Downregulation of retinoblastoma protein is involved in the enhanced cytotoxicity of 4-hydroxytamoxifen plus mifepristone combination therapy versus antiestrogen monotherapy of human breast cancer

In this study, human MCF-7 breast cancer cells, which express functional estrogen and progesterone receptors, were used to compare the efficacy of combined antiestrogen plus antiprogestin therapy to antiestrogen monotherapy. Cells were treated with the antiestrogen 4-hydroxytamoxifen (4-OHT) and/or the antiprogestin mifepristone (MIF) and effects on cell proliferation (cytostatic action), cell cycle phase, the phosphorylation state of the tumor suppressor retinoblastoma protein (Rb), and induction of active cell death (cytotoxic action) were determined. Combination hormonal therapy showed both increased cytostatic and cytotoxic activity as compared to either monotherapy. The increased cytostatic action was mediated by Rb activation; whereas, the cytotoxic (pro-apoptotic) action of combined hormonal therapy correlated to a significant reduction in Rb protein levels. To test the apparent role of Rb protein loss in the pro-apoptotic action of combined hormonal therapy, Rb was downregulated in MCF-7 cells using siRNA-targeting. The siRNA-mediated knockdown of Rb combined with 4-OHT therapy resulted in a pro-apoptotic action similar to that resulting from 4-OHT and MIF combination treatment, which included increased cell detachment from the monolayer, high-molecular-weight genomic DNA fragmentation, and cleavage of poly ADP-ribose polymerase (PARP) and lamin A. From these studies, we conclude that Rb protein downregulation is required for 4-OHT-treated, estrogen receptor positive (ER+) breast cancer cells to undergo active cell death. We discuss the potential of using an antiprogestin such as MIF plus antiestrogen treatment to more effectively downregulate Rb in ER+ breast cancer cells to increase the overall cytotoxic action of hormonal therapy.     

Compared effects of GnRH analogs and 4-hydroxytamoxifen on growth and steroid receptors in antiestrogen sensitive and resistant MCF-7 breast cancer cell sublines

Summary Antiestrogens, such as tamoxifen, have a direct antitumor action and are widely used in cancer therapy. The direct antitumor action of GnRH analogs has been documented in bothin vitro and clinical studies. GnRH analog direct action could therefore provide an alternative approach in postmenopausal patients developing antiestrogen resistance, a frequent cause of relapse during hormonal treatment of breast cancer. This study was carried out to compare the effects of two GnRH analogs (the agonist Decapeptyl and the antagonist BIM 21009C) and the antiestrogen 4-hydroxy-tamoxifen (OH-TAM) on proliferation in antiestrogen-responsive or antiestrogen-resistant human breast cancer cell lines (MCF-7, MCF-7 LY2). Ineffective when used without estrogen stimulation, both of the GnRH analogs and OH-TAM inhibit the 17-estradiol-stimulated growth of the estrogen-responsive cell line MCF-7. Compared with parental MCF-7 cell line responsiveness, the antiestrogen-resistant variant MCF-7 LY2 appears to be resistant to GnRH analogs. These findings indicate similarities between the two types of compounds with regard to their antiestrogenic effects on growth observedin vitro. However, since unlike OH-TAM, Decapeptyl displays no effect on steroid receptor levels of sensitive MCF-7 cells, the intracellular inhibitory mechanisms of these drugs are probably in part different. This study suggests that the direct effect of the studied GnRH analogs would not be a potent tool for treating antiestrogen-resistant breast tumors.     

Induction of acquired resistance to antiestrogen by reversible mitochondrial DNA depletion in breast cancer cell line

Although the net benefits of tamoxifen in adjuvant breast cancer therapy have been proven, the recurrence of the cancer in an aggressive and hormone independent form has been highly problematic. We previously demonstrated the important role mitochondrial DNA (mtDNA) plays in hormone-independence in prostate cancer. Here, the role of mtDNA in breast cancer progression was investigated. We established hydroxytamoxifen (4-OHT) resistant HTRMCF by growing MCF-7, human breast adenocarcinoma cells, in the presence of 4-OHT. HTRMCF was cross-resistant to 4-OHT and ICI182,780 concurrent with the depletion of mtDNA. To further investigate the role of mtDNA depletion, MCF-7 was depleted of mtDNA by treatment with ethidium bromide. MCF Rho 0 was resistant to both 4-OHT and ICI182,780. Furthermore, cybrid (MCFcyb) prepared by fusion MCF Rho 0 with platelet to transfer mtDNA showed susceptibility to antiestrogen. Surprisingly, after withdrawal of 4-OHT for 8 weeks, HTRMCF and their clones became susceptible to both drugs concurrent with a recovery of mtDNA. Herein, our results substantiated the first evidence that the depletion of mtDNA induced by hormone therapy triggers a shift to acquired resistance to hormone therapy in breast cancer. In addition, we showed that mtDNA depletion can be reversed, rendering the cancer cells susceptible to antiestrogen. The fact that the hormone independent phenotype can be reversed should be a step toward more effective treatments for estrogen-responsive breast cancer.     

