Estrogen-dependent growth and estrogen receptor (ER)-α concentration in T47D breast cancer cells are inhibited by VACM-1, a cul 5 gene

Vasopressin-activated calcium mobilizing receptor (VACM-1)/cullin 5 (cul 5) inhibits growth when expressed in T47D breast cancer cells by a mechanism that involves a decrease in MAPK phosphorylation and a decrease in the early growth response element (egr-1) concentration in the nucleus. Since both MAPK and egr-1 pathways can be regulated by 17β-estradiol, we next examined the effects of VACM-1 cDNA expression on estrogen-dependent growth in T47D cells and on estrogen receptor (ER) concentrations. Our results demonstrate that in T47D cells, both basal and 17β-estradiol-dependent increase in cell growth and MAPK phosphorylation were inhibited in cells transfected with VACM-1 cDNA. Further, Western blot and immunocytochemistry data analyses indicate that ER concentrations and its nuclear localization are significantly lower in cells transfected with VACM-1 cDNA when compared to controls. These data indicate that in the T47D cancer cell line VACM-1 inhibits growth by attenuating estrogen-dependent signaling responses. These findings may have implications in the development of cancer treatments.     

Resveratrol enhances anti-proliferative effect of VACM-1/cul5 in T47D cancer cells

Vasopressin-activated calcium-mobilizing (VACM-1) protein is a cul-5 gene product that forms complexes with a subclass of ubiquitin E3 ligases involved in proteasomal protein degradation. The expression of VACM-1 cDNA in the T47D breast cancer cell line inhibits growth and decreases phosphorylation of mitogen activated protein kinase. Factors that regulate expression or stability of VACM-1 protein have not been identified, however. In our search to identify drugs/substances that may control VACM-1 protein expression, we examined the effects of resveratrol (trans-3,5,4′-trihydroxystilbene), a natural component in the human diet which inhibits tumor initiation and promotion. CMV vector and VACM-1 cDNA stably transfected T47D breast cancer-derived cells were treated with resveratrol and cell growth and VACM-1 protein concentrations were measured. Since the cellular mechanism of resveratrol-dependent inhibition of cell growth also involves the regulation of estrogen receptors, the effect of 17-β−estradiol and resveratrol on ERα levels and on cell growth was examined in control and in VACM-1 cDNA transfected cells. Our results demonstrate that antiproliferative effect of resveratrol observed in the control T47D cancer cells was significantly enhanced in VACM-1 cDNA transfected T47D cells. Western blot results indicated that resveratrol increased VACM-1 protein concentration. Finally, treatment with resveratrol for 24 and 48 h attenuated 17-β−estradiol induced increase in cell growth both in control and in VACM-1 cDNA transfected cells. The effect was significantly higher in the VACM-1 cDNA transfected cells when compared to controls. These results indicate that the antiproliferative effect of resveratrol may involve induction of VACM-1/cul5.     

VACM-1, a cul-5 gene,inhibits cellular growth by a mechanism that involves MAPK and p53 signaling pathways

Vasopressin-activated Ca²⁺-mobilizing (VACM)-1 gene product is a 780-amino acid membrane protein that shares sequence homology with cullins, a family of genes involved in the regulation of cell cycle. However, when expressed in vitro, VACM-1 attenuates basal and vasopressin- and forskolin-induced cAMP production. Mutating the PKA-dependent phosphorylation site in the VACM-1 sequence (^S730AVACM-1) prevents this inhibitory effect. To further examine the biological role of VACM-1, we studied the effect of VACM-1 and ^S730AVACM-1 proteins on cellular proliferation and gene expression in Chinese hamster ovary and COS-1 cells. Cellular proliferation of VACM-1-expressing cell lines was significantly lower compared with that of the vector-transfected cells, whereas it was significantly increased in ^S730AVACM-1-derived cell lines. Furthermore, expression of VACM-1 but not ^S730AVACM-1 protein retarded cytokinesis and prevented MAPK phosphorylation. Screening with the Human PathwayFinder-1 GEArray system and subsequent Western blot analysis demonstrated that VACM-1 induces p53 mRNA and protein expression. In summary, VACM-1 inhibits cellular growth by a mechanism that involves cAMP, MAPK phosphorylation, and p53 expression. mitogen-activated protein kinase; cytokinesis; vasopressin-activated calcium-mobilizing receptor     

Progestins induce down-regulation of insulin-like growth factor-I (IGF-I) receptors in human breast cancer cells: potential autocrine role of IGF-II.

