Effect of estrogen and antiestrogens on cell proliferation and synthesis of secreted proteins in the human breast cancer cell line MCF-7 and a tamoxifen resistant variant subline, AL-1.

The human breast cancer cell line MCF-7 contains estrogen receptors and responds to estrogens with an increase in growth rate and to antiestrogens with a decrease in growth rate. Estrogen stimulation of cell proliferation is concomitant with an increase in the synthesis and secretion of three proteins with mol. wt 52 kDa, 61 kDa and 66 kDa and a decrease in the synthesis and secretion of a 42 kDa protein. The antiestrogen ICI 164,384 has a complete estrogen antagonistic effect on the synthesis of these secreted proteins, whereas the antiestrogen tamoxifen has an agonistic effect on the synthesis and secretion of the 52 kDa protein. We believe that the above mentioned estrogen regulated secreted proteins are either directly or indirectly involved in control of cell proliferation, and the less pronounced inhibitory effect of tamoxifen on cell proliferation compared to ICI 164,384 may be due to agonistic effects of tamoxifen. A tamoxifen resistant variant of the MCF-7 cell line, the AL-1 subline, can be growth inhibited by ICI 164,384, although a higher concentration is needed to inhibit the AL-1 cells compared to the parent MCF-7 cells. Tamoxifen has no effect on secreted proteins from the AL-1 cells, whereas ICI 164,384 has a complete estrogen antagonistic effect on secreted proteins, indicating that the mechanisms by which estrogens and antiestrogens influence cell proliferation may be via up and down regulation of secreted proteins with growth regulatory functions.     

EGFRvIII‐induced estrogen‐independence,tamoxifen‐resistance phenotype correlates with PgR expression and modulation of apoptotic molecules in breast cancer

The tumor‐specific, ligand‐independent, constitutively active epidermal growth factor receptor (EGFR) variant, EGFRvIII, remains understudied in breast cancer. Here, we report that expression of EGFRvIII in the ErbB‐2‐overexpressing, estrogen‐dependent MDA‐MB‐361 breast cancer cell line resulted in significant estrogen‐independent tumor growth in ovariectomized, athymic nude mice in comparison to MDA‐MB‐361/wt cells. MDA‐MB‐361/vIII breast cancer cells maintained estrogen‐induced tumor growth, but were tamoxifen‐resistant in the presence of estrogen, while MDA‐MB‐361/wt cells had a significant reduction in tumor growth in the presence of estrogen and tamoxifen. Tamoxifen alone did not have a significant effect on EGFRvIII‐mediated estrogen‐independent tumor growth. Constitutive signaling from the EGFRvIII receptor resulted in an increased activation of both the Akt and MAPK pathways. Compared to estrogen‐dependent, tamoxifen‐sensitive MCF‐7/vIII breast cancer cells, which had unchanged levels of ERα, but an increase in progesterone receptor (PgR) in comparison to MCF‐7/wt cells, MDA‐MB‐361/vIII cells had a reduction in ERα expression as well as a more pronounced reduction in PgR compared with MDA‐MB‐361/wt cells. EGFRvIII expression was also significantly associated with an absence of PgR protein in invasive human breast cancer specimens. Alterations of proapoptotic proteins and antiapoptotic proteins were observed in EGFRvIII transfectants. In conclusion, constitutive signaling through EGFRvIII and its downstream effector proteins crosstalks with the ERα pathway, resulting in loss of PgR expression and alterations in the apoptotic pathway, which may result in the estrogen‐independent, tamoxifen‐resistant phenotype conferred to EGFRvIII‐expressing breast cancer cells. © 2009 UICC     

Characterization of estrogen and progesterone receptors and the dissociated regulation of growth and progesterone receptor stimulation by estrogen in MDA-MB-134 human breast cancer cells