Upregulation of PKC-delta contributes to antiestrogen resistance in mammary tumor cells

Acquired resistance to tamoxifen (Tam) in breast cancer patients is a serious therapeutic problem. We have previously reported that protein kinase C-delta (PKC-delta) plays a major role in estrogen (E2)-mediated cell proliferation. To determine if PKC-delta is one of the major alternate signaling pathways that supports cell growth in the presence of Tam, we determined the levels of PKC isoforms in four different models of antiestrogen-resistant cells. Three out of four antiestrogen resistance cell lines (Tam/MCF-7, ICI/MCF-7 and HER-2/MCF-7) expressed significantly high levels of both total and activated PKC-delta levels compared to sensitive cells. Estrogen receptor (ER) alpha content and function are maintained in all the antiestrogen-resistant cell lines. Overexpressing active PKC-delta in Tam-sensitive MCF-7 cells (PKC-delta/MCF-7) led to Tam resistance both in vitro and in vivo. Inhibition of PKC-delta by rottlerin (a relatively specific inhibitor of PKC-delta) or siRNA significantly inhibited estrogen- and Tam-induced growth in antiestrogen-resistant cells. PKC-delta levels are significantly higher in Tam-resistant tumors compared to Tam-sensitive tumors in xenograft model (P<0.05). Taken together, these data suggest that PKC-delta plays a major role in antiestrogen resistance in breast tumor cells and thus provides a new target for treatment.     

4-Hydroxytamoxifen inhibits proliferation of multiple myeloma cells in vitro through down-regulation of c-Myc, up-regulation of p27Kip1, and modulation of Bcl-2 family members.

PURPOSE: Multiple myeloma is an incurable B-cell malignancy requiring new therapeutic strategies. Our approach was to analyze the in vitro effects of a selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT), on six multiple myeloma cell lines. EXPERIMENTAL DESIGN: Cultured multiple myeloma cells were treated with various 4-OHT concentrations and the cellular response was studied: cell proliferation, cell viability, induction of apoptosis, caspase activities, and expression of signaling proteins. RESULTS: We found that pharmacologic concentrations of 4-OHT inhibit cell proliferation (4 of 6 cell lines). This inhibition is achieved by two independent events: a block at the G(1) phase of the cell cycle and the induction of apoptotic death. The cellular response to 4-OHT depends on the presence of functional estrogen receptors. 4-OHT treatment activates an intrinsic mitochondrial caspase-9-dependent pathway but not the Fas/FasL death pathway. Signaling pathways known to be involved in the survival and/or proliferation of multiple myeloma cells are not affected by 4-OHT treatment. 4-OHT-induced G(1) arrest is accompanied by the up-regulation of the cell cycle inhibitor p27(Kip1) and the down-regulation of c-Myc. Among the Bcl-2 family members tested, the proapoptotic BimS protein is induced whereas the antiapoptotic protein Mcl-1 is decreased. CONCLUSIONS: Although the effects of 4-OHT are observed at micromolar concentrations, cellular mechanisms responsible for G(1) arrest, as well as apoptosis induction, are similar to those observed in breast cancer cells. Our data support the concept that 4-OHT may represent an alternative approach to inhibit proliferation and induce apoptosis of multiple myeloma cells.     

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.     

Induction of cell death in antiestrogen resistant human breast cancer cells by the protein kinase CK2 inhibitor DMAT

Protein kinase CK2 is involved in cell proliferation and survival, and found overexpressed in virtually all types of human cancer, including breast cancer. We demonstrate that inhibition of CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT), a potent and specific CK2 inhibitor, results in caspase-mediated killing of human breast cancer cells with acquired resistance to antiestrogens, while DMAT fails to kill parental MCF-7 cells. The antiestrogen resistant breast cancer cells express reduced levels of Bcl-2 compared to MCF-7 cells. Reduced Bcl-2 protein level is also found in a tamoxifen resistant human breast tumor grown as a xenograft. We show that re-expression of Bcl-2 partially rescues antiestrogen resistant MCF-7 sublines from DMAT-induced cell death. In summary, our data suggest a novel role of CK2 in antiestrogen resistance.     

Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

Introduction

In this pre-clinical in vitro study conducted in estrogen receptor positive (ER+) breast cancer cells, we have characterized the effects of insulin-like growth factor I (IGF-1) on the cytostatic and cytotoxic action of antiestrogen treatment when used as a single agent or in combination with the antiprogestin mifepristone (MIF). Our goal was to identify new molecular targets to improve the efficacy of hormonal therapy in breast cancer patients that have a poor response to hormonal therapy, in part, due to high circulating levels of unbound insulinIGF-1.

