Term Estrogen Deprivation Research Articles

Identification of estrogen receptor down-regulators for endocrine resistant breast cancer

Resistance to endocrine therapies remains an impediment for the treatment of estrogen receptor (ER) positive breast cancer. ER down regulator Fulvestrant has showed great activity to overcome the endocrine resistance. However, Fulvestrant has poor bioavailability due to the hydrophobicity. Identification of novel ER down regulator is still important. Compounds 172 and 183 are two steroidal compounds with androgen scaffold but significantly down regulated ER in multiple breast cancer cell lines. RT-PCR results indicated that both compounds did not affect ER gene expression. Proteasome inhibitor MG132 could attenuate ER down regulation effect of the compounds, suggesting that the ER down regulation was via ubiquitin-proteasomal pathway. Furthermore, compounds 172 and 183 could downregulate ER in endocrine resistant breast cancer cell model long term estrogen deprivation (LTED) MCF-7 cells. Hydrophobicity of compounds 172 and 183 were determined and showed improved solubility compared to Fulvestrant. All these results suggested that compounds 172 and 183 could be potential lead compounds for drug development for the treatment of endocrine resistance breast cancer.

Abstract 1076: Hormonal regulation of SEMA7A promotes therapeutic resistance in ER+ breast cancer

Abstract Background: The majority of all breast cancers (BC) are estrogen receptor positive (ER+). While ER-targeting endocrine therapies have improved patient survival, many tumors become drug resistant and recur within 20 years. Thus, novel targets are needed to treat recurrent ER+BC. Previous reports have described tumor-promotional effects of Semaphorin 7A (SEMA7A) in ER- disease; however, the role of SEMA7A in ER+ disease is not well-characterized. Hypothesis: SEMA7A promotes cell survival and drug resistance in ER+ BC. Methods: We measured expression of SEMA7A in drug resistant cells via immunoblotting. We then overexpressed SEMA7A and used ER-targeting drugs (fulvestrant and tamoxifen) to test how SEMA7A-expressing cells respond to endocrine therapy. In vitro, we utilized cell proliferation and survival assays. In vivo, we implanted ER+ BC cells, then treated the animals with fulvestrant to assess how SEMA7A affects primary tumor volume and metastasis. We also measured markers of “stemness” and multi-drug resistance via flow cytometry. Results: We found that SEMA7A expression correlated with decreased relapse free survival in patients who received endocrine therapy for ER+BC (Kmplotter; p=0.042). We also observe that SEMA7A can be hormonally regulated in ER+BC, but expression did not consistently decrease with endocrine therapy. Instead, long term estrogen deprivation and ER-targeting treatments increased SEMA7A expression, likely through signaling via other hormone receptors such as the androgen receptor, which also increases with long term estrogen deprivation. Additionally, overexpression of SEMA7A promoted in vitro growth and survival of ER+ cells during estrogen-deprivation, tamoxifen, or fulvestrant treatments. In vivo, SEMA7A promoted fulvestrant resistance in the primary tumor and also induced lung metastases. Finally, we identified pro-survival signaling as a therapeutic vulnerability of ER+SEMA7A+ tumors. Conclusion: These studies show that SEMA7A can promote drug resistance in ER+ BC. We suggest that targeting pro-survival signaling may prove efficacious for treating SEMA7A+ tumors, which are less likely be resistant to endocrine therapies. Citation Format: Lyndsey S. Crump, Garhett L. Wyatt, Taylor R. Rutherford, Jennifer K. Richer, Weston W. Porter, Traci R. Lyons. Hormonal regulation of SEMA7A promotes therapeutic resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1076.