We have examined the properties of the estrogen receptor and progesterone receptor in MDA-MB-134 human breast cells and have evaluated the effects of estrogen on cell proliferation and progesterone receptor levels in these cells as indices of hormonal sensitivity. These cells contain high levels of estrogen receptor (approximately 1.5 pmol/mg DNA) and low levels of progesterone receptor (0.15 pmol/mg DNA). More than 80% of the estrogen receptor is found in the nuclear fraction in the absence of estrogen, and the Kd of the receptor for estradiol is approximately 1.5 X 10(-10) M. Upon exposure to estradiol, the receptors become occupied, but there is no processing or apparent decrease in either nuclear or total cellular estrogen receptor content, as can be seen in MCF-7 human breast cancer cells. The nuclear estrogen receptor sediments as a 4.6 S species on high salt sucrose gradients, and it can be detected on sodium dodecyl sulfate-polyacrylamide gel immunoblot analysis as a species of molecular weight 65,000, identical to that of the MCF-7 estrogen receptor, using the monoclonal antibodies D75P3 gamma and H222Sp gamma prepared against the MCF-7 estrogen receptor. The estrogen receptor shows binding selectivity for estrogens and antiestrogens, and its affinity for ligands follows the order diethylstilbestrol (190%) greater than estradiol (100%) greater than estriol (13%) greater than tamoxifen (3%), as expected for estrogen receptor. Hence the receptor appears normal in many of its physicochemical properties and in terms of its binding affinity and specificity for estrogens and antiestrogens. Control cells contain low levels of progesterone receptor that display high affinity (Kd = 6 X 10(-9) M) for the synthetic progestin R5020, but exposure to estradiol (10(-11)-10(-7)M) fails to increase cellular progesterone receptor levels. In contrast, estradiol markedly stimulates the rate of cell proliferation, while tamoxifen suppresses the growth of control and of estradiol treated cells. Hence, our data show that these cells, which contain substantial levels of estrogen receptor, respond to estrogen with enhanced cell proliferation but fail to have their progesterone receptor level modulated by estradiol. These cells represent an interesting and unusual situation in which estrogenic regulation of proliferation and the stimulation of progesterone receptor are dissociated. These cells should prove useful in further evaluation of estrogenic regulation of cell proliferation and specific protein synthesis in human breast cancer.     

Biological activity of all-trans-retinoic acid with and without tamoxifen and alpha-interferon 2a in breast cancer patients

In addition to suppressing breast cancer cell growth, retinoids potentiate growth inhibition in human breast cancer when tested in vitro and in vivo with tamoxifen and/or interferon. The purpose of this study was to ascertain the biologic effects of all-trans-retinoic acid (ATRA) administered alone and with tamoxifen +/- interferon and to identify the relationship between ATRA plasma concentrations and optimal biological dose (the lowest dose that produces a biological response). Three consecutive groups of 15 patients with locally advanced operable breast cancer were treated, in accordance with good clinical practice (GCP) requirements, with ATRA at 3 dose levels alone or with tamoxifen +/- alpha-interferon 2a at flat doses. After 3 weeks, the tumors were surgically removed. Biological parameters measured at the beginning (in biopsy tissue) and end (in surgical tissue) of the study were compared. The optimal biological dose for ATRA was 15 mg/m2/day. Treatments influenced tumor grade but not cell cycle kinetics (G0-G1 phase) or proliferation (Ki67 levels). ATRA induced progesterone receptors independent of dose level and co-administered drugs, but did not induce estrogen receptors when administered alone. Retinoic acid receptor (RAR)-alpha was not affected by treatment and RAR-alpha was moderately influenced whereas RAR-beta (concomitantly with transforming growth factor-beta) was induced in 33% of patients by ATRA alone. ATRA pharmacokinetics were dose- and time-dependent. Neither the ATRA + tamoxifen nor the ATRA + tamoxifen + interferon combinations potentiated the ATRA-induced biological changes. Future studies evaluating the role of RAR-beta as a biological marker of retinoid activity are warranted.     

Combined effects of lonidamine and tamoxifen in human breast-cancer cell-lines

The antiproliferative activity of lonidamine, alone or in combination with the antiestrogen tamoxifen, was studied on estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines. Lonidamine by itself induced an appreciable cytotoxic effect on all five cell lines, with 50% inhibitory concentrations (IC50) ranging from 19.5 to 54 mu M. The combination of lonidamine and tamoxifen, simultaneously administered or in sequence, provided additive effects on the estrogen receptor-negative MCF/DX cell line and a sub-additive interaction in the estrogen receptor-positive MCF7 cells. The negative interference between the two drugs could be ascribed to the marked reduction induced by lonidamine in the expression of estrogen receptor in the MCF7 cells.     

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. III. Hormone receptor status and responsiveness

PMC42 is a new human breast carcinoma cell line. In this report the content of estrogen, progesterone, and glucocorticoid receptors in PMC42 has been determined. The estrogen receptor content (1,750 cytoplasmic sites and 350 nuclear sites) was lower than that described for MCF7 and T47D. The cells would not proliferate in serum-free medium without the addition of beta-estradiol (optimum concentration 10(-8) M) or progesterone (5 X 10(-8) M). The addition of both hormones induced a more than additive increase in proliferation (P less than .005, n = 18). Similarly, addition of insulin or hydrocortisone induced proliferation; however, in this case, the effect of the hormones together was only additive. The addition of tamoxifen (10(-6) M) led to a significant decrease in cell numbers and inhibited the stimulatory effects of 10(-8) M beta-estradiol.     

