Endocrine Therapy Resistance In Breast Cancer Research Articles

Endocrine therapy resistance and epithelial to mesenchymal transition are driven by Nicastrin and Notch4 cooperation in MCF7 breast cancer cells

Endocrine therapy resistant (ETR) tumors often display mesenchymal features, associated with aggressive and enhanced motility behaviour. Notch signalling is over-activated in ETR cells. By blocking it, it is possible to interfere with the cell growth. Notch is also implicated in regulating epithelial to mesenchymal transition (EMT) affecting both migration and invasion in breast cancer cells. We know that Nicastrin is the major component of the multi-subunit protease complex Gamma Secretase (GS) which executes intramembrane proteolysis of integral proteins such Notch. We used two models of Endocrine Therapy Resistant MCF7 breast cancer cell lines which acquired EMT feature and showed higher levels of Nicastrin and Notch targets. Moreover, they displayed higher Notch4 levels but lower Notch1 and Notch2, indicating a possible switch of the Notch signalling when cells acquire resistance. We demonstrated that Gamma Secretase inhibitor PF03084014 and anti-Nicastrin MAbs were particularly effective in partially inhibiting the EMT process by blocking Nicastrin-Notch4 contribution to resistance. Importantly, we also demonstrated that the stem cells-like population was reduced upon these treatments. Both Nicastrin and Notch4 genetic silencing lead to similar effects. Finally, stably overexpressing Nicastrin, was sufficient to activate Notch4 in MCF7 and render them unresponsive to tamoxifen. Here we highlight our recent study.

Targeting tyrosine-kinases and estrogen receptor abrogates resistance to endocrine therapy in breast cancer.

Despite numerous therapies that effectively inhibit estrogen signaling in breast cancer, a significant proportion of patients with estrogen receptor (ER)-positive malignancy will succumb to their disease. Herein we demonstrate that long-term estrogen deprivation (LTED) therapy among ER-positive breast cancer cells results in the adaptive increase in ER expression and subsequent activation of multiple tyrosine kinases. Combination therapy with the ER down-regulator fulvestrant and dasatinib, a broad kinase inhibitor, exhibits synergistic activity against LTED cells, by reduction of cell proliferation, cell survival, cell invasion and mammary acinar formation. Screening kinase phosphorylation using protein arrays and functional proteomic analysis demonstrates that the combination of fulvestrant and dasatinib inhibits multiple tyrosine kinases and cancer-related pathways that are constitutively activated in LTED cells. Because LTED cells display increased insulin receptor (InsR)/insulin-like growth factor 1 receptor (IGF-1R) signaling, we added an ant-IGF-1 antibody to the combination with fulvestrant and dasatinib in an effort to further increase the inhibition. However, adding MK0646 only modestly increased the inhibition of cell growth in monolayer culture, but neither suppressed acinar formation nor inhibited cell migration in vitro and invasion in vivo. Therefore, combinations of fulvestrant and dasatinib, but not MK0646, may benefit patients with tyrosine-kinase-activated, endocrine therapy-resistant breast cancer.

Development of mTOR inhibitor in endocrine therapy-resistant breast cancer

Development of mTOR inhibitor in endocrine therapy-resistant breast cancer

Efficacy of Histone Deacetylase and Estrogen Receptor Inhibition in Breast Cancer Cells Due to Concerted down Regulation of Akt

Hormonal therapy resistance remains a considerable barrier in the treatment of breast cancer. Activation of the Akt-PI3K-mTOR pathway plays an important role in hormonal therapy resistance. Our recent preclinical and clinical studies showed that the addition of a histone deacetylase inhibitor re-sensitized hormonal therapy resistant breast cancer to tamoxifen. As histone deacetylases are key regulators of Akt, we evaluated the effect of combined treatment with the histone deacetylase inhibitor PCI-24781 and tamoxifen on Akt in breast cancer cells. We demonstrate that while both histone deacetylase and estrogen receptor inhibition down regulate AKT mRNA and protein, their concerted effort results in down regulation of AKT activity with induction of cell death. Histone deacetylase inhibition exerts its effect on AKT mRNA through an estrogen receptor-dependent mechanism, primarily down regulating the most abundant isoform AKT1. Although siRNA depletion of AKT modestly induces cell death, when combined with an anti-estrogen, cytotoxicity is significantly enhanced. Thus, histone deacetylase regulation of AKT mRNA is a key mediator of this therapeutic combination and may represent a novel biomarker for predicting response to this regimen.