Hormonal Regulation of Semaphorin 7a Promotes Therapeutic Resistance in Breast Cancer

Background: The majority of all breast cancers (BC) are estrogen receptor positive (ER+). While ER-targeting endocrine therapies have improved patient survival, many of these tumors develop drug resistance and recur within 20 years. Therefore, novel targets are needed to predict for recurrence and to treat recurrent ER+BC. Previous reports describe a tumor-promotional role for Semaphorin 7A (SEMA7A) in ER- disease; yet, the role of SEMA7A in ER+ disease is poorly characterized. Hypothesis: SEMA7A promotes cell survival and drug resistance in ER+ BC. Methods: We overexpressed SEMA7A in ER+ BC cells, then used the ER-targeting agents tamoxifen and fulvestrant to test how SEMA7A-expressing cells respond to endocrine therapy. In vitro, we used proliferation and cell survival assays. In vivo, we implanted ER+ BC cells, then treated the animals with fulvestrant to measure how SEMA7A affects tumor growth and metastasis. We also utilized drug resistant cells, which have high endogenous SEMA7A levels, to measure markers of stemness and multi-drug resistance via flow cytometry. Results: We first found that SEMA7A expression correlates with decreased relapse free survival in patients with ER+BC who received endocrine therapy (Kmplotter; p=0.042). We also observe that SEMA7A is hormonally regulated in ER+BC, but its expression does not uniformly decrease with endocrine therapy agents. Instead, long term estrogen deprivation and ER-targeting drug treatments increase SEMA7A expression, likely through the action of other hormone receptors such as the androgen receptor, which also increases with long term estrogen deprivation. Further, in ER+ cell lines, overexpression of SEMA7A promotes in vitro growth in the face of estrogen-deprivation, tamoxifen, or fulvestrant treatments. In vivo, SEMA7A promotes fulvestrant resistance in the primary tumor and induces lung metastases. Finally, we report that pro-survival signaling is a therapeutic vulnerability of ER+SEMA7A+ tumors. Conclusion: These studies describe that SEMA7A promotes drug resistance in ER+ BC. We propose that targeting pro-survival signaling may prove efficacious for treating SEMA7A+ tumors, which are less likely to respond to endocrine therapies.

New insights into acquired endocrine resistance of breast cancer.

The translational research strategy of targeting estrogen receptor α (ERα) positive breast cancer and then using long term anti-hormone adjuvant therapy (5-10 years) has reduced recurrences and mortality. However, resistance continues to occur and improvements are required to build on the success of tamoxifen and aromatase inhibitors (AIs) established over the past 40 years. Further translational research has described the evolution of acquired resistance of breast cancer cell lines to long term estrogen deprivation that parallels clinical experience over years. Additionally, recent reports have identified mutations in the ERα obtained from the recurrences of AI treated patients. These mutations allow the ERα to activate without ligands and auto stimulate metastatic tumor growth. Furthermore, the new biology of estrogen-induced apoptosis in acquired resistant models in vitro and in vivo has been interrogated and applied to clinical trials. Inflammation and stress are emerging concepts occurring in the process of acquired resistance and estrogen-induced apoptosis with different mechanisms. In this review, we will present progress in the understanding of acquired resistance, focus on stress and inflammatory responses in the development of acquired resistance, and consider approaches to create new treatments to improve the treatment of breast cancer with endocrine resistance.

Abstract P3-05-10: Non-coding RNAs derived from near the ESR1 gene acts as a transcriptional regulator during estrogen deprivation adaptation of ER positive breast cancer cells

Abstract Endocrine therapies that blocks estrogen production are effective for estrogen receptor (ER)-positive breast cancer. However, endocrine therapy treated patients eventually experience relapse after a long period of estrogen deprivation. The mechanism underlying acquisition of estrogen independent growth by ER positive breast cancer cells remains unclear. To understand such molecular mechanism, we used a cell model LTED (long term estrogen deprivation) which MCF7 cells were cultured under estrogen deprivation for 4-10 months. In LTED cells, we found that ER encoded gene ESR1 was up-regulated and ER overproduction was essential for estrogen-independent cell growth. We also revealed that RNA transcriptions of the ESR1 and several neighbor genes were co-induced from both coding and non-coding regions in LTED cells, using RNA-sequence. These highly transcribed regions were corresponded to active histone modifications and transcription factor bindings according to publically available genome-wide analyses data. Fluorescence in situ hybridization (FISH) analyses indicated that RNA from the chromatin domain region nearby ESR1 were co-localized and made foci in nucleus. We found non-coding regions that are particularly highly transcribed. FISH analyses indicated that RNAs from these regions might interact with the parental ESR1 gene locus. Recent studies have shown that non-coding RNAs are involved in transcriptional regulation and chromatin regulation. To understand the role of the non-coding RNA, we have generated MCF7 cells lines that lack the non-coding site, using CRISPR/CAS9 system. We found that mRNA transcription of multiple genes including ESR1 were impaired by the deletion. These findings suggested that these non-coding RNAs may be involved in chromatin regulation of the chromatin domain nearby ESR1. In this study, we found non-coding RNAs that control transcription of chromatin domain genes in ER positive breast cancer cells. Such non-coding RNA mediated transcriptional regulation might be critical for endocrine therapy resistance adaptation. Citation Format: Fujiwara S, Saitoh N, Tomita S, Abdalla MO, Iwase H, Nakao M. Non-coding RNAs derived from near the ESR1 gene acts as a transcriptional regulator during estrogen deprivation adaptation of ER positive breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-10.

