Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer.

Aromatase inhibitors (AIs) reduce estrogen levels up to 98% as the standard practice to treat postmenopausal women with estrogen receptor-positive (ER+) breast cancer. However, approximately 30% of ER+ breast cancers develop resistance to treatment. Enhanced interferon-alpha (IFNα) signaling is upregulated in breast cancers resistant to AIs, which drives expression of a key regulator of survival, interferon-induced transmembrane protein 1 (IFITM1). However, how upregulated IFNα signaling mediates AI resistance is unknown. In this study, we utilized MCF-7:5C cells, a breast cancer cell model of AI resistance, and demonstrate that these cells exhibit enhanced IFNα signaling and ligand-independent activation of the estrogen receptor (ERα). Experiments demonstrated that STAT1, the mediator of intracellular signaling for IFNα, can interact directly with ERα. Notably, inhibition of IFNα signaling significantly reduced ERα protein expression and ER-regulated genes. In addition, loss of ERα suppressed IFITM1 expression, which was associated with cell death. Notably, chromatin immunoprecipitation experiments validated that both ERα and STAT1 associate with ERE sequences in the IFITM1 promoter. Overall, hyperactivation of IFNα signaling enhances ligand-independent activation of ERα, which promotes ER-regulated, and interferon stimulated gene expression to promote survival in AI-resistant breast cancer cells.

Disrupting interferon-alpha and NF-kappaB crosstalk suppresses IFITM1 expression attenuating triple-negative breast cancer progression.

Overexpression of interferon induced transmembrane protein-1 (IFITM1) enhances tumor progression in multiple cancers, but its role in triple-negative breast cancer (TNBC) is unknown. Here, we explore the functional significance and regulation of IFITM1 in TNBC and strategies to target its expression. Immunohistochemistry staining of a tissue microarray demonstrates that IFITM1 is overexpressed in TNBC samples which is confirmed by TCGA analysis. Targeting IFITM1 by siRNA or CRISPR/Cas9 in TNBC cell lines significantly inhibits proliferation, colony formation, and wound healing in vitro. Orthotopic mammary fat pad and mammary intraductal studies reveal that loss of IFITM1 reduces TNBC tumor growth and invasion in vivo. RNA-seq analysis of IFITM1/KO cells reveals significant downregulation of several genes involved in proliferation, migration, and invasion and functional studies identified NF-κB as an important downstream target of IFITM1. Notably, siRNA knockdown of p65 reduces IFITM1 expression and a drug-repurposing screen of FDA approved compounds identified parthenolide, an NFκB inhibitor, as a cytotoxic agent for TNBC and an inhibitor of IFITM1 in vitro and in vivo. Overall, our findings suggest that targeting IFITM1 by suppressing interferon-alpha/NFκB signaling represents a novel therapeutic strategy for TNBC treatment.

Deciphering the role of interferon alpha signaling and microenvironment crosstalk in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most rare and aggressive subtype of breast cancer characterized by clusters of tumor cells invading lymph vessels, high rates of metastasis, and resistance to systemic chemotherapy. While significant progress has been made in understanding IBC, survival among IBC patients is still only one half that among patients with non-IBC. A major limitation to the development of more specific and effective treatments for IBC is a lack of identifiable molecular alterations that are specific to IBC. Emerging evidence suggests that the aggressive nature of IBC is not specific to IBC cells but instead driven by the interplay between autonomous signaling and context-dependent cytokine networks from the surrounding tumor microenvironment. Recently, the type I interferon, specifically the interferon alpha signature, has been identified as a pathway upregulated in IBC but few studies have addressed its role. Activation of the interferon alpha signaling pathway has been shown to contribute to apoptosis and cellular senescence but is also attributed to increased migration and drug resistance depending on the interferon-stimulated genes transcribed. The mechanisms promoting the increase in interferon alpha expression and the role interferon alpha plays in IBC remain speculative. Current hypotheses suggest that immune and stromal cells in the local tumor microenvironment contribute to the interferon alpha signaling cascade within the tumor cell and that this activation may further alter the immune and stromal cells within the microenvironment. This review serves as an overview of the role of interferon alpha signaling in IBC. Ideally, future experiments should investigate the mechanistic interplay of interferons in IBC to develop more efficacious treatment strategies for IBC patients.

Interaction Between MUC1 and STAT1 Drives IFITM1 Overexpression in Aromatase Inhibitor-Resistant Breast Cancer Cells and Mediates Estrogen-Induced Apoptosis.