Genome-wide reprogramming of the chromatin landscape underlies endocrine therapy resistance in breast cancer

The estrogen receptor (ER)α drives growth in two-thirds of all breast cancers. Several targeted therapies, collectively termed endocrine therapy, impinge on estrogen-induced ERα activation to block tumor growth. However, half of ERα-positive breast cancers are tolerant or acquire resistance to endocrine therapy. We demonstrate that genome-wide reprogramming of the chromatin landscape, defined by epigenomic maps for regulatory elements or transcriptional activation and chromatin openness, underlies resistance to endocrine therapy. This annotation reveals endocrine therapy-response specific regulatory networks where NOTCH pathway is overactivated in resistant breast cancer cells, whereas classical ERα signaling is epigenetically disengaged. Blocking NOTCH signaling abrogates growth of resistant breast cancer cells. Its activation state in primary breast tumors is a prognostic factor of resistance in endocrine treated patients. Overall, our work demonstrates that chromatin landscape reprogramming underlies changes in regulatory networks driving endocrine therapy resistance in breast cancer.

Abstract P2-09-02: Epigenetic Regulation of histone variants - a role in hormone therapy resistant breast cancer?

Abstract OBJECTIVE To utilize a genome wide approach to characterize epigenetic changes associated with resistance to estrogen deprivation, thus mimicking resistant to AIs. BACKGROUND Mechanisms of endocrine resistance include the activation of growth factor receptor pathways, overexpression of ER co-activators, and metabolic resistance due to polymorphisms in metabolizing enzymes. Recently, epigenetic mechanisms have been investigated to explain endocrine resistance. The majority of mechanisms for acquired resistance have been described following tamoxifen therapy, but the mechanisms for resistance to aromatase inhibitors (AI) remain less well known. HYPOTHESIS Estrogen deprivation results in altered methylation and altered expression of critical target genes in breast cancer cells, which helps the cell to survive in the absence of estrogen, contributing to acquired AI resistance in breast cancer patients. MATERIALS AND METHODS We utilized derivatives of the ER_ MCF-7 cell line which were isolated after being cultured in estrogen-free media for 9 months (C4-12 cell line) and 18–24 months (LTED, Long term estrogen deprivation cell line). A genome-wide methylation screen was performed using Methyl CpG binding Domain (MBD) pull down assay followed by hybridization into Affymetrix Human Promoter 1.0R Array. Altered DNA methylation was validated by bisulfite genomic sequencing assays while gene expression of candidate genes was studied by qRT-PCR and Western blotting. RESULTS There were a significant amount of differentially methylated genes in C4-12 and LTED when compared to MCF7. Intriguingly, among the genes which were significantly hypomethylated in both endocrine resistant cell lines were a number of histone genes, with the homomorphic variant HIST1H2BE showing the strongest hypomethylation. Canonical histones have both homomorphic and heteromorphic variants, which differ in their timing of expression and similarity to the canonical histone protein's amino acid sequence. Overexpression of various heteromorphic histone variants have been implicated in carcinogenesis, but to date, there have been no studies on homomorphic variants, and specifically H2B variants, in regards to their role in tumorigenesis. We show that hypomethylation of HIST1H2BE was associated with its upregulation in C4-12 and LTED cells. Furthermore, knockdown of of HIST2BE resulted in decreased growth in the endocrine resistant cells, but not in the parental MCF-7 cell lines. CONCLUSION & FUTURE DIRECTIONS The study is novel in two ways; first, this is the first study to show a potential role for histone variants in endocrine resistance. Second, this is one of very few examples where hypomethylation (instead of hypermethylation) results in altered expression of genes critical in tumorigenesis. Current studies include the detailed characterization of the phenotype following over-expression and knockdown of HIST1H2BE, the examination of the timing of expression of this variant in the S phase, and finally we are measuring methylation and expression of HIST2BE in a large collection of tumors from AI-treated breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-09-02.