Abstract P3-05-05: Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant breast cancer

Abstract AIM To target tumor re-wiring by combined mTORC1 inhibition plus hormonal treatment with or without co-blockade of ERBB signaling in endocrine resistant models of human breast cancer (BC). BACKGROUND Around 80% of BCs are estrogen receptor positive (ER+). Endocrine therapies target estrogenic stimulation of tumor growth but resistance remains problematic. Several strategies have shown that resistance often depends on the acquisition of enhanced cross-talk between ER and growth-factor pathways, allowing the disease to circumvent the need for steroid hormones. We have previously reported the antiproliferative effects of the combination of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signaling were observed. We hypothesised that combined targeting of three signaling pathways, namely ER, ERBB and mTORC1 may provide enhanced anti-tumor activity. METHODS ER+ BC cell lines (MCF7, SUM44 and HCC1428) adapted to long term estrogen-deprivation (LTED) which model relapse on an aromatase inhibitor, along with their wild-type (wt) cell lines were treated with neratinib, a pan-ERBB tyrosine kinase inhibitor, in combination with RAD001 ± estradiol (E2), tamoxifen or fulvestrant. End points included proliferation, cell signaling, cell cycle and effect on ER-mediated transactivation and recruitment by ChIP. RESULTS All cell lines showed a concentration-dependent decrease in proliferation in response to RAD001 (IC50 0.6-50nM in absence of E2 and 1-10nM in presence of E2). A wide range of IC50 values (300-1000nM) was observed with neratinib treatment in the presence of E2. However, in the absence of E2, wt cell lines showed IC50 values in excess of 1800nM with hormetic dose response curves, in which lower concentrations induced cell proliferation. In contrast, LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in all wt cell lines and their LTED derivatives, but the maximum effect was observed when a triple combination of RAD001, neratinib and ER-blockade was used. Expression of pS6 was dramatically suppressed by RAD001 ± neratinib in all cell lines tested, whilst neratinib caused a cell line specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines following treatment with RAD001, indicating both inhibitors were effectively suppressing their respective targets. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and significantly reduced cell cycle progression. In the absence of E2, RAD001 caused a reduction in ER-mediated transcription and decrease in recruitment of ER and the CREB-binding protein (CBP) to the TFF1 promoter. In contrast, neratinib induced a marked increase in ER-recruitment and concomitant rise in ER-mediated transactivation, which was reduced by the addition of RAD001. CONCLUSION Targeting tumor re-wiring by triple blockade of ERBB, ER and mTORC1 signaling pathways significantly reduces cell proliferation supporting the potential combination in patients who have relapsed on endocrine therapy and retain a functional ER. Citation Format: Nikitorowicz-Buniak J, Ribas R, Rani A, Pancholi S, Guest SK, Cutler Jr RE, Lalani A, Dowsett M, Johnston SR, Martin L-A. Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-05.