Insulin-like growth factor-I (IGF-I) receptors are present in breast cancer cells and may play a role in breast cancer cell growth. We have studied the effect of progestins on IGF-I receptors in T47D human breast cancer cells. T47D cells constitutively express high levels of progesterone receptors and are a model for studying the regulation of cellular functions by progestins. Treatment of T47D cells with either progesterone or the synthetic progestin promegestone (R5020) decreased IGF-I receptor content by approximately 50%, as measured by Scatchard analysis and receptor biosynthesis studies. In contrast to progestins, estradiol, dexamethasone, and dihydrotestosterone did not influence IGF-I receptor content. No effect of R5020 was seen after 12 h of incubation, a near-maximal effect was seen after 24 h, and greatest effects were seen after 72 h. R5020 decreased IGF-I receptor mRNA abundance, indicating that progestins acted at the level of gene expression. However, progestins also increased the secretion of IGF-II, a ligand for the IGF-I receptor. In contrast to IGF-II, T47D cells did not express IGF-I. The addition of exogenous IGF-II to T47D cells down-regulated both IGF-I receptor binding and IGF-I receptor mRNA abundance. This study indicates, therefore, that progestins regulate IGF-I receptors in breast cancer cells and suggests that this regulation occurs via an autocrine pathway involving enhanced IGF-II secretion.     

Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells.

The expression of vascular endothelial growth factor (VEGF) by breast tumors has been previously correlated with a poor prognosis in the pathogenesis of breast cancer. Furthermore, VEGF secretion is a prerequisite for tumor development. Although most of the effects of VEGF have been shown to be attributable to the stimulation of endothelial cells, we present evidence here that breast tumor cells are capable of responding to VEGF. We show that VEGF stimulation of T-47D breast cancer cells leads to changes in cellular signaling and invasion. VEGF increases the cellular invasion of T-47D breast cancer cells on Matrigel/ fibronectin-coated transwell membranes by a factor of two. Northern analysis for the expression of the known VEGF receptors shows the presence of moderate levels of Flt-1 and low levels of Flk-1/KDR mRNAs in a variety of breast cancer cell lines. T-47D breast cancer cells bind 125I-labeled VEGF with a Kd of 13 x 10(-9) M. VEGF induces the activation of the extracellular regulated kinases 1,2 as well as activation of phosphatidylinositol 3'-kinase, Akt, and Forkhead receptor L1. These findings in T-47D breast cancer cells strongly suggest an autocrine role for VEGF contributing to the tumorigenic phenotype.     

Extracellular matrix enhances heregulin-dependent BRCA1 phosphorylation and suppresses BRCA1 expression through its C terminus