Chromatin Landscape and Endocrine Response in Breast Cancer

Over two-thirds of breast cancers rely on estrogen receptor α (ERα) for their growth. Endocrine therapies antagonize estrogen-dependent ERα activation but resistance to these treatments occurs and is associated with poor prognosis. Crosstalk between alternative survival pathways and ERα are currently held as the primary cause of resistance. However, blocking these pathways does not cure endocrine therapy resistant breast cancer suggesting the existence of additional mechanisms. While cancer is commonly considered a genetic disease, the importance of epigenetic events in promoting tumor initiation and progression is increasingly recognized. Here, we consider how epigenetic modifications and alterations to the chromatin landscape contribute to endocrine therapy resistance by modulating ERα expression or altering its genomic activity.

Abstract 5753: Potential therapeutic use of ER beta modulators in treating endocrine therapy resistant breast cancers

Abstract Although systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) such as letrozole or block actions of estrogen/ER actions by anti-estrogens (AEs) like tamoxifen are well tolerated, unfortunately, essentially all breast cancers in women with advanced breast cancer develop resistance to these agents, and the benefit of adjuvant therapy is also limited by the development of resistance. In addition, side effects associated with the systemic inhibition of aromatase with current compounds limits their long-term utility of them as chemopreventative agents. Using in vitro and preclinical postmenopausal breast cancer models, we have investigated whether treatment of endocrine therapy resistant (AI or AE) breast cancer cells with selective plant-derived ER beta agonists (Liquiritigenin; LIQ or Nyasol; NYA) alone or in combination with AE or AI affects the growth of resistant breast cancer cells, thereby restores sensitivity to AE or AI. To test whether combination therapy restores sensitivity to letrozole or tamoxifen, we have treated resistant breast cancer cells with either letrozole or tamoxifen alone or with a combination of AE or AI and LIQ/NYA, active ingredient of MF101 used for treatment hot flashes in postmenopausal women. In addition, an effect of these compounds on cell growth was also tested using different breast cancer cells with and without ERα including triple negative breast cancer cells. Efficacy of these drugs was also tested in these model cells with or without ERβ expression. Compared to single agents, combination treatment not only restored sensitivity to letrozole but also resulted in decreased cell proliferation and increased apoptosis as well as increased ERβ and decreased ERα levels in resistant cells. Combination of LIQ or NYA not only diminished cell growth various breast cancer cells (that express endogenous aromatase) but also affected the expression of aromatase. Detailed investigations indicated that LIQ/NYA affects the induction of aromatase by specifically inhibiting breast cancer specific aromatase promoter 1.3 as well as its activity through inflammatory cytokine-mediated actions. ERβ-mediated inhibition of tumor cell growth appears to be regulated by different molecular mechanisms. Some of these molecular pathways are different than that are known to be responsible for resistance to hormonal therapeutic agents. These studies suggest the therapeutic benefit of LIQ or NYA ERβ agonist to resensitize breast cancer cells that are resistant to AEs or AI and their ability to selectively inhibit tumor induction of aromatase. Our study suggests a novel role of natural ER beta agonists as valuable therapeutic modulators to treat endocrine sensitive as well as resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5753. doi:1538-7445.AM2012-5753

Abstract 2702: Effects of Src and IGF1R inhibition on acquired endocrine therapy resistant breast cancer

Abstract Effective standard therapy for hormone receptor-positive (HR+) breast cancer targets the estrogen receptor (ER). However, a significant proportion of women with HR+ breast cancer relapse. Furthermore, the majority of women with metastatic HR+ breast cancer develop resistance to endocrine therapy. Adaptive cross-talk between ER and growth factor receptor pathways including IGF1R and Src is involved in acquired endocrine therapy resistance. To determine effects of adding dasatinib (D), Src inhibitor, and/or anti-IGF1R Ab MK0646 (M) to the ER antagonist fulvestrant (F) in an acquired endocrine therapy resistant breast cancer, we used long-term estrogen-deprived (LTED) derivative of ER+ breast cancer cell lines, MCF-7/LTED and HCC-1428/LTED. Parental and LTED cells were treated with variable combinations of F, D and M, followed by determinations of drug effects on cell growth, migration, and invasion in vitro and tumor growth in vivo. We demonstrated that LTED cells were resistant to F alone, comparing parental cells (P<0.05) in cell growth, migration and invasion. Addition of D to F appeared to be synergistic in all systems including cell growth, cell migration and invasion. However, the addition of M to F or to F+D appeared inhibition on cell growth in monolayer with low-serum medium, but not to significantly add inhibitory effects on cell growth in 3D and cell migration and invasion. The effects of IGF-1R antibody, MK0646, on cells growth in monolayer showed not consistent with 3D, which might be due to growth factors in the matrigel, although we used growth factors decreased matrigel. To verify the effects of the drugs on tumor growth in vivo, we developed xenograft models using MCF-7/LTED cells and treated the mice with F, D, and M along or variable combinations. The study demonstrated that all the combinations of F+D+M, F+D, and F+M (from P< 0.05 to <0.001 in viable time points) show inhibited the tumor growth comparing vehicle or F alone. The inhibitory effect of F+D+M is higher than F+D and F+M. In summary, the addition of dasatinib to fulvestrant appeared to be synergistic in all systems including cell growth, cell migration and invasion and tumor growth in vivo. The addition of MK0646 to fulvestrant mildly inhibited MCF-7/LTED cell growth and xenografts growth, but not cell migration and cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2702. doi:1538-7445.AM2012-2702