The human oncoprotein, mucin 1 (MUC1), drives tumorigenesis in breast carcinomas by promoting epithelial-to-mesenchymal transition (EMT), epigenetic reprogramming, and evasion of immune response. MUC1 interacts with STAT1, through JAK/STAT signaling, and stimulates transcription of IFN-stimulated genes, specifically IFN-induced transmembrane protein 1 (IFITM1). Our laboratory has previously shown that IFITM1 overexpression in aromatase inhibitor (AI)-resistant breast cancer cells promotes aggressiveness. Here, we demonstrate that differential regulation of MUC1 in AI-sensitive (MCF-7 and T-47D) compared with AI-resistant (MCF-7:5C) cells is critical in mediating IFITM1 expression. A tumor microarray of 94 estrogen receptor-positive human breast tumors correlated coexpression of MUC1 and IFITM1 with poor recurrence-free survival, poor overall survival, and AI-resistance. In this study, we investigated the effects of MUC1/IFITM1 on cell survival and proliferation. We knocked down MUC1 levels with siRNA and pharmacologic inhibitors, which abrogated IFITM1 mRNA and protein expression and induced cell death in AI-resistant cells. In vivo, estrogen and ruxolitinib significantly reduced tumor size and decreased expression of MUC1, P-STAT1, and IFITM1. IMPLICATIONS: MUC1 and IFITM1 overexpression drives AI resistance and can be targeted with currently available therapies.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/5/1180/F1.large.jpg.

Correction to: Buthionine sulfoximine sensitizes antihormone-resistant human breast cancer cells to estrogen-induced apoptosis.

After the publication of this work [1] an error was noticed in Fig. 3a and Fig. 5a.

IFITM1 suppression blocks proliferation and invasion of aromatase inhibitor-resistant breast cancer in vivo by JAK/STAT-mediated induction of p21.

Interferon induced transmembrane protein 1 (IFITM1) belongs to a family of interferon stimulated genes (ISGs) that is associated with tumor progression and DNA damage resistance; however, its role in endocrine resistance is not known. Here, we correlate IFITM1 expression with clinical stage and poor response to endocrine therapy in a tissue microarray consisting of 94 estrogen receptor (ER)-positive breast tumors. IFITM1 overexpression is confirmed in the AI-resistant MCF-7:5C cell line and not found in AI-sensitive MCF-7 cells. In this study, the orthotopic (mammary fat pad) and mouse mammary intraductal (MIND) models of breast cancer are used to assess tumor growth and invasion in vivo. Lentivirus-mediated shRNA knockdown of IFITM1 in AI-resistant MCF-7:5C cells diminished tumor growth and invasion and induced cell death, whereas overexpression of IFITM1 in wild-type MCF-7 cells promoted estrogen-independent growth and enhanced their aggressive phenotype. Mechanistic studies indicated that loss of IFITM1 in MCF-7:5C cells markedly increased p21 transcription, expression and nuclear localization which was mediated by JAK/STAT activation. These findings suggest IFITM1 overexpression contributes to breast cancer progression and that targeting IFITM1 may be therapeutically beneficial to patients with endocrine-resistant disease.

Everolimus downregulates estrogen receptor and induces autophagy in aromatase inhibitor-resistant breast cancer cells.

BACKGROUND: mTOR inhibition of aromatase inhibitor (AI)-resistant breast cancer is currently under evaluation in the clinic. Everolimus/RAD001 (Afinitor®) has had limited efficacy as a solo agent but is projected to become part of combination therapy for AI-resistant breast cancer. This study was conducted to investigate the anti-proliferative and resistance mechanisms of everolimus in AI-resistant breast cancer cells. METHODS: In this study we utilized two AI-resistant breast cancer cell lines, MCF-7:5C and MCF-7:2A, which were clonally derived from estrogen receptor positive (ER+) MCF-7 breast cancer cells following long-term estrogen deprivation. Cell viability assay, colony formation assay, cell cycle analysis and soft agar anchorage-independent growth assay were used to determine the efficacy of everolimus in inhibiting the proliferation and tumor forming potential of MCF-7, MCF-7:5C, MCF-7:2A and MCF10A cells. Confocal microscopy and transmission electron microscopy were used to evaluate LC3-II production and autophagosome formation, while ERE-luciferase reporter, Western blot, and RT-PCR analyses were used to assess ER expression and transcriptional activity. RESULTS: Everolimus inhibited the proliferation of MCF-7:5C and MCF-7:2A cells with relatively equal efficiency to parental MCF-7 breast cancer cells. The inhibitory effect of everolimus was due to G1 arrest as a result of downregulation of cyclin D1 and p21. Everolimus also dramatically reduced estrogen receptor (ER) expression (mRNA and protein) and transcriptional activity in addition to the ER chaperone, heat shock protein 90 protein (HSP90). Everolimus restored 4-hydroxy-tamoxifen (4OHT) sensitivity in MCF-7:5C cells and enhanced 4OHT sensitivity in MCF-7 and MCF-7:2A cells. Notably, we found that autophagy is one method of everolimus insensitivity in MCF-7 breast cancer cell lines. CONCLUSION: This study provides additional insight into the mechanism(s) of action of everolimus that can be used to enhance the utility of mTOR inhibitors as part of combination therapy for AI-resistant breast cancer.

Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner.

BACKGROUND: Inflammatory breast cancer (IBC) is a very aggressive and lethal subtype of breast cancer that accounts for about 4 % of all breast cancers diagnosed in the United States. Despite the efforts of several investigators to identify the molecular factors driving the aggressive phenotype of IBC, a great deal is still unknown about the molecular underpinnings of the disease. In the present study, we investigated the role of interferon-induced transmembrane protein 1 (IFITM1), a well-known interferon-stimulated gene (ISG), in promoting the aggressiveness of SUM149 IBC cells. METHODS: Western blot and real-time polymerase chain reaction analyses were performed to assess the protein and messenger RNA (mRNA) levels of IFITM1 and other ISGs in three IBC cell lines: SUM149, MDA-IBC-3, and SUM190. IFITM1 expression and cellular localization were assessed by using immunofluorescence, while the tumorigenic potential was assessed by performing cell migration, invasion, and colony formation assays. Small interfering RNA and short hairpin RNA knockdowns, enzyme-linked immunosorbent assays, and luciferase assays were performed to determine the functional significance of IFITM1 and signal transducers and activators of transcription 1 and 2 (STAT1/2) in SUM149 cells. RESULTS: We found that IFITM1 was constitutively overexpressed at the mRNA and protein levels in triple-negative SUM149 IBC cells, but that it was not expressed in SUM190 and MDA-IBC-3 IBC cells, and that suppression of IFITM1 or blockade of the IFNα signaling pathway significantly reduced the aggressive phenotype of SUM149 cells. Additionally, we found that knockdown of STAT2 abolished IFITM1 expression and IFITM1 promoter activity in SUM149 cells and that loss of STAT2 significantly inhibited the ability of SUM149 cells to proliferate, migrate, invade, and form 2-D colonies. Notably, we found that STAT2-mediated activation of IFITM1 was particularly dependent on the chromatin remodeler brahma-related gene 1 (BRG1), which was significantly elevated in SUM149 cells compared with SUM190 and MDA-IBC-3 cells. CONCLUSIONS: These findings indicate that overexpression of IFITM1 enhances the aggressive phenotype of triple-negative SUM149 IBC cells and that this effect is dependent on STAT2/BRG1 interaction. Further studies are necessary to explore the potential of IFITM1 as a novel therapeutic target and prognostic marker for some subtypes of IBCs.

Mechanisms of endocrine resistance in breast cancer: an overview of the proposed roles of noncoding RNA.

Endocrine therapies such as tamoxifen and aromatase inhibitors are the standard treatment options for estrogen receptor-positive breast cancer patients. However, resistance to these agents has become a major clinical obstacle. Potential mechanisms of resistance to endocrine therapies have been identified, often involving enhanced growth factor signaling and changes in the expression or action of the estrogen receptor, but few studies have addressed the role of noncoding RNA (ncRNA). Two important types of ncRNA include microRNA (miRNA) and long noncoding RNA (lncRNA). miRNAs are small RNA molecules that regulate gene expression via translational inhibition or degradation of mRNA transcripts, while lncRNAs are larger RNA molecules that have been shown to play a role in multiple cellular maintenance functions such as protein scaffolding, chromatin looping, and regulation of mRNA stability. Both miRNA and lncRNA have recently impacted the field of breast cancer research as important pieces in the mechanistic puzzle of the genes and pathways involved in breast cancer development and progression. This review serves as an overview of the roles of miRNA and lncRNA in breast cancer progression and the development of endocrine resistance. Ideally, future experiments in the field should include identification of ncRNAs that could be potential therapeutic targets in endocrine-resistant tumors, as well as ncRNA biomarkers that facilitate more tumor-specific treatment options for endocrine-resistant breast cancer patients.

Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death.