Abstract 25: Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells

Abstract Estrogen deprivation using aromatase inhibitors (AIs) is the standard of care for postmenopausal women with hormone receptor-positive breast cancer. However, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. We have previously reported the development of an AI-resistant breast cancer cell line, MCF-7:5C, which was clonally derived from parental MCF-7 breast cancer cells following long term estrogen deprivation. A unique phenotype of AI-resistant MCF-7:5C cells is that they grow in the absence of estrogen; however, when they are treated with estradiol they undergo apoptosis/cell death through activation of the mitochondrial death pathway. More recently, we have also discovered that AI-resistant MCF-7:5C cells constitutively overexpress the interferon stimulated gene IFITM1. IFITM1 is a cell surface membrane protein that is most well-known for its ability to block viral replication, however, its role in breast cancer and AI-resistance is not known. We found that IFITM1 was constitutively overexpressed at the protein (> 20-fold) and mRNA (> 15-fold) level in AI-resistant MCF-7:5C breast cancer cells compared to parental MCF-7 cells and that its overexpression strongly correlated with enhanced growth and increased aggressiveness. Clinical data also indicated that IFITM1 was overexpressed in 36 out of 40 AI-resistant breast tumor samples compared to AI-sensitive tumors. In this study, we assessed the functional role of IFITM1 in AI-resistant MCF-7:5C cells. Notably, we found that knockdown of IFITM1 significantly induced cell death in MCF-7:5C cells and it reduced cell proliferation. The induction of cell death as a result of IFITM1 knockdown was associated with increases in p21, Bax, Noxa, and Puma expression; however, p53 was not altered in these cells. To validate the effect observed with IFITM1 knockdown in our resistant cells we used a second shRNA targeting IFITM1. We found that shRNA knockdown of IFITM1 induced PARP cleavage and p21, reduced cell proliferation, and induced cell death in AI-resistant MCF-7:5C cells. Moreover, we found that knockdown of IFITM1 dramatically enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells which was associated with induction of pro-apoptotic proteins and p21 via a p53-independent mechanism. Further analysis revealed that knockdown of STAT1 and STAT2, which are critical regulators of IFITM1, also significantly enhanced estradiol-induced apoptosis in MCF-7:5C cells. Taken together, these findings indicate that overexpression of IFITM1 plays a critical role in breast cancer and AI-resistance and that targeting this gene can sensitize AI-resistant cells to estrogen-induced death. Furthermore, these results suggest that IFITM1 can regulate the mitochondrial pathway through its interactions with anti-apoptotic proteins. Citation Format: Hye Joung Choi, Joan Lewis-Wambi. Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 25. doi:10.1158/1538-7445.AM2015-25

Abstract 5488: Everolimus inhibits the proliferation of aromatase inhibitor-resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) are currently the standard treatment for estrogen receptor positive breast cancer in postmenopausal women. Unfortunately, the majority of patients treated with AIs eventually develop resistance to these drugs. The mechanism by which AI resistance occurs is still not known, however, there is strong evidence that upregulation of the PI3K/Akt/mTOR pathway might play an important role. As such, mTOR inhibition is now under study in clinical trials as a breast cancer treatment. The newest mTOR inhibitor, Everolimus (Afinitor®), has been FDA approved for the treatment of metastatic breast cancer. We have previously reported the development of two AI-resistant breast cancer cell lines (MCF-7:5C and MCF-7:2A) which were clonally derived from parental MCF-7 cells following long term estrogen deprivation. Both MCF-7:5C and MCF-7:2A cells have been shown to overexpress members of the PI3K/Akt/mTOR pathway. In this study, we examined the therapeutic benefits of everolimus in AI-resistant MCF-7:5C and MCF-7:2A cells compared to endocrine sensitive MCF-7 breast cancer cells and normal breast epithelium MCF10A cells. Results from cell viability assay showed that everolimus markedly inhibited the proliferation of endocrine sensitive MCF-7 cells and AI-resistant MCF-7:5C and MCF-7:2A cells in a time-dependent and concentration-dependent manner with IC50s of 30 nM, 40 nM and 10 nM, respectively. Notably, the MCF-7:2A cells were the most sensitive to everolimus which caused a 70% reduction in proliferation at 24 hours whereas in MCF-7 and MCF-7:5C cells only 30% inhibition was observed at the same time point. The inhibitory effect of everolimus was due primarily to G1 arrest and was associated with significant downregulation of phospho-mTOR, phospho-p70S6K, phospho-Akt, GSK3β, cyclin D1 and p21. Colony formation assay in soft agar indicated that AI-resistant MCF-7:5C and MCF-7:2A cells formed significantly more colonies than MCF-7 cells and that treatment with everolimus notably reduced the size and number of colonies for all three cell lines. Finally, we found that exposure of AI-resistant MCF-7:5C and MCF-7:2A cells to a single dose of gamma-irradiation (6 Gy) significantly enhanced the anti-proliferative effect of everolimus on these cells. In summary, these studies showed that everolimus significantly inhibited the proliferation and tumorigenicity of AI-resistant breast cancer cells and that everolimus might be an effective treatment option for patients with AI-resistant breast cancer. Citation Format: Asona Lui, Joan Lewis-Wambi. Everolimus inhibits the proliferation of aromatase inhibitor-resistant breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5488. doi:10.1158/1538-7445.AM2014-5488