Highlights of This Issue

The role of G-protein coupled–receptors (GPCR) in cancer and how to best target their function remains incompletely understood. Using The Cancer Genome Atlas, primary tumor specimens, and in vitro studies, Woerner and coworkers found that expression of G-protein coupled–receptor kinases (GRK), critical regulators of GPCR function, is altered in glioblastoma in a molecular subtype–specific fashion. Decreased GRK3 expression in classical glioblastoma was associated with a growth advantage, which was blocked by reconstitution of GRK3 expression. These data indicate that GPCRs function to regulate glioblastoma growth and suggest that modulation of GPCR phosphorylation may be an important therapeutic strategy.The antioxidant enzyme extracellular superoxide dismutase EcSOD is downregulated in a large proportion of human lung cancers. To explore its role in cancer, Teoh-Fitzgerald and colleagues examined EcSOD gene expression, as well as the biological effects of its reexpression in EcSOD-negative tumor cells. Epigenetic silencing by aberrant cytosine methylation of the promoter was the most frequent cause of expression loss in human lung adenocarcinoma compared with normal lung. Reexpression of EcSOD inhibited the in vitro invasiveness of human lung adenocarcinoma cells. This study identifies EcSOD as an epigenetically regulated lung cancer target and a modifier of the malignant phenotype.TRAIL induces apoptosis in basal/triplenegative breast cancer (TNBC) cell lines, whereas other subtypes are relatively resistant. Inhibition of WEE1, a cell-cycle checkpoint regulator, causes increased cell death in cancer cell lines. Here, Garimella and colleagues show that pretreatment with WEE1 inhibitor increased the toxicity of TRAIL in TNBC cell lines. This effect is attributed to increased surface expression of death receptors resulting in increased caspase activation initiated by caspase-8. These data suggest that the combination of WEE1 inhibitor and TRAIL could provide a novel approach to targeted therapy for TNBC.Although largely unknown, the abnormalities in DNA repair that underlie the genomic instability in sporadic cancers are potential therapeutic targets. Tobin and colleagues have characterized a novel defect in DNA double-strand break repair in hormone therapy–resistant breast cancer that can be identified in breast cancer cell lines and tumor biopsies and can be selectively targeted by a combination of PARP and DNA ligase inhibitors. These preclinical studies provide the rationale for the development of effective therapeutic strategies for hormone therapy–resistant breast cancer that may be applicable to other cancers, including leukemias, with similar DNA repair abnormalities.

P4-01-08: The Role of Ser991 PELP1 Phosphorylation in Therapy Resistance and Metastasis of Breast Cancer.