INTRODUCTION: Estrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance. METHODS: Real-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression. RESULTS: We found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells. CONCLUSION: Overall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.

Loss of pigment epithelium-derived factor: a novel mechanism for the development of endocrine resistance in breast cancer.

INTRODUCTION: Despite the benefits of endocrine therapies such as tamoxifen and aromatase inhibitors in treating estrogen receptor (ER) alpha-positive breast cancer, many tumors eventually become resistant. The molecular mechanisms governing resistance remain largely unknown. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that displays broad anti-tumor activity based on dual targeting of the tumor microenvironment (anti-angiogenic action) and the tumor cells (direct anti-tumor action). Recent studies indicate that PEDF expression is significantly reduced in several tumor types, including breast cancer, and that its reduction is associated with disease progression and poor patient outcome. In the current study, we investigated the role of PEDF in the development of endocrine resistance in breast cancer. METHODS: PEDF mRNA and protein levels were measured in several endocrine-resistant breast cancer cell lines including MCF-7:5C, MCF-7:2A, and BT474 and in endocrine-sensitive cell lines MCF-7, T47D, and ZR-75-1 using real-time PCR and western blot analyses. Tissue microarray analysis and immunohistochemistry were used to assess the PEDF protein level in tamoxifen-resistant breast tumors versus primary tumors. Lentiviruses were used to stably express PEDF in endocrine-resistant breast cancer cell lines to determine their sensitivity to tamoxifen following PEDF re-expression. RESULTS: We found that PEDF mRNA and protein levels were dramatically reduced in endocrine-resistant MCF-7:5C, MCF-7:2A, and BT474 breast cancer cells compared with endocrine-sensitive MCF-7, T47D, and ZR-75-1 cells, and that loss of PEDF was associated with enhanced expression of pSer167ERα and the receptor tyrosine kinase rearranged during transfection (RET). Importantly, we found that silencing endogenous PEDF in tamoxifen-sensitive MCF-7 and T47D breast cancer cells conferred tamoxifen resistance whereas re-expression of PEDF in endocrine-resistant MCF-7:5C and MCF-7:2A cells restored their sensitivity to tamoxifen in vitro and in vivo through suppression of RET. Lastly, tissue microarray studies revealed that PEDF protein was reduced in ~52.4% of recurrence tumors (31 out of 59 samples) and loss of PEDF was associated with disease progression and poor patient outcome. CONCLUSION: Overall, these findings suggest that PEDF silencing might be a novel mechanism for the development of endocrine resistance in breast cancer and that PEDF expression might be a predictive marker of endocrine sensitivity.

Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time.

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17ß-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

The selective estrogen receptor modulator bazedoxifene inhibits hormone-independent breast cancer cell growth and down-regulates estrogen receptor α and cyclin D1.

Bazedoxifene (BZA) is a third-generation selective estrogen receptor modulator (SERM) that has been approved for the prevention and treatment of postmenopausal osteoporosis. It has antitumor activity; however, its mechanism of action remains unclear. In the present study, we characterized the effects of BZA and several other SERMs on the proliferation of hormone-dependent MCF-7 and T47D breast cancer cells and hormone-independent MCF-7:5C and MCF-7:2A cells and examined its mechanism of action in these cells. We found that all of the SERMs inhibited the growth of MCF-7, T47D, and MCF-7:2A cells; however, only BZA and fulvestrant (FUL) inhibited the growth of hormone-independent MCF-7:5C cells. Cell cycle analysis revealed that BZA and FUL induced G(1) blockade in MCF-7:5C cells; however, BZA down-regulated cyclin D1, which was constitutively overexpressed in these cells, whereas FUL suppressed cyclin A. Further analysis revealed that small interfering RNA knockdown of cyclin D1 reduced the basal growth of MCF-7:5C cells, and it blocked the ability of BZA to induce G(1) arrest in these cells. BZA also down-regulated estrogen receptor-α (ERα) protein by increasing its degradation and suppressing cyclin D1 promoter activity in MCF-7:5C cells. Finally, molecular modeling studies demonstrated that BZA bound to ERα in an orientation similar to raloxifene; however, a number of residues adopted different conformations in the induced-fit docking poses compared with the experimental structure of ERα-raloxifene. Together, these findings indicate that BZA is distinct from other SERMs in its ability to inhibit hormone-independent breast cancer cell growth and to regulate ERα and cyclin D1 expression in resistant cells.

The Conformation of the Estrogen Receptor Directs Estrogen-Induced Apoptosis in Breast Cancer: A Hypothesis.