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.

Abstract 2293: Weak or partial agonism of ERα during long term estrogen deprivation drives unique mechanisms of estrogen-independent growth

Abstract Endocrine therapy remains the cornerstone of treatment of estrogen-receptor positive breast cancer, with aromatase inhibitors (AIs) the current front-line therapy in post-menopausal women. Despite extending disease-free survival, 15-20% of patients receiving adjuvant AIs will relapse within 5 years. Long term estrogen deprivation (LTED) of breast cancer cells in culture has been successfully used to mimic AI-induced estrogen depletion to dissect mechanisms of AI resistance, implicating several pathways in estrogen-independent growth. However, patients treated with the AI anastrozole on the ATAC trial were reported to have average serum estrogen (E2) concentrations of 3pM. Importantly, in vitro growth assays with MCF-7 cells show that 3pM E2 is sufficient to induce growth. Further, 5α-androstane-3β,17β-diol (3βAdiol) is an androgen metabolite generated independent of aromatase, and thus is insensitive to AI therapy. We have demonstrated that 3βAdiol is an ERα agonist and can induce the growth of breast cancer cells. We sought to expand upon established LTED models to account for these observations. We hypothesize that weak or partial agonism of ERα during LTED by low concentrations of E2 or by 3βAdiol may result in the development of estrogen-independence different to that seen during complete estrogen deprivation. To model the effects of long term low concentrations of E2 or 3βAdiol, MCF-7 cells were grown in medium with charcoal stripped serum supplemented with defined hormone concentrations. Cells were selected in the EC10 and EC90 of E2 (1pM (LE) and 50pM (HE), respectively) or 3βAdiol (50pM (L3) and 1nM (H3), respectively), or estrogen deprived (ED – with 0.05% ethanol vehicle) for 7 months. Estrogen-independence was evaluated throughout selection by assessing cell growth in the absence or presence of E2 or 3βAdiol. ED cells developed estrogen-independence at 9-12 weeks, and are minimally responsive to E2 or 3βAdiol. H3, L3 and LE cells developed the ability to grow in estrogen-free conditions by 3-4 months, 4-5 months, and 6-7 months, respectively. L3 and LE cells are minimally responsive to E2/3βAdiol, while H3 cells remain fully responsive to E2/3βAdiol compared to parental MCF-7. HE cells continue to mimic parental cells after selection. Estrogen-free growth is blocked by anti-estrogen in LE and L3 cells; ED and H3 cells are anti-estrogen resistant. Western blot analysis demonstrated that LE and L3 cells maintain ERα activation (via ERα phosphorylation) in the absence of hormone, correlating with activated MAPK and Akt. cDNA expression analysis suggests distinct mechanisms of estrogen-independence in ED versus H3 cells. The presence of low concentrations of E2 or 3βAdiol selected unique mechanisms of estrogen-independence versus traditional LTED. These long term selections may represent novel and clinically relevant models of AI resistance. 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 2293. doi:10.1158/1538-7445.AM2011-2293

In vitro and in vivo effects of a cyclooxygenase-2 inhibitor nimesulide analog JCC76 in aromatase inhibitors-insensitive breast cancer cells