Abstract BACKGROUND: Growth factor induced activation of MAPK pathway is suggested to play a critical role both in invasive and hormone therapy resistant breast cancer via ligand independent activation of ERα. Proline Glutamic acid Leucine Rich Protein (PELP1) is a well established ER-coregulator that plays a critical role in ER's nuclear and extra-nuclear functions. PELP1 functions as a proto-oncogene and deregulation of PELP1 expression is linked to increased proliferation, metastasis and therapy resistance. The objective of this study is to examine, whether growth factor signaling modulates PELP1 mediated ER coactivation functions via MAPK pathway. METHODS: Bioinformatics and phosphopeptide specific antibodies were employed in this study. In vitro kinase assays and MAPK inhibitors were used to confirm MAPK phosphorylation of PELP1. Site directed mutants and ERE reporter gene assays were used to demonstrate the significance of MAPK site in PELP1. Growth factor and estrogen regulation of PELP1 phosphoryaltion at Ser991 was validated in therapy sensitive (MCF7, ZR75) and resistant (MCF-7 HER2, MCF7-Tam and MCF-7Ca-LTLT cells using PELP1 specific phosphoantibody. Breast cancer model cells stably expressing PELP1 mutant or PELP1 peptides encompassing MAPK phosphorylation was used to test the significance of this MAPK site in PELP1 using proliferation, migration, and invasion assays. IHC analysis using PELP1 phospho antibodies was performed using murine xenograft tumors and human breast tumor arrays (TMA). RESULTS: Growth factors (EGF, Heregulin) induced Ser991 phosphorylation of PELP1 in murine mammary epithelial cells and also in human breast cancer cell lines. Growth factor mediated Ser991PELP1 phosphorylation was abrogated by ERK1/2 MAPK pathway inhibitors including PD98059 and U0126. In ERE-reporter luciferase assays using ZR75 cells, PELP1 (WT) but not the ser991Ala mutant of PELP1 corroborated growth factor-ER crosstalk. Therapy resistant model cells (MCF-7-Tam, MCF-7-LTLT-ca and MCF-7-HER2) exhibited enhanced Ser991PELP1 phosphoryation compared to parental MCF7. TAT-Peptide encompassing Ser991 inhibited growth factor mediated phosphorylation of PELP1 and revealed a potent ability to inhibit the growth of therapy resistant cells. Xenograft tumors of hormone therapy model cell lines showed increased Ser991PELP1 phosphorylation and enhanced nuclear localization compared to hormonal sensitive xenograft tumors. IHC analysis of human breast invasive TMA revealed enhanced PELP1 ser991 phosphorylation in advanced metastatic tumors with prominent nuclear/nucleolar localization compared to normal and benign tumors. CONCLUSIONS: In this study, we identified that growth factor-MAPK pathway modulate ER coregulator PELP1 functions via phosphorylation and demonstrated that Ser991 phosphorylation of PELP1 has the potential to govern the growth Factor-ER cross talk leading to therapy resistance and metastatic phenotype of breast cancer. Peptides encompassing Ser991PELP1 site and Phosphoantibodies targeting Ser991 represent a novel therapeutic target and diagnostic markers in breast cancer. Supported by DOD Fellowship W81XWH-09-1-0010 (BCN) and DOD W81XWH-08-1-0604 (RKV) Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-08.

Roscovitine confers tumor suppressive effect on therapy-resistant breast tumor cells

IntroductionCurrent clinical strategies for treating hormonal breast cancer involve the use of anti-estrogens that block estrogen receptor (ER)α functions and aromatase inhibitors that decrease local and systemic estrogen production. Both of these strategies improve outcomes for ERα-positive breast cancer patients, however, development of therapy resistance remains a major clinical problem. Divergent molecular pathways have been described for this resistant phenotype and interestingly, the majority of downstream events in these resistance pathways converge upon the modulation of cell cycle regulatory proteins including aberrant activation of cyclin dependent kinase 2 (CDK2). In this study, we examined whether the CDK inhibitor roscovitine confers a tumor suppressive effect on therapy-resistant breast epithelial cells.MethodsUsing various in vitro and in vivo assays, we tested the effect of roscovitine on three hormonal therapy-resistant model cells: (a) MCF-7-TamR (acquired tamoxifen resistance model); (b) MCF-7-LTLTca (acquired letrozole resistance model); and (c) MCF-7-HER2 that exhibit tamoxifen resistance (ER-growth factor signaling cross talk model).ResultsHormonal therapy-resistant cells exhibited aberrant activation of the CDK2 pathway. Roscovitine at a dose of 20 μM significantly inhibited the cell proliferation rate and foci formation potential of all three therapy-resistant cells. The drug treatment substantially increased the proportion of cells in G2/M cell cycle phase with decreased CDK2 activity and promoted low cyclin D1 levels. Interestingly, roscovitine also preferentially down regulated the ERα isoform and ER-coregulators including AIB1 and PELP1. Results from xenograft studies further showed that roscovitine can attenuate growth of therapy-resistant tumors in vivo.ConclusionsRoscovitine can reduce cell proliferation and survival of hormone therapy-resistant breast cancer cells. Our results support the emerging concept that inhibition of CDK2 activity has the potential to abrogate growth of hormonal therapy-resistant cells.