BACKGROUND: Estrogens are classified as type I (planar) and type II (angular) based on their structures. In this study we have used triphenylethylenes (TPEs) compounds related to 4OHT to address the hypothesis that the conformation of the liganded estrogen receptor (ERα) may dictate the E2-induced apoptosis of the ER+ breast cancer cells. MATERIALS AND METHODS: ERα positive MCF7:5C cells were used to study the apoptosis induced by E2, 4OHT and TPEs. Growth and apoptosis assay were used to evaluate apoptosis and the ability to reverse the E2-induced apoptosis. ERα protein were measured by western blotting to investigate the destruction of ERα by TPEs in MCF7 cells. ChIP assay were performed to study the in-vivo recruitment of ERα and SRC3 at classical E2-responsive promoter TFF1 (PS2) by TPEs. Molecular modeling was used to predict the binding mode of the TPE to the ERα. RESULTS: TPEs were not only unable to induce efficient apoptosis in MCF7:5C cells but also reversed the E2-induced apoptosis similar to 4OHT. Furthermore, the TPEs and 4OHT did not reduce the ERα protein levels unlike E2. ChIP assay confirmed very weak recruitment of SRC3 despite modest recruitment of ERα in the presence of TPEs. Molecular modeling suggested the TPE would bind in antagonistic mode with the ERα. CONCLUSION: Our results advances the hypothesis that the TPE liganded ERα complex structurally resembles the 4OHT bound ERα and cannot efficiently recruit co-activator SRC3. As a result, the TPE complex cannot induce apoptosis of ER+ breast cancer cells although it may cause growth of the breast cancer cells. The conformation of the estrogen-ER complex differentially controls growth and apoptosis.

Structure-function relationships of estrogenic triphenylethylenes related to endoxifen and 4-hydroxytamoxifen.

Estrogens can potentially be classified into planar (class I) or nonplanar (class II) categories, which might have biological consequences. 1,1,2-Triphenylethylene (TPE) derivatives were synthesized and evaluated against 17beta-estradiol (E2) for their estrogenic activity in MCF-7 human breast cancer cells. All TPEs were estrogenic and, unlike 4-hydroxytamoxifen (4OHTAM) and Endoxifen, induced cell growth to a level comparable to that of E2. All the TPEs increased ERE activity in MCF-7:WS8 cells with the order of potency as followed: E2 > 1,1-bis(4,4'-hydroxyphenyl)-2-phenylbut-1-ene (15) > 1,1,2-tris(4-hydroxyphenyl)but-1-ene (3) > Z 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (7) > E 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (6) > Z(4-(1-(4-ethoxyphenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl)phenol (12) > 4-OHTAM. Transient transfection of the ER-negative breast cancer cell line T47D:C4:2 with wild-type ER or D351G ER mutant revealed that all of the TPEs increased ERE activity in the cells expressing the wild-type ER but not the mutant, thus confirming the importance of Asp351 for ER activation by the TPEs. The findings confirm E2 as a class I estrogen and the TPEs as class II estrogens. Using available conformations of the ER liganded with 4OHTAM or diethylstilbestrol, the TPEs optimally occupy the 4OHTAM ER conformation that expresses Asp351.

New hypotheses and opportunities in endocrine therapy: amplification of oestrogen-induced apoptosis.

AIMS: To outline the progress being made in the understanding of acquired resistance to long term therapy with the selective oestrogen receptor modulators (SERMs, tamoxifen and raloxifene) and aromatase inhibitors. The question to be addressed is how we can amplify the new biology of oestrogen-induced apoptosis to create more complete responses in exhaustively antihormone treated metastatic breast cancer. METHODS AND RESULTS: Three questions are posed and addressed. (1) Do we know how oestrogen works? (2) Can we improve adjuvant antihormonal therapy? (3) Can we enhance oestrogen-induced apoptosis? The new player in oestrogen action is GPR30 and there are new drugs specific for this target to trigger apoptosis. Similarly, anti-angiogenic drugs can be integrated into adjuvant antihormone therapy or to enhance oestrogen-induced apoptosis in Phase II antihormone resistant breast cancer. The goal is to reduce the development of acquired antihormone resistance or undermine the resistance of breast cancer cells to undergo apoptosis with oestrogen respectively. Finally, drugs to reduce the synthesis of glutathione, a subcellular molecule compound associated with drug resistance, can enhance oestradiol-induced apoptosis. CONCLUSIONS: We propose an integrated approach for the rapid testing of agents to blunt survival pathways and amplify oestrogen-induced apoptosis and tumour regression in Phase II resistant metastatic breast cancer. This Pharma platform will provide rapid clinical results to predict efficacy in large scale clinical trials.

