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The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGFbeta1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling., (Copyright (c) 2009 UICC.)

Estrogen acts via its receptor (ER) to stimulate cell growth and differentiation in the mammary gland. ER and progesterone receptor (PR), which is regulated by estrogen via ER, have been used as prognostic markers in clinical management of breast cancer patients. Patients with ER- breast tumors have a poorer prognosis than patients with ER+ tumors. The aim of the present study was the identification of tumor-associated genes differentially expressed in breast tumors regarding the presence or absence of ER and PR hybridized with cDNA microarrays containing 4,500 tumor-derived expressed sequence tags generated using the ORESTES technique. Samples of human primary breast carcinomas from 38 patients were analyzed. The experiments were performed in triplicates and data from each element were acquired by phosphoimage scanning. Data acquisition was performed using the ArrayVision software. After normalization statistical analysis was applied. In a preliminary analysis, 98 differentially expressed transcripts were identified, 46 were found to be more expressed in ER+/PR+ and 52 were found to be more expressed in ER-/PR- breast tumors. The biochemical functions of the genes in the reported expression profile are diverse and include metabolic enzymes, protein kinases, helicases, transcription factors, cell cycle regulators and apoptotic factors. ER-/PR- breast tumors displayed increased levels of transcripts of genes associated with neurodegeneration and genes associated with proliferation were found in ER+/PR+ tumors.

We have investigated the effects of laminin, on the plasminogen-activator system of MCF-7 breast-carcinoma cells. MCF-7 cells were incubated on plastic or laminin-coated wells, and medium and cell lysate aliquots were assayed for tissue-type (tPA) and urokinase-type plasminogen activator (uPA) by a chromogenic assay in combination with anti-uPA antibodies. Cells cultured on laminin displayed a 5-fold increase in tPA activity and a 2-fold decrease in uPA activity relative to cells on plastic. These effects could be mimicked by laminin fragment P1 but not by collagen I or fibronectin. tPA activity of cells treated with estradiol (10 nM) was 3-fold higher, that of cells on laminin treated with estradiol was 15-fold higher, than that of control. Northern-blot analysis showed that tPA mRNA levels were up-regulated by estradiol and laminin, whereas PAI-1 mRNA levels were down-regulated by laminin and not affected by E2. Concomitant treatment with laminin and estradiol, decreased PAI-1 mRNA and increased tPA mRNA levels, accounting for the synergistic increase in tPA activity. Laminin exerted only a modest (approx. 2-fold) inhibitory effect on uPA mRNA levels. In the breast-carcinoma cell line MDA-MB-231, down-regulation of PAI-1 and uPA mRNA by laminin was not observed. Adhesion assays indicated that alpha2beta1 is the predominant receptor for laminin in MCF-7 cells. MDA-MB-231 cells expressed alpha2 (54%) but this integrin is not used as a laminin receptor. These results support a role for alpha2beta1 in mediating interactions of MCF-7 with LN.

The relative expression of estrogen receptor (ER) and progesterone receptor (PR) mRNA transcripts was measured in 71 primary breast-cancer biopsies. ER and PR binding activity were estimated in parallel by the dextran-coated-charcoal method. There was a close correlation between the amount of ER mRNA and estradiol binding activity. Tumors from post-menopausal patients contained higher levels of ER mRNA than those from pre-menopausal patients. Northern-blot analysis indicated the presence of a major band of 6.3 kb in all ER mRNA-positive tumors. Some tumors showed, in addition, 3.7- and 2.4-kb transcripts. PR binding activity and overall PR mRNA levels correlated moderately. PR mRNA and ER mRNA were associated. Four PR mRNA species with estimated sizes of 11.4, 4.5, 3.7 and 2.5 kb were detected in 14% of the PR mRNA-positive tumors. The 3.7-kb transcript was detected to varying degrees in all PR mRNA-positive biopsies, accompanied in some tumors by the 2.5-kb species. ER and PR mRNA levels > or = 50 pg/5 micrograms total RNA correlated with prolonged survival of the patients. In addition, high ER mRNA levels were associated with absence or minimal necrosis and vascular invasion together with absence or minimal level of tumor lymphocytic infiltration, but not with age, clinical stage, tumor size or overexpression of c-myc or c-erbB-2 mRNA. PR mRNA was not statistically associated with any of the above clinicopathological features. A bivariate analysis showed that both ER and PR mRNA levels were able to predict overall survival independently of the lymph-node status.