Methods

IGF-1-mediated effects on cytostasis and apoptotic cell death were determined with cell counts conducted in the presence and absence of trypan blue; enzyme-linked immunosorbent assays to determine the intracellular levels of cleaved cytokeratin 18, a marker of epithelial cancer cell apoptosis; and immunoblot analysis to determine the levels of cleaved poly-ADP ribose polymerase (PARP) and lamin A that result from caspase-dependent apoptosis. Cytotoxicity was further characterized by determination of the levels of reactive oxygen species (ROS) and the percent of mitochondrial membrane depolarization in cell populations treated with the different hormones in the presence and absence of IGF-1. Small molecule inhibitors of the dual-specificity protein kinase MEK1, MEK1 siRNA, Bim siRNA, and vectors overexpressing MEK1 wild type and mutant, dominant negative cDNA were used to identify key IGF-1 downstream prosurvival effectors.

Results

IGF-1, at physiologically relevant levels, blocked the cytotoxic action(s) of the antiestrogens 4-hydroxytamoxifen (4-OHT) and tamoxifen (TAM) when used as single agents or in combination with the antiprogestin MIF. The antiapoptotic action of IGF-1 was mediated primarily through the action of MEK1. MEK1 expression reduced the levels of ROS and mitochondrial membrane depolarization induced by the hormonal treatments via a mechanism that involved the phosphorylation and proteasomal turnover of the proapoptotic BH3-only Bcl-2 family member Bim. Importantly, small-molecule inhibitors of MEK1 circumvented the prosurvival action of IGF-1 by restoring Bim to levels that more effectively mediated apoptosis in ER⁺ breast cancer cells.

Conclusion

his study provides strong support for the use of MEK1 inhibitors in combination with hormonal therapy to effectively affect cytostasis and activate a Bim-dependent apoptotic pathway in ER⁺ breast cancer cells. We discuss that MEK1 blockade may be a particularly effective treatment for women with high circulating levels of IGF-1, which have been correlated to a poor prognosis.     

Estrogen Receptor Expression and Estrogen Receptor-independent Cytotoxic Effects of Tamoxifen on Malignant Rhabdoid Tumor Cells in vitro

Recent studies have shown that the antiestrogen tamoxifen (TAM) can be used in the treatment of malignant neoplasms other than breast cancer. In the present study, we investigated the expression of estrogen receptor (ER) in six malignant rhabdoid tumor (MRT) cell lines. Alterations in MRT cell growth in response to estrogen or antiestrogens (4-hydroxytamoxifen (4-OHT), TAM, and ICI 182 780) were also investigated. RT-PCR and western blotting showed that ER-a was expressed in three of the six MRT cell lines. While 17-β-estradiol (E2) did not significantly alter MRT cell line proliferation, the hydroxylated tamoxifen metabolite 4-OHT significantly inhibited the growth of all 6 MRT cell lines. However, the steroidal antiestrogen ICI 182 780 did not alter the proliferation of any of the MRT cell lines. 4-OHT induced apoptosis in both ER-α-negative and ER-α-positive MRT cell lines, as assessed by nuclear morphology and DNA fragmentation. Neither growth inhibition nor induction of apoptosis due to 4-OHT was blocked by the addition of excess E2. Our data suggested that 4-OHT induced cytotoxic effects against MRT cells, and that these effects were independent of ER expression.     

Induction of antiproliferation and apoptosis in estrogen receptor negative MDA-231 human breast cancer cells by mifepristone and 4-hydroxytamoxifen combination therapy: a role for TGFbeta1

Mifepristone (MIF) is an antiprogestin with potent anti-glucocorticoid and anti-androgen activity. MIF also appears to have anti-tumor activity independent of its ability to bind to nuclear receptors. In this study, we tested the ability of MIF to inhibit the growth of ER and PR negative breast cancer cells. In addition, because high-dose anti-estrogen treatment has been shown to inhibit ER and PR negative breast cancer cells, we compared the anti-proliferative activity of MIF to that of the anti-estrogen 4-hydroxytamoxifen (TAM) or combination hormonal therapy (MIF + TAM). MIF and TAM therapy induced a significant time- and dose-dependent growth inhibition and, ultimately, induced cell death in MDA-231 cells as evidenced by increased DNA fragmentation, cytochrome c release from the mitochondria, and the activation of caspase-3. The anti-proliferative activity of TAM plus MIF combination treatment was at least additive as compared to either monotherapy. The earliest indicator of TAM and MIF cytostatic and cytotoxic action on MDA-231 cells was a significant (p<0.05) induction of TGFbeta1 secretion into the growth medium within 4 h of treatment. Secreted TGFbeta1 levels at 24 and 48 h were significantly higher in the TAM plus MIF treatment group as compared to cells treated with TAM or MIF alone. TGFbeta1 neutralizing antibody or addition of mannose-6-phosphate (M6P), a reagent also used to inhibit TGFbeta1, significantly attenuated the TAM and/or MIF-induced cell growth inhibition and cell death. In summary, our results indicate that MIF used in combination with TAM can effectively kill estrogen-insensitive human breast cancer cells. Our study further implies that agents that effectively increase TGFbeta1 levels in ER negative breast cancer cells may be one treatment approach for hormone-independent breast cancers.     