The Paradox of Oestradiol-Induced Breast Cancer Cell Growth and Apoptosis.

High dose oestrogen therapy was used as a treatment for postmenopausal patients with breast cancer from the 1950s until the introduction of the safer antioestrogen, tamoxifen in the 1970s. The anti-tumour mechanism of high dose oestrogen therapy remained unknown. There was no enthusiasm to study these signal transduction pathways as oestrogen therapy has almost completely been eliminated from the treatment paradigm. Current use of tamoxifen and the aromatase inhibitors seek to create oestrogen deprivation that prevents the growth of oestrogen stimulated oestrogen receptor (ER) positive breast cancer cells. However, acquired resistance to antihormonal therapy does occur, but it is through investigation of laboratory models that a vulnerability of the cancer cell has been discovered and is being investigated to provide new opportunities in therapy with the potential for discovering new cancer-specific apoptotic drugs. Laboratory models of resistance to raloxifene and tamoxifen, the selective oestrogen receptor modulators (SERMs) and aromatase inhibitors demonstrate an evolution of drug resistance so that after many years of oestrogen deprivation, the ER positive cancer cell reconfigures the survival signal transduction pathways so oestrogen now becomes an apoptotic trigger rather than a survival signal. Current efforts are evaluating the mechanisms of oestrogen-induced apoptosis and how this new biology of oestrogen action can be amplified and enhanced, thereby increasing the value of this therapeutic opportunity for the treatment of breast cancer. Several synergistic approaches to therapeutic enhancement are being advanced which involve drug combinations to impair survival signaling with the use of specific agents and to impair bcl-2 that protects the cancer cell from apoptosis. We highlight the historical understanding of oestrogen's role in cell survival and death and specifically illustrate the progress that has been made in the last five years to understand the mechanisms of oestrogen-induced apoptosis. There are opportunities to harness knowledge from this new signal transduction pathway to discover the precise mechanism of this oestrogen-induced apoptotic trigger. Indeed, the new biology of oestrogen action also has significance for understanding the physiology of bone remodeling. Thus, the pathway has a broad appeal in both physiology and cancer research.

Protective effects of dietary antioxidants on proton total-body irradiation-mediated hematopoietic cell and animal survival.

Abstract Dietary antioxidants have radioprotective effects after gamma-radiation exposure that limit hematopoietic cell depletion and improve animal survival. The purpose of this study was to determine whether a dietary supplement consisting of l-selenomethionine, vitamin C, vitamin E succinate, alpha-lipoic acid and N-acetyl cysteine could improve survival of mice after proton total-body irradiation (TBI). Antioxidants significantly increased 30-day survival of mice only when given after irradiation at a dose less than the calculated LD(50/30); for these data, the dose-modifying factor (DMF) was 1.6. Pretreatment of animals with antioxidants resulted in significantly higher serum total white blood cell, polymorphonuclear cell and lymphocyte cell counts at 4 h after 1 Gy but not 7.2 Gy proton TBI. Antioxidants significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGFbeta1 and increased bone marrow cell counts and spleen mass after TBI. Maintenance of the antioxidant diet resulted in improved recovery of peripheral leukocytes and platelets after sublethal and potentially lethal TBI. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival after proton TBI.

Estrogen regulation of apoptosis: how can one hormone stimulate and inhibit?

The link between estrogen and the development and proliferation of breast cancer is well documented. Estrogen stimulates growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Interestingly, there is strong evidence that estrogen induces apoptosis in breast cancer and other cell types. Forty years ago, before the development of tamoxifen, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in post-menopausal women. While the mechanisms by which estrogen induces apoptosis were not completely known, recent evidence from our laboratory and others demonstrates the involvement of the extrinsic (Fas/FasL) and the intrinsic (mitochondria) pathways in this process. We discuss the different apoptotic signaling pathways involved in E2 (17beta-estradiol)-induced apoptosis, including the intrinsic and extrinsic apoptosis pathways, the NF-kappaB (nuclear factor-kappa-B)-mediated survival pathway as well as the PI3K (phosphoinositide 3-kinase)/Akt signaling pathway. Breast cancer cells can also be sensitized to estrogen-induced apoptosis through suppression of glutathione by BSO (L-buthionine sulfoximine). This finding has implications for the control of breast cancer with low-dose estrogen and other targeted therapeutic drugs.