Effects of type I and II interferons on cultured human breast cells: interaction with estrogen receptors and with tamoxifen

The combined effects of tamoxifen, a competitive inhibitor of estrogen, and type I or II interferons on the proliferation of several human breast cancer cell lines in vitro were examined. Additive antiproliferative effects were observed with interferons and tamoxifen, in two estrogen receptor positive cell lines, MCF-7 and T-47D. In MCF-7 cells neither beta ser interferon, gamma interferon, nor alpha 6 interferon were able to significantly alter estrogen receptor levels. Antigrowth activities of beta ser interferon and gamma interferon in an estrogen receptor negative cell line, HS578T, were equivalent to those in estrogen receptor-positive cell lines. Consistent with the antiproliferative effects of interferons, high affinity beta ser interferon receptors and interferon inducible 2'5'-oligoadenylate synthetase were present in both MCF-7 and HS578T cell lines. Thus steroid hormone receptor status did not influence the antiproliferative effects of interferons on breast carcinoma cells in vitro.     

Cell proliferation,apoptosis, and expression of cyclin D1 and cyclin E as potential biomarkers in tamoxifen-treated mammary tumors

Tamoxifen has been widely used for treatment, and more recently, for the prevention of breast cancer. Since breast carcinomas are composed of heterogeneous populations of estrogen receptor-positive (ER+) cells, we hypothesized that tamoxifen may suppress tumor growth by differentially affecting cell proliferation and apoptosis. ER+ mammary tumors were induced in Sprague–Dawley rats by N-methyl-N-nitrosourea (MNU) and when they became palpable, the animals were treated for 5, 10, or 20 days with tamoxifen, 1.0 mg/kg body weight. Tamoxifen induced a time-dependent decrease in proliferating (BrdU-labeled) cells, arrested the cells in G1/0 phase, and differentially decreased the cyclin E and cyclin D1 expression at mRNA and protein levels. In the same tumors, apoptotic cells increased during the first 10 days of treatment, but their number remained unchanged with extension of the treatment to 20 days. Thus, we provide data that tamoxifen may differentially affect cell proliferation and apoptosis in mammary tumors and that the expression levels of cyclin D1 and cyclin E might also be considered potential intermediate biomarkers of response of mammary tumors to tamoxifen and possibly to other selective estrogen receptor modulators (SERMs).     

Herstatin inhibits heregulin-mediated breast cancer cell growth and overcomes tamoxifen resistance in breast cancer cells that overexpress HER-2

Ligands of the ErbB family of receptors and estrogens control the proliferation of breast cancer cells. Overexpression of human EGF receptor HER-2 (erbB2) leads to amplified heregulin (HRG) signaling, promoting more aggressive breast cancer that is nonresponsive to estrogen and the antiestrogenic drug tamoxifen. Herstatin (Hst), a secreted HER-2 gene product, binds to the HER-2 receptor ectodomain blocking receptor activation. The aim of this study was to investigate the impact of this HER-2 inhibitor on HRG-induced signaling, proliferation, and sensitivity to tamoxifen in breast cancer cells with and without HER-2 overexpression. The expression of Hst in MCF7 cells eliminated HRG signaling through both mitogen-activated protein kinase and Akt pathways and prevented HRG-mediated proliferation. The loss in signaling corresponded to downregulation of the HRG receptors, HER-3 and HER-4, whereas HER-2 overexpression strongly stimulated the levels of both HRG receptors. Although Hst blocked HRG signaling in both parental and HER-2 transfected cells, it enhanced sensitivity to tamoxifen only in the MCF7 cells that overexpressed HER-2. To evaluate further the efficacy of Hst as an anticancer agent, His-tagged Hst was expressed in transfected insect cells, purified, and added to the breast cancer cells. As in the transfected cells, purified Hst inhibited HER-3 levels and suppressed HRG-induced proliferation of MCF7 and BT474 breast cancer cells. In contrast, the HER-2 monoclonal antibody, herceptin, downregulated HER-2, but not HER-3. These results suggest the potential use of Hst against HRG-mediated growth of breast cancers with high and low levels of HER-2 and against tamoxifen resistance in HER-2 overexpressing breast cancer.     

Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens

We have examined the effect of short-term and long-term growth in the absence of estrogens on the proliferation rate and estrogen and antiestrogen responsiveness of MCF-7 human breast cancer cells. The removal of phenol red, the pH indicator in tissue culture medium that is weakly estrogenic (Y. Berthois et al., Proc. Natl. Acad. Sci. USA, 83:2496-2500, 1986), immediately slows the cell proliferation rate, and MCF-7 cells grown in phenol red-free medium with charcoal dextran-treated serum for periods up to 1 mo maintain this reduced rate of cell proliferation. In these short-term phenol red-withdrawn cells, estradiol stimulates proliferation markedly and reproducibly, and antiestrogens inhibit estrogen-stimulated proliferation. Antiestrogens by themselves appear as partial agonists/antagonists; at low concentrations they stimulate proliferation weakly, but they show no stimulation at the high concentrations where they fully inhibit estrogen-stimulated proliferation. In contrast to the short-term phenol red-withdrawn cells, cells maintained for several months (5 to 6 mo) in the apparently complete absence of estrogens (no phenol red, with charcoal dextran-treated calf serum) show a markedly increased basal rate of cell proliferation; estradiol is unable to increase this rate of proliferation further, but antiestrogens are able to decrease proliferation. This change in growth pattern is associated with a 3-fold increase in cellular estrogen receptor levels. Despite their differing basal growth rates, cells grown in either the short-term (less than 1 mo) or long-term (greater than 6 mo) absence of estrogens both have progesterone receptor levels that are very low and, in both cases, estradiol increases progesterone receptor levels markedly. Thus, under long-term estrogen-free conditions, there is a dissociation between the stimulation of cell proliferation and of specific protein synthesis (progesterone receptor) by estrogen. The increase in the cell proliferation rate observed in cells grown in the long-term absence of estrogen may reflect altered regulation of growth factor production or altered sensitivity to growth factors in the medium or produced by the cells themselves. Hence, these breast cancer cells adapt significantly to long-term growth in estrogen-free conditions, an observation that may be relevant to understanding the growth of hormone-responsive human breast cancers in vivo.     

Novel senescence associated gene, YPEL3, is repressed by estrogen in ER+ mammary tumor cells and required for tamoxifen-induced cellular senescence

Estrogen signaling plays an important role in breast carcinogenesis. An increased understanding of estrogen gene targets and their effects will allow for more directed and effective therapies for individuals with breast cancer, particularly those with estrogen receptor positive tumors resistant to tamoxifen therapy. Here, we identify YPEL3 as a growth suppressive protein downregulated by estrogen in estrogen receptor positive breast cancer cell lines. Estrogen repression of YPEL3 expression was found to be independent of p53 but dependent on estrogen receptor alpha expression. Importantly, YPEL3 expression, which is induced by the removal of estrogen or treatment with tamoxifen triggers cellular senescence in MCF-7 cells while loss of YPEL3 increases the growth rate of MCF-7 cells. Taken together these findings suggest that YPEL3 may represent a potential target for directed hormonal therapy for estrogen receptor positive breast cancer patients.     

Macroautophagy inhibition sensitizes tamoxifen-resistant breast cancer cells and enhances mitochondrial depolarization

Macroautophagy (autophagy), a process for lysosomal degradation of organelles and long-lived proteins, has been linked to various pathologies including cancer and to the cellular response to anticancer therapies. In the human estrogen receptor positive MCF7 breast adenocarcinoma cell line, treatment with the endocrine therapeutic tamoxifen was shown previously to induce cell cycle arrest, cell death, and autophagy. To investigate specifically the role of autophagy in tamoxifen treated breast cancer cell lines, we used a siRNA approach, targeting three different autophagy genes, Atg5, Beclin-1, and Atg7. We found that knockdown of autophagy, in combination with tamoxifen in MCF7 cells, results in decreased cell viability concomitant with increased mitochondrial-mediated apoptosis. The combination of autophagy knockdown and tamoxifen treatment similarly resulted in reduced cell viability in the breast cancer cell lines, estrogen receptor positive T-47D and tamoxifen-resistant MCF7-HER2. Together, these results indicate that autophagy has a primary pro-survival role following tamoxifen treatment, and suggest that autophagy knockdown may be useful in a combination therapy setting to sensitize breast cancer cells, including tamoxifen-resistant breast cancer cells, to tamoxifen therapy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells

The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage-independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2-specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.     