Third generation aromatase inhibitors (AIs) are more effective than tamoxifen in the treatment of estrogen receptor (ER) positive breast cancer. However, long-term use of AIs commonly results in resistance. We examined whether compound JCC76{Cyclohexanecarboxylic acid [3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-amino)-phenyl]-amide}, an analog of Cyclooxygenase-2 (COX-2) inhibitor nimesulide, can inhibit the growth of AI-insensitive breast cancer cells and the mechanisms by which the compound affects cell proliferation. LTEDaro (long term estrogen deprived MCF-7aro cell) cells, which are a model for AI resistance, were used in this study. JCC76 effectively inhibited LTEDaro cell proliferation with an IC 50 of 2.75 ± 0.31 μM. Further investigations reveal that the compound significantly induced apoptosis in LTEDaro cells by decreasing pAKT, BCL-2 and pBad protein levels, which were all up regulated in the cells after long term estrogen deprivation. LTEDaro tumor size and weight were decreased in ovariectomized nude mice treated with the compound, and cell apoptosis in the tumor tissue was increased compared to the control. The animal weight remained almost unchanged which indicated the low toxicity of the compound. These results suggest that JCC76 overcame AI resistance by inducing cell apoptosis as illustrated in the in vitro and in vivo models. Collectively, results from this study provide data to support that nimesulide analog JCC76 may be a new drug candidate to treat AI-resistant breast cancers. It could be also used as a lead to design and synthesize more potent derivatives.

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 P4-02-12: Genomic Evolution of Endocrine-Resistant Breast Cancer Cell Lines Reveals Molecular Aberrations Consistent with Biological Phenotype

Abstract Suppression of estrogen synthesis using aromatase inhibitors (AIs) is highly effective in the treatment of postmenopausal women with estrogen receptor alpha-positive (ER+) breast cancer. Third generation AIs are superior to adjuvant tamoxifen resulting in improved disease-free survival and a lower incidence of side effects. Unfortunately, one of the consequences of long-term estrogen deprivation or exhaustive endocrine therapy is the development of drug resistance. We and others have shown that acquisition of resistance to long term estrogen deprivation or to selective estrogen receptor modulators (SERMs) in breast cancer cells is accompanied by an increase in malignant cell behavior. The Jordan Laboratory has developed in vitro and in vivo models of endocrine-resistant breast cancer that mimic pathobiology observed in anti-hormone refractory breast tumors. MCF-7:5C and MCF-7:2A are two ER+ human breast cancer cell lines derived from long term estrogen deprivation of hormone-dependent MCF-7 cells. MCF-7:TAM2 and MCF-7:RAL2 are also ER+ derivatives of MCF-7 that are resistant to tamoxifen and raloxifene, respectively. These models of resistance share several phenotypic and molecular characteristics, which differ dramatically from the isogenic parental line. Differences include robust growth in estrogen-deprived medium, mesenchymal morphology, and increased invasiveness and motility in vitro. They also exhibit elevated AKT activity, loss of E-cadherin, and additional molecular markers consistent with malignant progression. We have conducted an array-based genomic study to elucidate molecular mechanisms associated with development of endocrine resistance in each model. We observed a high degree of genomic evolution in all endocrine-resistant models. Several regions of common genomic aberration were observed in the MCF-7:5C and MCF-7:2A cells that indicate significant deregulation of glycolysis and glucose metabolism, a metabolic process known to be driven by activated AKT. Gene ontology analysis of genes differentially expressed by both MCF-7:5C and MCF-7:2A also highlighted deregulated AKT signaling and cell cycle control in these cells. Interestingly, amplification of the estrogen receptor 1 (ESR1) gene, which encodes estrogen receptor alpha, was observed in MCF-7:5C and MCF-7:2A models but not in the SERM-resistant models supporting diversity underlying mechanisms of endocrine resistance depending on therapies used. This study indicates that biological drivers of endocrine resistance can be identified using integrated genomic and bioinformatic approaches. We are currently prioritizing the likely molecular drivers of endocrine-resistant disease using high throughput RNAi technology. Grant support: Department of Defense Breast Program under award number BC050277 Center of Excellence (V.C. Jordan). Views and opinions of and endorsements by the author(s) do not reflect those of the U.S. Army or the Department of Defense. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-12.

Estrogen Deprivation Results in Altered DNA Methylation Profile in Breast Cancer Cells – Role in Endocrine Resistance?