Germ line mutations in the breast cancer susceptibility gene BRCA1 account for the increased risk of early onset of familial breast cancer, whereas overexpression of the ErbB family of receptor tyrosine kinases has been linked to the development of nonfamilial or sporadic breast cancer. To analyze whether there is a link between these two regulatory molecules, we studied the effects of ErbB-2 activation by heregulin (HRG) on BRCA1 function. It was previously demonstrated that HRG induced the phosphorylation of BRCA1, which was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Since altered interaction between cells and the surrounding extracellular matrix (ECM) is a common feature in a variety of tumors and since ECM modulates intracellular signaling, we hypothesized that ECM may affect the expression and HRG-dependent phosphorylation of BRCA1. Following stimulation by HRG, a strong increase in [(3)H]thymidine incorporation was observed in human T47D breast cancer cells seeded on plastic (PL). When T47D cells were seeded on laminin (LAM) or Matrigel, HRG induced a significantly higher proliferation than it did in cells seeded on PL. T47D cells seeded on poly-L-lysine had an abrogated mitogenic response, indicating the involvement of integrins in this process. HRG treatment induced a transient phosphorylation of BRCA1 that was enhanced in T47D cells grown on LAM. LAM-enhanced BRCA1 phosphorylation was mediated through alpha(6) integrin upon HRG stimulation. Accordingly, T47D cells grown on LAM had the greatest increase in ErbB-2 activation, PI3K activity, and phosphorylation of Akt. A similar pattern of BRCA1 mRNA expression was observed when T47D cells were seeded on PL, LAM, or COL4. There was a significant decrease in the steady state of the BRCA1 mRNA level on both the LAM and COL4 matrices compared to that for cells seeded on PL. In addition, HRG stimulation caused a significant decrease in BRCA1 mRNA expression that was dependent on protein synthesis. Pretreatment with both the calpain inhibitor ALLN (N-acetyl-Leu-Leu-norleucinal) and the proteosome inhibitor lactacystin inhibited the HRG-induced down-regulation of BRCA1 mRNA expression. Likewise, there was a strong decrease in the protein level of BRCA1 in T47D cells 4 h after treatment with HRG compared to its level in control nontreated T47D cells. Pretreatment with the proteosome inhibitors ALLN, lactacystin, and PSI [N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal] inhibited also the HRG-induced down-regulation of BRCA1 protein in breast cancer cells. Interestingly, BRCA1 mRNA expression in HCC-1937 breast cancer cells, which express C-terminally truncated BRCA1, was not affected by either LAM or CL4. No phosphorylation of BRCA1 from HCC-1937 cells was observed in response to HRG. While Cdk4 phosphorylated wild-type BRCA1 in response to HRG in T47D cells, Cdk4 failed to phosphorylate the truncated form of BRCA1 in HCC-1937 cells. Furthermore, overexpression of wild-type BRCA1 in HCC-1937 cells resulted in the phosphorylation of BRCA1 and decreased BRCA1 expression upon HRG stimulation while overexpression of truncated BRCA1 in T47D cells resulted in a lack of BRCA1 phosphorylation and restoration of BRCA1 expression. These findings suggest that ECM enhances HRG-dependent BRCA1 phosphorylation and that ECM and HRG down-regulate BRCA1 expression in breast cancer cells. Furthermore, ECM suppresses BRCA1 expression through the C terminus of BRCA1.     

Complement component 1, q subcomponent binding protein is a marker for proliferation in breast cancer

Complement component 1, q subcomponent binding protein (C1QBP), is a multi-compartmental protein with higher mRNA expression reported in breast cancer tissues. This study evaluated the association between immunohistochemical expression of the C1QBP protein in breast cancer tissue microarrays (TMAs) and clinicopathological parameters, in particular tumor size. In addition, an in vitro study was conducted to substantiate the breast cancer TMA findings. Breast cancer TMAs were constructed from pathological specimens of patients diagnosed with invasive ductal carcinoma. C1QBP protein and proliferating cell nuclear antigen (PCNA) immunohistochemical analyses were subsequently performed in the TMAs. C1QBP immunostaining was detected in 131 out of 132 samples examined. The C1QBP protein was predominantly localized in the cytoplasm of the breast cancer cells. Univariate analysis revealed that a higher C1QBP protein expression was significantly associated with older patients (P = 0.001) and increased tumor size (P = 0.002). Multivariate analysis showed that C1QBP is an independent predictor of tumor size in progesterone-positive tumors. Furthermore, C1QBP was also significantly correlated with expression of PCNA, a known marker of proliferation. Inhibition of C1QBP expression was performed by transfecting C1QBP siRNA into T47D breast cancer cells, a progesterone receptor-positive breast cancer cell line. C1QBP gene expression was analyzed by real-time RT-PCR, and protein expression by Western blot. Cell proliferation assays were also performed by commercially available assays. Down-regulation of C1QBP expression significantly decreased cell proliferation and growth in T47D cells. Taken together, our findings suggest that the C1QBP protein could be a potential proliferative marker in breast cancer.     

VACM-1/cul5 expression in vascular tissue in vivo is induced by water deprivation and its expression in vitro regulates aquaporin-1 concentrations

VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24?h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24?h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.     

S14 protein in breast cancer cells: direct evidence of regulation by SREBP-1c, superinduction with progestin, and effects on cell growth

Most breast cancers exhibit brisk lipogenesis, and require it for growth. S14 is a lipogenesis-related nuclear protein that is overexpressed in most breast cancers. Sterol response element-binding protein-1c (SREBP-1c) is required for induction of lipogenesis-related genes, including S14 and fatty acid synthase (FAS), in hepatocytes, and correlation of SREBP-1c and FAS expression suggested that SREBP-1c drives lipogenesis in tumors as well. We directly tested the hypothesis that SREBP-1c drives S14 expression and mediates lipogenic effects of progestin in T47D breast cancer cells. Dominant-negative SREBP-1c inhibited induction of S14 and FAS mRNAs by progestin, while active SREBP-1c induced without hormone and superinduced in its presence. Changes in S14 mRNA were reflected in protein levels. A lag time and lack of progestin response elements indicated that S14 and FAS gene activation by progestin is indirect. Knockdown of S14 reduced, whereas overexpression stimulated, T47D cell growth, while nonlipogenic MCF10a mammary epithelial cells were not growth-inhibited. These data directly demonstrate that SREBP-1c drives S14 gene expression in breast cancer cells, and progestin magnifies that effect via an indirect mechanism. This supports the prediction, based on S14 gene amplification and overexpression in breast tumors, that S14 augments breast cancer cell growth and survival.     

