Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells

Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17beta-estradiol (E2) up-regulates PI3K in an ERalpha-dependent manner, but not ERbeta, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ERalpha-positive MCF-7 cells and ERalpha-negative MDA-MB-231 cells with 10nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP(3) level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ERalpha-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ERalpha-dependent mechanism in MCF-7 cells.     

Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein

Knowledge regarding the expression of the recently cloned estrogen receptor beta (ERbeta) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ERbeta mRNA, but lacked ERalpha mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while genistein possessed slight growth inhibitory effects on HT29, Colo320 and Lovo cells at 10 microM, at which concentration is stimulated the growth of ERalpha-positive human breast cancer MCF-7 cells. Tamoxifen inhibited the growth of HT29 and Colo320 cells, dose-dependently, as well as MCF-7 cells. A transfected reporter plasmid containing a vitellogenin estrogen response element could be activated by estradiol in Colo320 cells. Taken together with previous reports, these data suggest that ERalpha and ERbeta may have different biological functions in colon cells.     

Estradiol rapidly activates Akt via the ErbB2 signaling pathway

Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-alpha (ERalpha) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ERalpha into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ERalpha. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ERalpha, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.     

Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R2=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line.     

Effects of SDF-1-CXCR4 signaling on microRNA expression and tumorigenesis in estrogen receptor-alpha (ER-α)-positive breast cancer cells

The majority of breast cancer cases ultimately become unresponsive to endocrine therapies, and this progression of breast cancer from hormone-responsive to hormone-independent represents an area in need of further research. Additionally, hormone-independent carcinomas are characterized as being more aggressive and metastatic, key features of more advanced disease. Having previously shown the ability of the stromal-cell derived factor-1 (SDF-1)–CXCR4 signaling axis to promote primary tumorigenesis and hormone independence by overexpressing CXCR4 in MCF-7 cells, in this study we further examined the role of SDF-1/CXCR4 in the endogenously CXCR4-positive, estrogen receptor α (ER-α)-positive breast carcinoma cell line, MDA–MB-361. In addition to regulating estrogen-induced and hormone-independent tumor growth, CXCR4 signaling stimulated the epithelial-to-mesenchymal transition, evidenced by decreased CDH1 expression following SDF-1 treatment. Furthermore, inhibition of CXCR4 with the small molecule inhibitor AMD3100 induced CDH1 gene expression and inhibited CDH2 gene expression in MDA–MB-361 cells. Further, exogenous SDF-1 treatment induced ER-α-phosphorylation in both MDA–MB-361 and MCF-7–CXCR4 cells, demonstrating ligand-independent activation of ER-α through CXCR4 crosstalk. qPCR microRNA array analyses of the MDA–MB-361 and MCF-7–CXCR4 cell lines revealed changes in microRNA expression profiles induced by SDF-1, consistent with a more advanced disease phenotype and further supporting our hypothesis that the SDF-1/CXCR4 signaling axis drives ER-α-positive breast cancer cells to a hormone independent and more aggressive phenotype. In this first demonstration of SDF-1–CXCR4-induced microRNAs in breast cancer, we suggest that this signaling axis may promote tumorigenesis via microRNA regulation. These findings represent future potential therapeutic targets for the treatment of hormone-independent and endocrine-resistant breast cancer.     

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.     

Effects of the environmental estrogens bisphenol A, o,p'-DDT, p-tert-octylphenol and coumestrol on apoptosis induction, cell proliferation and the expression of estrogen sensitive molecular parameters in the human breast cancer cell line MCF-7

In the presented study, we have analysed effects of the environmental estrogens bisphenol A (BPA), p-tert-octylphenol (OCT), o,p'-DDT (DDT) and coumestrol (COU) on cell proliferation, apoptosis induction, progesterone receptor (PR) and androgen receptor (AR) mRNA expression and ER alpha protein expression in comparison to estradiol (E2) and the selective ER modulator (SERM) raloxifene (RAL) and the pure antiestrogen faslodex (ICI 182780) in the human breast cancer cell line MCF-7. A dose dependent analysis of the cell cycle distribution of MCF-7 cells after administration of OCT, DDT and COU revealed a significant induction of cell proliferation and reduced rate of apoptosis. Maximum induction of cell proliferation and the lowest rate of apoptosis could be observed at a dose of 10(-6)M. Interestingly, administration of BPA reduces the rate of apoptosis, but does not enhance proliferation at any dose analysed. PR mRNA expression in MCF-7 cells was up regulated after administration of COU and DDT, whereas treatment with BPA and OCT did not effect PR mRNA expression. AR mRNA expression was down regulated by COU, but not effected by BPA, DDT and OCT. The expression of ER alpha protein in the breast cancer cells was slightly down regulated by COU and DDT, but unaffected by BPA and OCT. In summary and in comparison to the effects observed after administration of E2, RAL and ICI our data indicate that none of the analysed compounds exhibit properties comparable to RAL and ICI. COU and DDT exhibit properties which are very similar to E2. Administration of BPA and OCT did not effect any of the estrogen sensitive molecular parameters analysed. Nevertheless OCT is a very potent stimulator of cell proliferation in MCF-7 cells. Surprisingly, BPA is not able to induce the proliferation of MCF-7 breast cancer cells, but turns out to be a very potent inhibitor of apoptosis. For this reason and in agreement to the effects of BPA on the molecular parameters analysed, we conclude that BPA does not act in a classical estrogen like manner in MCF-7 breast cancer cells.     

Phosphorylation by p38 mitogen-activated protein kinase promotes estrogen receptor α turnover and functional activity via the SCF(Skp2) proteasomal complex

The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCF(Skp2) proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers.     

Macrophages, estrogen and the microenvironment of breast cancer

Estrogen is a major mitogenic stimulus to established breast cancer. Estrogen sources include ovarian, extraglandular sites and breast tissue. Which source primarily maintains benign and breast cancer tissue estrogen concentrations remains unclear. While macrophages may comprise up to 50% of the mass of breast carcinomas, previous studies neglected to study them as possible sources of estrogen. We present evidence that breast macrophages constitute an in situ source of estradiol and that the amount produced is sufficient to mediate cellular proliferation. We utilized immunohistochemistry and RT-PCR to study cell-specific aromatase expression in (i) 29 breast biopsies, (ii) human monocytes/macrophages and (iii) a myeloid cell line (THP-1) capable of differentiating into macrophages. Use of a breast cancer cell line (MCF-7) provided biologic confirmation of the role of aromatization in cell proliferation. We demonstrated considerable amounts of immunoreactive-aromatase (irARO) in breast tissue macrophages and a positive correlation between the proportion of irARO present in macrophages and lesion severity. Using in vitro techniques, we demonstrated that monocytes and THP-1 cells require differentiation into macrophages to produce aromatase in amounts approaching placental levels. The amount of estrogen produced by THP-1 cells stimulated MCF-7 cells to proliferate, an effect blocked by aromatase inhibitors. Estrogen production by macrophages in breast tissue appears sufficient to stimulate the proliferation of adjacent epithelial cells and to autoregulate cytokine production. These findings represent a new dimension of cellular regulation in breast tissue with major biologic implications, amenable to pharmacological manipulation.