Killing the second messenger: targeting loss of cell cycle control in endocrine-resistant breast cancer

The majority (∼70%) of breast cancers are steroid hormone receptor (SR) positive at the time of diagnosis. Endocrine therapies that target estrogen receptor α (ERα) action (tamoxifen, toremifene, fulvestrant) or estrogen synthesis (aromatase inhibitors: letrozole, anastrozole, exemestane; or ovarian suppression) are a clinical mainstay. However, up to 50% of SR+ breast cancers exhibit de novo or acquired resistance to these clinical interventions. Mechanisms of resistance to endocrine therapies often include upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Cyclin D1, the regulatory subunit of cyclin-dependent protein kinases four and six (CDK4/6) serves as a convergence point for multiple signaling pathways. In a recent paper entitled 'Therapeutically Activating Retinoblastoma (RB): Reestablishing Cell Cycle Control in Endocrine Therapy-Resistant Breast Cancer', Thangavel et al. reported maintenance of cyclin D1 expression and RB phosphorylation in the face of ER ablation in multiple breast cancer cell line models of endocrine resistance. RB-dysfunction defined a unique gene signature that was associated with luminal B-type breast cancer and predictive of poor response to endocrine therapies. Notably, a new CDK4/6 inhibitor (PD-0332991) was capable of inducing growth arrest by a mechanism that was most consistent with cellular senescence. In this review, these findings are discussed in the context of SRs as important mediators of cell cycle progression, and the frequent loss of cell cycle checkpoint control that typifies breast cancer progression. These studies provide renewed hope of effectively stabilizing endocrine-resistant breast cancers using available complementary (to endocrine-based therapies) cytostatic agents in the form of CDK4/6 inhibitors.

A phase II study of the histone deacetylase inhibitor vorinostat combined with tamoxifen for the treatment of patients with hormone therapy-resistant breast cancer

Background:Histone deacetylases (HDACs) are crucial components of the oestrogen receptor (ER) transcriptional complex. Preclinically, HDAC inhibitors can reverse tamoxifen/aromatase inhibitor resistance in hormone receptor-positive breast cancer. This concept was examined in a phase II combination trial with correlative end points.Methods:Patients with ER-positive metastatic breast cancer progressing on endocrine therapy were treated with 400 mg of vorinostat daily for 3 of 4 weeks and 20 mg tamoxifen daily, continuously. Histone acetylation and HDAC2 expression in peripheral blood mononuclear cells were also evaluated.Results:In all, 43 patients (median age 56 years (31–71)) were treated, 25 (58%) received prior adjuvant tamoxifen, 29 (67%) failed one prior chemotherapy regimen, 42 (98%) progressed after one, and 23 (54%) after two aromatase inhibitors. The objective response rate by Response Evaluation Criteria in Solid Tumours criteria was 19% and the clinical benefit rate (response or stable disease >24 weeks) was 40%. The median response duration was 10.3 months (confidence interval: 8.1–12.4). Histone hyperacetylation and higher baseline HDAC2 levels correlated with response.Conclusion:The combination of vorinostat and tamoxifen is well tolerated and exhibits encouraging activity in reversing hormone resistance. Correlative studies suggest that HDAC2 expression is a predictive marker and histone hyperacetylation is a useful pharmacodynamic marker for the efficacy of this combination.