Adenovirus-directed expression of dominant negative estrogen receptor induces apoptosis in breast cancer cells and regression of tumors in nude mice.

BACKGROUND: Estrogen receptors (ER) are expressed in about two thirds of human breast cancer, and are an important pharmacological target for treatment of these tumors. Dominant negative forms of the ER have been suggested as an alternative method to disrupt ER function. In this study, we examined the effect of dominant negative ER mutants (ER1-536 and L540Q) on ER-positive breast cancer cells in vitro and in vivo. MATERIALS AND METHODS: ER-positive T47D breast cancer cells were infected with adenoviral vectors expressing ER1-536 and L540Q to examine the effects of the mutants on gene expression and cell growth. Adenoviral vectors containing the wild type ER (AdwtER) and beta-galactosidase gene (AdGal) were used as controls. RESULTS: Ad1-536 or AdL540Q infection inhibited T47D cell growth and induced apoptosis, increasing Bax protein and phosphorylation of p38 mitogen-activated-protein kinase (MAPK). Consistent with the apoptotic effects in vitro, pre-infection of T47D cells with Ad1-536 or AdL540Q inhibited tumor formation when these cells were introduced into nude mice. In addition, injection of Ad1-536 and AdL540Q into pre-established T47D tumors induced tumor regression. Apoptosis, in conjunction with the activation of caspase-3 and phosphorylation of p38 MAPK, was detected in the shrinking tumors. Overexpression of wild-type ER by AdwtER infection also produced antiproliferative and apoptotic effects, but to a lesser extent than the ER1-536 and L540Q mutants. CONCLUSIONS: These results indicate that dominant negative ER mutants have the potential to induce apoptosis of T47D cells and regression of tumors. The delivery of dominant negative ERs by adenoviral vectors may provide a useful tool for targeted therapy of ER-positive breast cancer.     

Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 (^S730AVACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with ^S730AVACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with ^S730AVACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.     

Src Drives Growth of Antiestrogen Resistant Breast Cancer Cell Lines and Is a Marker for Reduced Benefit of Tamoxifen Treatment

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.     

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.     

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.     

A mechanism of drug resistance to tamoxifen in breast cancer

Drug resistance to tamoxifen (Tam) is a significant clinical problem but the mechanism through which this occurs remains elusive. We have developed a number of xenograft models of Tam-stimulated growth that model breast cancer progression using estrogen receptor positive MCF-7 or T47D breast cancer cells. When estrogen-stimulated T47D:E2 tumors are treated long term with Tam, Tam-stimulated tumors develop (T47D:Tam) that are stimulated by both estrogen and Tam. When HER-2/neu status is determined, it is clear that the T47D:Tam tumors express significantly higher levels of HER-2/neu protein by immunohistochemistry and mRNA as measured by real-time RT-PCR. The T47D:Tam tumors also express higher levels of estrogen receptor and progesterone receptor protein than their estrogen-stimulated T47D:E2 counterparts. We compared out results to the MCF-7 model of Tam-stimulated growth. The MCF-7:Tam ST (estrogen- and Tam-stimulated) and MCF-7:Tam LT (estrogen-inhibited, Tam-stimulated) were bilaterally transplanted to account for any mouse to mouse variation and characteristic growth patterns were observed. TUNEL staining was performed on MCF-7:Tam LT treated with either estrogen or Tam and it was concluded that estrogen-inhibited tumor growth was a result of increased apoptosis. Three phases of tumor progression are described that involve increases in HER-2/neu expression, de-regulation of estrogen receptor expression and increases in apoptosis which in concert determine the phenotype of drug resistance to Tam.     

Expression of estrogen and progesterone receptors and estrogen metabolizing enzymes in different breast cancer cell lines

Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.