situ detection of different subpopulations in MCF-7 cell cultures. Using this technology, we demonstrate the heterogeneity of the MCF-7 cell line in terms of both nuclear size and estrogen-receptor content. Analysis of the organization (topography) of the different subpopulations in culture reveals a nonrandom distribution of cells. When studying the development of these cell subpopulations as a function of time of culture, we observe modifications of their topography associated with an increase of estrogen-receptor-expressing cells. Moreover, the use of cluster analysis allows study of the local organization of these subpopulations. These changes appear to be independent of cell proliferation.

Differential regulation of normal and tumoral breast epithelial cell growth by fibroblasts and 1,25-dihydroxyvitamin D3.

Mesenchymal‐epithelial interactions are of paramount importance during normal and tumoral breast developments. We have investigated the paracrine growth regulation of normal and tumoral breast epithelial cells by fibroblasts derived from normal or pathological breast tissues. In some cases, breast cancer MCF‐7 cells or normal epithelial cells in primary culture were cocultured with fibroblasts in a Transwell system allowing diffusible factor exchanges. Alternatively, conditioned medium produced by fibroblast cultures was added to epithelial cell cultures. Fibroblasts were shown to stimulate the proliferation of normal and carcinoma cells through paracrine mechanisms. However, the paracrine exchanges appeared to be different in normal versus tumoral breast epithelial cell growth regulation. Moreover, vitamin D‐related compounds that have been proposed as anti‐tumoral drugs were studied for their ability to affect normal and tumoral mammary epithelial cell proliferation and to interfere with the growth‐regulatory activity of fibroblasts. Whereas vitamin D compounds inhibited MCF‐7 cell growth, they led to a marked stimulation of the proliferation of normal mammary epithelial cells. Moreover, it was shown that the vitamin D analog EB 1089 can block the mitogenic effect of fibroblast‐conditioned medium on tumoral but not normal breast epithelial cells. The differential effects of vitamin D compounds on cell proliferation provide further data in favor of the different behaviours of normal and tumoral mammary epithelial cells. The potential therapeutic use of vitamin D derivatives in the treatment of breast cancer is supported by these results but their growth‐stimulatory properties on normal epithelial cells cannot be overlooked.