Abstract 1733: Therapeutic significance of ERα—PELP1 axis in blocking endocrine therapy resistance

Abstract Breast cancer therapies target the estrogen receptor-alpha (ERα) with letrozol an aromatase inhibitor or anti-estrogens such tamoxifen to limit circulating estrogen levels and alter ERα functions. Many women suffering from recurrent or advanced disease develop resistance to current targeted endocrine treatments. Emerging data suggests therapeutic resistance is precipitated through dysfunction of ERα coregulatory proteins and crosstalk between ERα and growth factor signaling. Deregulated expression of proline-, glutamic acid- and leucine-rich protein 1 (PELP1), an ERα coregulator, was observed in a subset of human breast tumors. Recently, PELP1 was identified as an independent prognostic biomarker of shorter survival in ERα positive/luminal-like breast cancer patients. Epigenetic changes of target gene promoters are modulated by PELP1 via an interaction with lysine specific demethylase 1 (KDM1). Additionally, PELP1 promotes crosstalk between ERα and Src kinase leading to enhanced activation of extranuclear signaling pathways. Therapeutic potential of targeting the ERα-PELP1 axis in vivo was examined using a pharmacological approach in well-established preclinical models of endocrine therapy resistant breast cancer. We utilized (1) siRNA liposomes to down regulate PELP1 expression; (2) pargyline and NCL-1, KDM1 inhibitors, to block PELP1-mediated epigenetic modifications and (3) dasatinib, a Src kinase inhibitor, to prevent PELP1-mediated extranuclear actions in both pre- and post-menopausal conditions. In xenograft-based assays, treatment of ERα positive breast cancer cells with liposomes containing PELP1-siRNA, dasatinib or KDM1 inhibitors significantly reduced proliferation and tumor growth. Combinatorial therapies of letrozol or tamoxifen with PELP1 axis blockers substantially impeded xenograft tumor growth by blocking both ERα nuclear and extranuclear signaling and promoting apoptosis in therapy resistant cells. Taken together, our results suggest targeting the ERα-PELP1 axis in combination with current endocrine therapies will enhance therapeutic efficacy and may inhibit or delay development of endocrine therapy resistance. This study is funded by Komen grant KG090447 and NIH grant CA095681. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2011-1733

Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy-resistant breast cancer

The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.

Abstract P2-19-01: Modulating Response to Phosphoinositol-3-Kinase/mTOR Inhibition in ER Positive Breast Cancer Cell Lines after Acquired Resistance to Estrogen Deprivation

Abstract Background: We have recently shown that acquired resistance to aromatase inhibitors (AI) can, in some instances, be reversed low-dose estradiol treatment until progression followed by repeat treatment with an aromatase inhibitor (Ellis, M. JAMA 302:774, 2009). We have also demonstrated that simultaneous inhibition of phosphoinositol-3-kinase (PI3K) and ER (though estrogen deprivation) activates cell death through apoptosis, which is reversed by estradiol, revealing a link between ER activation and the efficacy of PI3 kinase inhibition (Crowder, R. Cancer Res 69:3955, 2009). Consistent with this concept we have also shown that long term estrogen deprived (LTED) cells are co-resistant to both classes of agent (SABCS 2009 Abs#3131). We therefore studied ways to overcome resistance to PI3 kinase inhibitors in LTED cell lines using second line endocrine therapy strategies that could be offered to patients with AI resistant advanced disease. Methods: ER+ MCF7 and T47D (PIK3CA mutant) cells were cultured in phenol-red free media containing charcoal-stripped serum for 12 months to create long term estrogen deprivation (LTED) variants that mimicked clinical endocrine therapy resistance. A variant of the MCF7 LTED line that regained estrogen-dependent proliferation was created by re-exposure to estradiol for 4 months (MCF7 LTED-R). The effects of the PI3K isoform inhibitor BKM120, the mTOR inhibitor RAD001 and the dual PI3K/mTOR inhibitor BGT226 in MCF7 LTED and T47D LTED were assessed in combination with estrogen deprivation and fulvestrant. MCF7 LTED-R cells were treated with PI3 kinase pathway inhibitors after acute estradiol withdrawal. Apoptosis was measured using TUNEL flow cytometry. Results: MCF7 LTED were resistant to all three PI3 kinase inhibitors, but combination treatment with fulvestrant restored apoptosis to levels seen with parental MCF7 cells after acute estrogen-deprivation. Acute estrogen deprivation in MCF7 LTED-R cells was also effective in restoring an apoptotic response to BGT226. T47D LTED cells, which, unlike MCF7 cells, have markedly down-regulated ER expression, become more sensitive to PI3 kinase monotherapy than the parental T47D cell line, particularly in response to BKM120, and fulvestrant did not increase the apoptotic response. Conclusions: The MCF7 cell results suggest that PI3 kinase inhibitors will be effective in endocrine therapy resistant advanced breast cancer if these agents are administered with appropriate second line endocrine strategies. Potentially efficacious approaches include fulvestrant combinations and second-line aromatase inhibition, but only after restoration of AI sensitivity with estradiol. The T47D experiments suggest that in the setting of ER+ tumors that lose ER upon disease progression PI3 kinase inhibitors may be very effective. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-19-01.