Linkage studies have indicated that a gene on chromosome arm 17q, designated BRCA1, confers susceptibility to familial breast and ovarian cancer. To investigate the possible involvement of the BRCA1 gene in sporadic breast cancer we have analysed loss of heterozygosity (LOH) in a panel of 100 sporadic primary breast tumours using 10 PCR-based polymorphic markers from 17q12-21. Allele losses were detected in 40 of 100 tumours informative for at least one of the markers analysed. Of these 40 deleted tumours, 27 showed partial or interstitial loss on 17q. The pattern of LOH in the tumours with partial or interstitial LOH revealed three putative distinct deleted regions on 17q12-21. The first lies on the proximal long arm between D17S250 and THRA1; the second one lies between D17S776 and D17S579, the region containing the BRCA1 gene; and the third is telomeric to D17S733. The most frequently deleted region overlaps with the minimal region containing the BRCA1 gene, suggesting that this gene might also be associated with the development or progression of a proportion of sporadic breast tumours.

Variation of laminin receptor levels (LNR) during myeloid-cell differentiation and in acute leukemia were investigated by 125I-laminin-binding determination during HL60 cell differentiation and in cells of patients with different types of leukemia, characterized according to the FAB classification. LNR levels in HL60 cells increased during differentiation, being significantly higher in cells exposed to phorbol myristate acetate (PMA) and ethanol (55,391 +/- 27,845 and 29,314 +/- 6,435 sites/cell respectively) as compared with HL60 controls (8,549 +/- 4,000 sites/cell). The control cells do not adhere to laminin-coated surfaces, but differentiation with PMA results in their rapid adherence on this substratum. Short treatment with PMA does not increase the number of adherent cells or the receptor expression. Granulocytes also presented equally high LNR concentration (29,739 +/- 13,516 sites/cell). The lymphoid cells (lymphocyte, acute lymphoid leukemia and chronic lymphocytic leukemia) shared low LNR numbers (less than 6,500 sites/cell). Myeloid cells displayed a wide range of LN receptors with higher levels being associated with the more differentiated FAB subgroups. 125I-laminin binding to lymphoid or myeloid leukemic cells was mainly inhibited by P1 fragments, whereas granulocytes and differentiated HL60 cells displayed a dual binding pattern for laminin fragments P1 and E8. These results were confirmed by assays using 125I-labelled P1 and E8 fragments. We conclude that magnitude of LNR levels and variation in expression of P1 and E8 receptors appear to be linked to lineage and maturation status in hematopoietic cells.

The MEL-85 human melanoma cell line was used to investigate the effects of both estradiol and dexamethasone on expression of laminin (LM) receptors and cell adhesion capacity. Immunoblotting of eluates from whole-cell extracts applied to LM Sepharose indicates the presence of an LM-binding protein of 116-130 kDa that reacted with an anti-beta 1 integrin antibody, suggesting that the putative LM receptor of MEL-85 cells is a member of the integrin family. Analysis of 125I-LM binding to whole cells indicates the existence of low-affinity components which display positive co-operativity. LM-fragment-8 competes for this binding to the same extent as unlabelled LM (75%), while fragment PI is inactive and fibronectin (FN) competes by about 30% only. Binding of labelled fragment-8 exhibits a pattern similar to that of intact LM. Cell adhesion to substrates coated with LM and LM fragments closely parallels binding to cells in suspension. MEL-85 cells were estradiol-receptor-negative. Estradiol treatment did not stimulate LM receptor levels or attachment to LM. Growth rate also remained unaltered. To characterize the glucocorticoid dependence of MEL-85 cells, we first established the presence of glucocorticoid receptors and an inhibitory effect on growth rate. Dexamethasone treatment resulted in marked enhancement of adhesion to LM, without altering LM receptor number or affinity. In addition, dexamethasone changed the morphology of MEL-85 cells in conjunction with higher LM expression as evaluated by immunofluorescence.

