Inducible upregulation of oestrogen receptor-beta1 affects oestrogen and tamoxifen responsiveness in MCF7 human breast cancer cells

To investigate the effect of altered oestrogen receptor (ER)alpha and ERbeta expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERbeta1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERbeta1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERbeta1 did not affect endogenous levels of ERalpha but increased progesterone receptor (PR) levels. Over-expression of ERbeta1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERalpha. In oestrogen-replete conditions, over-expression of ERbeta1 but not ERalpha reduced proliferation. Over-expression of ERbeta1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERbeta1 in the presence of an endogenously expressed ERalpha was associated with tamoxifen sensitivity but may negatively modulate ERalpha-mediated growth. However, not all ERalpha activities were inhibited since endogenous PR expression was increased by both ERalpha and ERbeta1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERbeta expression as well as ERbeta expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERbeta1 expression in ER-positive breast cancer.     

Resistance to different antiestrogens is caused by different multi-factorial changes and is associated with reduced expression of IGF receptor Ialpha

Development of antiestrogen resistance is a major clinical problem, and therefore it is crucial to elucidate the mechanisms involved. To investigate whether gain-of-function or loss-of-function mechanisms was most likely to be involved, cell fusion between the antiestrogen-sensitive MCF-7 and the ICI 164384- and ICI 182780-resistant MCF-7/164(R)-5 cell lines was performed. Furthermore, a fusion cell line between the tamoxifen-resistant MCF-7/TAM(R)-1 and the MCF-7/164(R)-5 cell line was established. A thorough investigation of growth parameters and expression of selected proteins (estrogen receptor-alpha (ERalpha), progesterone receptor (PR), Bcl-2, IGF-binding protein-2 (IGFBP2) and IGF receptor Ialpha (IGF-IRalpha)) in the fusion partners and fusion cells revealed that both gain- and loss-of-function changes occurred, and that the mechanisms resulting in resistance to the two antiestrogens were different. This multi-factoriality of antiestrogen resistance is promising in relation to sequential treatment of breast cancer patients with different types of endocrine therapy. Furthermore, we found an association between antiestrogen resistance and reduced IGF-IRalpha expression. Overall, the data presented in this report support the usefulness of cell fusion to clarify the mechanisms involved in development of resistance to the pure antiestrogens ICI 182780 and ICI 164384 and the selective ER modulator tamoxifen and suggest IGF-IRalpha as a new sensitive marker for response to antiestrogen treatment.     

Activation of adenosine deaminase in MCF-7 cells through IGF-estrogen receptor alpha crosstalk

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.     

Transcriptional activation of heat shock protein 27 gene expression by 17beta-estradiol and modulation by antiestrogens and aryl hydrocarbon receptor agonists

Heat shock protein 27 (Hsp 27) is expressed in mammary tumors and may play a role in tumor growth and response to anti-neoplastic drug therapy. 17beta-Estradiol (E2) induces Hsp 27 mRNA levels in MCF-7 human breast cancer cells, and we have investigated the comparative inhibitory mechanisms using the aryl hydrocarbon receptor (AhR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the direct-acting antiestrogen ICI 164,384. TCDD inhibited E2-induced Hsp 27 gene expression and analysis of the Hsp 27 gene promoter showed that the inhibitory response was associated with AhR interactions with a pentanucleotide motif at -3 to +2 in the promoter that corresponded to the core sequence of a dioxin responsive element. In contrast, ICI 164,384 induced Hsp 27 gene expression and reporter gene activity in MCF-7 cells and this represents one of the few examples of the estrogen receptor-alpha (ERalpha) agonist activity of the 'pure' antiestrogen ICI 164,384.     

Role of estrogen receptor (ER) alpha in insulin-like growth factor (IGF)-I-induced responses in MCF-7 breast cancer cells

Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that induces proliferation of MCF-7 breast cancer cells, and cotreatment with the phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 and the antiestrogen ICI 182780 inhibits IGF-I-induced growth. The role of estrogen receptor alpha (ERalpha) in mediating responses induced by IGF-I was investigated in cells transfected with small inhibitory RNA for ERalpha (iERalpha). The results showed that IGF-I-dependent phosphorylation of Akt and mitogen-activated protein kinase, induction of G(1)-S-phase progression and enhanced expression of cyclin D1 and cyclin E were dependent on ERalpha. Moreover, these same IGF-I-induced responses were also inhibited by the antiestrogen ICI 182780 and this was in contrast to a previous report suggesting that ICI 182780 did not affect IGF-I-dependent activation of PI3-K or induction of cyclin D1 expression. ICI 182780 exhibits antimitogenic activity and iERalpha inhibits G(1)-S-phase progression and proliferation of MCF-7 cells treated with IGF-I, suggesting that the effects of the antiestrogen are primarily related to downregulation of ERalpha.     