Abstract 1031: Sphingolipid metabolism as a therapeutic target in chemotherapy resistant and hormone therapy resistant breast cancer

Abstract Endocrine- and chemo-resistance are major causes for failure of treatment in recurrent breast cancer. Normal apoptotic pathways targeted by chemotherapeutic agents become altered, diminishing their efficacy and increasing resistance mechanisms. The proapoptotic sphingolipid compound N-Octanoyl-D-erythro-sphingosine (C8-ceramide) mimics endogenous long chain ceramide and is a potent drug in endocrine-/chemo-resistant breast cancer cells. In this study, we examined basal expression levels of modifier enzymes for endogenous ceramide regulation across drug sensitive, chemoresistant and hormone therapy- resistant breast cancer cells lines. Differences were found among UDP-galactose ceramide galactosyltransferase, sphingosine kinase-1, and acid sphingomyelinase gene expression in: MCF-10A, MCF-7, MDA-MB-231 and MCF-7TN-R cell lines. Using C8-ceramide as the lead compound, 8 novel ceramide analogs were synthesized with either enol or amide backbone substitutions and tested for biological activity. The enol substitution was found to be the most potent, with (2S,3R,E)-2-((E)benzylideneamino)octadec-4-ene1,3-diol (Analog C) displaying the greatest reduction in proliferation. Analog B appeared to be the most potent cytotoxic drug in MCF-7 and MDA-MB-231 cell lines with IC50 values of 6.2uM and 3.0 uM respectively. In the cytotoxicity assay MCF-7TN-R cell line, Analog A appeared to be the most potent with an IC50 of 2.4uM. The clonogenic proliferation assay provided very different, yet promising results. Analog D had the lowest IC50 of 460nM in MDA-MB-231, Analog C had an IC50 of 100nM in MCF-7TN-R, and Analog A had an IC50 of 1.48uM in the MCF-7 breast cancer cell line. All of the IC50s for proliferation assays were at least an order of magnitude more potent than the control drug C8 (IC50 of 5uM). Due to the potency of these drugs, these results exemplify the potential of novel ceramide-targeted therapies in chemo-resistant and endocrine-resistant breast cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1031.

MTOR inhibition reverses acquired endocrine therapy resistance of breast cancer cells at the cell proliferation and gene‐expression levels

Activation of the Akt/mammalian target of rapamycin (mTOR) pathway has been shown to be associated with resistance to endocrine therapy in estrogen receptor alpha (ERalpha)-positive breast cancer patients. Utmost importance is attached to strategies aimed at overcoming treatment resistance. In this context, this work aimed to investigate whether, in breast cancer cells, the use of an mTOR inhibitor would be sufficient to reverse the resistance acquired after exposure to endocrine therapy. The ERalpha-positive human breast adenocarcinoma derived-MCF-7 cells used in this study have acquired both cross-resistance to hydroxy-tamoxifen (OH-Tam) and to fulvestrant and strong activation of the Akt/mTOR pathway. Cell proliferation tests in control cells demonstrated that the mTOR inhibitor rapamycin enhanced cell sensitivity to endocrine therapy when combined to OH-Tam or to fulvestrant. In resistant cells, rapamycin used alone greatly inhibited cell proliferation and reversed resistance to endocrine therapy by blocking the agonist-like activity of OH-Tam on cell proliferation and bypassing fulvestrant resistance. Reversion of resistance by rapamycin was associated with increased ERalpha protein expression levels and modification of the balance of phospho-ser167 ERalpha/total ERalpha ratio. Pangenomic DNA array experiments demonstrated that the cotreatment of resistant cells with fulvestrant and rapamycin allowed the restoration of 40% of the fulvestrant gene-expression signature. Taken together, data presented herein strongly support the idea that mTOR inhibitor might be one of the promising therapeutic approaches for patients with ERalpha-positive endocrine therapy-resistant breast cancers.