G1P3, an interferon- and estrogen-induced survival protein contributes to hyperplasia, tamoxifen resistance and poor outcomes in breast cancer

Hormonally regulated survival factors can have an important role in breast cancer. Here we elucidate G1P3, a survival protein induced by interferons (IFNs), as a target of estrogen signaling and a contributor to poor outcomes in estrogen receptor-positive (ER(+)) breast cancer. Compared with normal breast tissue, G1P3 was upregulated in the malignant epithelium (50 × higher) and was induced by estrogen ex vivo. In accord with its overexpression in early stages of breast cancer (hyperplasia and ductal carcinoma in situ), in morphogenesis assays G1P3 enhanced the survival of MCF10A acinar luminal cells causing hyperplasia by suppressing detachment-induced loss of mitochondrial potential and apoptosis (anoikis). In cells undergoing anoikis, G1P3 attenuated the induction of Bim protein, a proapoptotic member of the Bcl-2 family and reversed the downmodulation of Bcl-2 protein. Downregulation of G1P3 induced spontaneous apoptosis in BT-549 breast cancer cells and significantly reduced the growth of ER(+) breast cancer cell MCF7 (P≤0.01), further suggesting its prosurvival activity. In agreement with its induction by estrogen, G1P3 antagonized tamoxifen, an inhibitor of ER in MCF7 cells. More importantly, elevated expression of G1P3 was significantly associated with decreased relapse-free and overall survival in ER(+) breast cancer patients (P≤0.01). Our studies suggest that elevated expression of G1P3 may perturb canonical tumor-suppressing activity of IFNs partly by affecting the balance of pro- and antiapoptotic members of Bcl-2 family proteins, leading to breast cancer development and resistance to therapies.     

cJun overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype

We have previously demonstrated decreased Jun/AP-1 activity in the breast cancer cell line MCF-7 when compared to normal or immortalized mammary epithelial cells. In this paper, we overexpress Jun in MCF-7 cells (MCF7Jun) and demonstrate that it results in diverse biologic and biochemical changes, which mimic those seen clinically in breast cancer. Overexpression of Jun causes significant alterations in the composition of AP-1, decreased junB and increased fra-1 expression and results in an increased biologic aggressiveness. MCF7Jun cells exhibit increased cellular motility, increased expression of a matrix degrading enzyme MMP-9, increased in vitro chemoinvasion and tumor formation in nude mice in the absence of exogenous estrogens. Furthermore, MCF7Jun cells are unresponsive to the growth stimulating effects of estrogen and growth inhibitory effects of tamoxifen. Analysis of the estrogen receptor (ER) expression and activity showed that the MCF7Jun cells have no detectable ER. MCF-7 cells overexpressing mutant forms of cJun were responsive to the growth stimulatory effects of estrogen indicating that full-length cJun is required to acquire the estrogen-independent phenotype in breast cancer cells.     

Idoxifene antagonizes estradiol-dependent MCF-7 breast cancer xenograft growth through sustained induction of apoptosis.

Idoxifene is a novel selective estrogen (E2) receptor (ER) modulator that is currently in clinical development for the treatment of breast cancer. Compared to tamoxifen, idoxifene is metabolically more stable, with a higher relative binding affinity for the ER and reduced agonist activity on breast and uterine cells. Idoxifene also inhibits calmodulin, a calcium-binding protein that is involved in cell signal transduction pathways. In this study, the abilities of idoxifene and tamoxifen to antagonize E2-dependent MCF-7 xenograft growth in oophorectomized athymic mice were compared. The basis for idoxifene's antitumor activity was examined by comparing the effectiveness of the clinically used transisomer (referred to here as idoxifene) with its cis-isomer, which has a 50-fold lower relative binding affinity for ER than idoxifene but similar calmodulin-inhibitory activity. Changes in tumor cell proliferation, apoptosis, and ER-dependent protein expression were studied. Both idoxifene and tamoxifen significantly inhibited E2-dependent tumor growth, whereas cis-idoxifene had little effect. Withdrawal of E2 support induced significant tumor regression due to impaired cell proliferation (Ki-67 score, 9 versus 51% compared to E2 controls) and induction of apoptosis (3.6 versus 0.9% compared to E2 controls). Both anti-E2s inhibited cell proliferation and caused a significant 3-fold induction of apoptosis in E2 supported tumors after 1 week, which was maintained for 3 months with idoxifene (3.1 versus 0.48% compared to E2 controls) but decreased back to baseline in tumors treated with tamoxifen (0.69%). In contrast, cis-idoxifene had no effect on either cell proliferation or apoptosis. Both tamoxifen and idoxifene initially induced ER expression, whereas prolonged therapy with tamoxifen significantly reduced progesterone receptor levels. In conclusion, idoxifene resulted in similar inhibition of E2-dependent MCF-7 xenograft growth compared with tamoxifen, an effect that is mediated via ER rather than through calmodulin. Sustained induction of apoptosis may contribute to prolonged antagonism of E2-dependent growth, and it occurred to a greater extent following 3 months of idoxifene, compared to tamoxifen.