Determinations of estradiol receptor (ER), progesterone receptor (PR), total lactate dehydrogenase activity (LDH) and electrophoretic separation of LDH isoenzymes were performed in cytosols from 118 samples of primary infiltrating ductal mammary carcinoma. ER + PR+ tumors demonstrated a significant increase in the proportion of the LDH muscle-type isoenzyme (LDH5) as compared to ER-PR- samples (p less than 0.002). Tumors lacking one of the receptors presented intermediate LDH5 percentages. As total LDH activity bore no relation to either the presence or absence of receptors, the increased proportion represents an absolute elevation of LDH5, suggesting that LDH5 may be a promising hormone-dependence marker. As an in vitro model to study whether LDH5 was induced by estradiol via ER, we have used two human breast cancer cell lines, MCF-7 and T47D. In both cell lines LDH5 was the sole isoenzyme. Total ER and PR have been determined by a whole-cell method. In MCF-7 cells (with high ER levels), after incubation with 10(-10) M estradiol, maximal induction of LDH had already been achieved. In relation to T47D (low ER levels) estradiol did not evoke an induction of LDH5 at any concentration examined. In MCF-7 cells, the level of LDH5 induction paralleled processing of ER. The processing effect was rapid, beginning within 5 min of estradiol addition, and was completed within 1 hr. With 10(-6) M tamoxifen, LDH5 induction was suppressed and this effect was reversed by estradiol. Such antiestrogenic effects of tamoxifen on LDH5 have not been observed in T47D cells. Agonistic effects of low doses of tamoxifen on LDH5 were not observed. Our studies suggest that estrogen stimulation of LDH5 involves ER.

In order to evaluate the relationship of plasminogen activator (PA) activity to the expression of estrogen (ER) and progesterone (PR) receptors, we assayed primary tumor specimens from 121 cases of female breast cancer. Other clinical and histopathological variables were also investigated with respect to their possible association with PA activity in the samples. Statistical correlations were examined by stratified analysis techniques and by multiple regression methods. PA activity was higher in tumors which were ER+PR+ than in those exhibiting other subsets of joint receptor expression, a finding which was particularly predominant in tumors from post-menopausal women. Of the other variables examined, only disease stage, nodal status and vascular infiltration presented marginal positive statistical associations with PA activity, although this was not confirmed by multivariate analysis. The latter technique showed that the presence of PR was sufficient to explain the variation in PA levels. However, ER emerged as the sole significantly explanatory factor when ER+PR- patients were removed from analysis. Both PR and age (negatively) were independent contributory factors for PA activity in the analysis of post-menopausal women. None of the variables examined emerged as being significantly associated with PA when data from pre-menopausal patients were used. These findings indicate that PA activity in breast tumor samples is statistically associated with the expression of functional estradiol receptors, although to a lesser extent than PR.

Two hundred and forty-two primary breast cancers were assayed for estrogen receptors (ER). Of these, 202 were analyzed for progesterone receptors (PR) and 155 for glucocorticoid receptors (GR). ER was positive in 58% of the specimens; PR and GR were positive in 57%. A positive association was found between ER but not PR or GR frequency and age. Frequency of ER, PR, and GR positivity was approximately the same in premenopausal and postmenopausal women but ER content was much higher in postmenopausal women. About 70% of ER+ patients were also PR+ and GR+. Both frequency of PR positivity as well as average concentration, and frequency of GR positivity as well as average concentration were positively correlated with ER.

Sixty-six human ductal infiltrative breast carcinomas were examined for their estrogen receptor (ER) by the dextran-coated charcoal procedure and for the presence of carcinoembryonic antigen (CEA) by immunofluorescence with the aid of monoclonal antibodies. Statistically significant differences in CEA positivity were found between stage III (33%) and stage II (19%) tumors (Z = 2.08; p less than 0.02) and between ER + (22%) and ER- (37%) tumors (Z = 2.083; p less than 0.02). An inverse correlation was observed between positivity and receptor concentrations. No difference in CEA positivity was observed between pre- and post-menopausal women.

Androgens have been frequently used in the treatment of breast cancer. However, objective responses seem to vary according to the steroid hormone receptor expression of the tumor. We have studied the relationship between concentrations of androgen receptors (AR) and those of estrogen (ER) and progesterone (PR) receptors by multiple regression and stratified analysis techniques in 154 cases of primary breast carcinomas and 39 cases of benign tumors. Both the proportion of AR-positive tumors and the concentration of AR were dependent upon the coexpression of ER and PR by the specimens. This association was evident for malignant tumors with predominance of the positive correlation between AR and ER over that of AR and PR in post-menopausal patients. In premenopausal women, ER and PR concentrations were similarly correlated with AR levels. The PR, but not the ER concentration, was positively correlated to AR levels in benign breast tumors. These findings were confirmed by multiple regression, taking into consideration additional information about the patients to build statistical models allowing prediction of AR. Among the other variables considered in building these models--age, menopausal status, weight, height, and clinical stage--only height (using data from all patients) and age (data from post-menopausal women) emerged in addition to ER and PR as significantly explaining the variability of AR as the dependent variable.