Clusterin: a potential target for improving response to antiestrogens

Antiestrogens represent the first line of therapy in the treatment of estrogen receptor-positive (ER+) breast cancer patients. Unfortunately, up to 40% of patients develop resistance associated with progression and frequently die for metastatic breast cancer. The molecular events leading to pharmacological resistance are not completely understood. We attempted to verify in an experimental model the role of cytoplasmic clusterin (CLU), a cytoprotective protein found to be up-regulated in antiestrogen-resistant patients, following neoadjuvant treatment with toremifene. The role of cytoplasmic clusterin in modulating response to two antiestrogens (toremifene and tamoxifen) was studied in two ER+ anti-estrogen-sensitive cell lines (MCF-7, 734B) and one ER+ antiestrogen-resistant cell line (T47D) using siRNA strategy. Resistant cells were characterised by higher levels of cytoplasmic clusterin than sensitive cells, and antiestrogen treatments up-regulated clusterin levels in both sensitive and resistant cell lines. Treatment with siRNA completely abolished cytoplasmic clusterin expression in all cell lines, but its down-regulation resulted in a significant decrease of cell growth only in the resistant line. We therefore concluded that: i) basal clusterin levels are higher in antiestrogen resistant cells, ii) clusterin is up-regulated following antiestrogen treatment independently of the sensitivity of the cell line, iii) down-regulation of cytoplasmic clusterin restores sensitivity to toremifene in the antiestrogen-resistant cell line. Such results support the concept that targeting CLU could represent a promising therapeutic strategy in association with antiestrogen treatment in breast cancer patients.     

Estrogen receptor expression and estrogen receptor-independent cytotoxic effects of tamoxifen on malignant rhabdoid tumor cells in vitro.

Recent studies have shown that the antiestrogen tamoxifen (TAM) can be used in the treatment of malignant neoplasms other than breast cancer. In the present study, we investigated the expression of estrogen receptor (ER) in six malignant rhabdoid tumor (MRT) cell lines. Alterations in MRT cell growth in response to estrogen or antiestrogens (4-hydroxytamoxifen (4-OHT), TAM, and ICI 182 780) were also investigated. RT-PCR and western blotting showed that ER-alpha was expressed in three of the six MRT cell lines. While 17-beta-estradiol (E2) did not significantly alter MRT cell line proliferation, the hydroxylated tamoxifen metabolite 4-OHT significantly inhibited the growth of all 6 MRT cell lines. However, the steroidal antiestrogen ICI 182 780 did not alter the proliferation of any of the MRT cell lines. 4-OHT induced apoptosis in both ER-alpha-negative and ER-alpha-positive MRT cell lines, as assessed by nuclear morphology and DNA fragmentation. Neither growth inhibition nor induction of apoptosis due to 4-OHT was blocked by the addition of excess E2. Our data suggested that 4-OHT induced cytotoxic effects against MRT cells, and that these effects were independent of ER expression.     

Regulation of human estrogen receptor gene, epidermal growth factor receptor gene, and oncogenes by estrogen and antiestrogen in MCF-7 breast cancer cells

It is generally believed that estrogen may act either as an initiator or as a promoter in carcinogenesis of human breast cancer. This estrogenic action is generally dependent on the estrogen receptor. In the human estrogen receptor, cDNA has a homology to V-erb-A oncogene. Experiments using MCF-7 human breast cancer cells were carried out to study the regulatory effect of estrogen and antiestrogen on RNA activities of oncogenes, estrogen receptor gene, and epidermal growth factor (EGF) receptor gene. The effect of estradiol on activation of estrogen and EGF receptor genes and myc, ras, and fos oncogenes was positive in relation to the concentrations of supplemented estradiol. In addition, the effects of antiestrogen (tamoxifen) were investigated. Tamoxifen suppressed MCF-7 cell growth, and spot hybridization of the RNA of MCF-7 cells revealed that RNA activities of estrogen and EGF receptor genes and myc, ras, and fos oncogenes were suppressed by tamoxifen. These results suggest that the three oncogenes and two receptor genes are partly regulated by estrogen and antiestrogen (tamoxifen) in MCF-7 human breast cancer cells. This regulatory system may have a role in carcinogenesis and in the treatment of human breast cancer.     

Inhibition of estrogen-dependent breast cell responses with phenylacetate

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.