Cadmium induces mitogenic signaling in breast cancer cell by an ERalpha-dependent mechanism

Breast cancer (BC) is linked to estrogen exposure. Estradiol (E2) stimulates BC cells proliferation by binding the estrogen receptor (ER). Hormone-related cancers have been linked to estrogenic environmental contaminants. Cadmium (Cd) a toxic pollutant, acts as estrogens in BC cells. Purpose of our study was to evaluate whether Cd regulates MCF-7 cell proliferation by activating ERK1/2, Akt and PDGFRalpha kinases. Cd increased cell proliferation and the ER-antagonist ICI 182,780 blunted it. To characterize an ER-dependent mechanism, ERalpha/beta expression was evaluated. Cd decreased ERalpha expression, but not ERbeta. Cd also increased ERK1/2, Akt and PDGFRalpha phosphorylation while ICI blocked it. Since stimulation of phosphorylation was slower than expected, c-fos and c-jun proto-oncogenes, and PDGFA were analyzed. Cd rapidly increased c-jun, c-fos and PDGFA expression. Cells were also co-incubated with the Cd and specific kinases inhibitors, which blocked the Cd-stimulated proliferation. In conclusion, our results indicate that Cd increases BC cell proliferation in vitro by stimulating Akt, ERK1/2 and PDGFRalpha kinases activity likely by activating c-fos, c-jun and PDGFA by an ERalpha-dependent mechanism.     

Oestrogen-induced apoptosis in colonocytes expressing oestrogen receptor beta

Epidemiological studies of postmenopausal hormone replacement therapy show a reduction in the risk of developing colon cancer, and animal studies using 17beta-oestradiol (E(2)) demonstrate a decreased incidence of chemically-induced colon cancer. Using the colon cancer cell line, COLO205, we found that E(2) induced a dose-dependent increase in DNA fragmentation and nuclear condensation, significant effects being seen at 10(-12 )mol/l. BSA-conjugated E(2), which cannot enter cells, was ineffective at inducing apoptosis in COLO205 cells, indicating that E(2) was not acting through a cell-membrane receptor. E(2) did not induce the morphological changes characteristic of differentiation. Using RT-PCR we found that the oestrogen receptor alpha (ERalpha) isoform was absent in the COLO205 cell line in contrast to CACO-2, LoVo and SW620 cells, but mRNAs for ERbeta1, -beta2, -beta5 and -beta6 isoforms were detected. Western immunoblotting results showed full-length ERbeta protein but no detectable ERalpha in COLO205 cells. In normal human colon tissue samples immunoreactive ERbeta was found but ERalpha was barely detectable. Expression of ERbeta was lost in some colon cancer specimens and reduced in others. We conclude that E(2), through ERbeta, at concentrations found during replacement therapy, may inhibit the development of colon cancer by inducing apoptosis.     

Both estrogen and raloxifene cause G1 arrest of vascular smooth muscle cells

The proliferation of vascular smooth muscle cells (VSMC) is a crucial pathophysiological process in the development of atherosclerosis. Although estrogen is known to inhibit the proliferation of VSMC, the mechanism responsible for this effect remains to be elucidated. In addition, the effect of raloxifene on VSMC remains unknown. We have shown here that 17beta-estradiol (E(2)) and raloxifene significantly inhibited the platelet-derived growth factor (PDGF)-stimulated proliferation of cultured human VSMC. Flow cytometry demonstrated that PDGF-stimulated S-phase progression of the cell cycle in VSMC was also suppressed by E(2) or raloxifene. We found that PDGF-induced phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by E(2) and raloxifene. These effects were associated with a decrease in cyclin D1 expression, without a change in cyclin-dependent kinase 4 or cyclin-dependent kinase inhibitor, p27(kip1) expression. ICI 182,780 abolished the inhibitory effects of E(2) and raloxifene on PDGF-induced pRb phosphorylation. Next, we examined which estrogen receptor (ER) is necessary for these effects of E(2) and raloxifene. Since VSMC express both ERalpha and ERbeta, A10, a rat aortic smooth muscle cell line that expresses ERbeta but not ERalpha, was used. The dose-dependent stimulation of A10 cell proliferation by PDGF was not inhibited by E(2) or raloxifene in contrast to the results obtained in VSMC. Moreover, E(2) and raloxifene significantly inhibited the PDGF-induced cyclin D1 promoter activity in A10 cells transfected with cDNA for ERalpha but not in the parental cells. These results suggested that E(2) and raloxifene exert an antiproliferative effect in VSMC treated with PDGF, at least in part through inhibition of pRb phosphorylation, and that the inhibitory effects of E(2) and raloxifene may be mainly mediated by ERalpha.