Abstract Postmenopausal breast cancer patients benefit from aromatase inhibitors (AIs) that reduce the levels of estrogens which are critical for tumor growth. To date, epigenetic contributions to hormonal therapy resistance have not been well characterized, with only a limited understanding of antiestrogen mediated epigenetic modification of chromatin and altered gene expression. Furthermore, no genome-wide studies have been undertaken to identify altered DNA methylation patterns due to estrogen deprivation, which mimic AI therapy. We hypothesized that estrogen deprivation results in altered methylation, and therefore altered expression of critical target genes in breast cancer cells, which ultimately helps the cell to survive in the absence of estrogen and thus would contribute to acquire AI resistance in breast cancer patients.We used previously established MCF-7 cell clones, termed C4-12 and LTED (Long Term Estrogen Deprivation) that were isolated after being cultured in estrogen-free media for 9 months and 18-24 months, respectively. A genome-wide methylation screen was done using Methyl CpG binding Domain (MBD) pull down assay followed by hybridization into Affymetrix Human Promoter 1.0R Array. Altered DNA methylations were validated by bisulfite genomic sequencing assays and gene expression of some selected genes were studied by qRT PCR.From the array, we found that long term estrogen deprivation results in widespread genomic hyper- and hypomethylation events. 267 and 301 genes were hypermethylated in C4-12 and LTED respectively. In both resistant cell lines, the number of hypermethyled genes was more prominent than hypomethylated genes since only 82 and 97 genes were hypomethylated in C4-12 and LTED respectively. While hypomethylation of genes generally correlated with increased gene expression, hypermethylation did not in many cases. A gene which was heavily methylated in both cell line system, possibly reflecting a more general and important pathway in estrogen-deprivation, was HOXC10. Transient and stable silencing of HOXC10 in MCF-7 cells resulted in increased cell proliferation in estrogen deprived medium, suggesting a potential role of HOXC10 in developing resistance to endocrine therapy.This is the first genome-wide approach to identify gene-specific epigenetic alterations, and the functional consequences of these changes in developing resistance to estrogen deprivation therapy. This approach allows the development of epigenetic signatures of therapeutic resistance to be used concurrently with genomic signatures to better understand the molecular mechanism of endocrine resistance. This study might provide the basis for future clinical trials such as combination therapy of DNA demethylating agents and aromatase inhibitors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5127.

The Effect of the IGF1R Antibody R1507 on ER Positive Breast Cancer Cell Lines Growth and Survival.

Abstract Background: The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF-1 and IGF-2 and is expressed in most breast cancer cells, where receptor activation induces proliferative, cell-survival, and transforming activities. Because crosstalk exists between ER and IGF1R blockade of both pathways could increase antitumor activity. Further, IGF signaling may be enhanced in ER+ cells that have become refractory to long term estrogen deprivation (LTED) explaining resistance to aromatase inhibitor therapy. The effect of the selective IGF1R antibody (R1507) was therefore assessed in a series of breast cancer cell lines in both estrogen-dependent and estrogen-independent growth phases.Methods: The ER positive breast cancer cells lines MCF7, T47D and HCC712 were kept in phenol red-free medium containing 5% charcoal-stripped serum. Several doses of R1507 were used to evaluate effects on cell proliferation, signaling and apoptosis. Western blotting with phospho-specific antibodies was used to observe effects on cell signaling, cell growth was measured using resazurin reduction and apoptosis was quantified by flow cytometry using the TUNEL assay. STED (short-term estrogen deprived) cells were treated with R1507 between 2 and 3 weeks after estrogen depletion and LTED were treated with R1507 after 6+ months of estrogen depletion.Results: R1507 suppressed IGF1R expression and inhibited IGF-1 stimulated IGF1R and AKT phosphorylation in all cell lines. R1507 had anti-proliferative effects in MCF7 cells in both estrogen-dependent and independent growth phases and the magnitude of the effect was similar to fulvestrant. R1507 had a smaller effect on cell proliferation in HCC712 cells and no effects on proliferation were observed in T47D cells. Antibody treatment resulted in significant induction of apoptosis in MCF7 STED but MCF7 LTED cells were resistant to the apoptotic effects of IGF1R blockade.Conclusions: R1507 had an inhibitory effect on IGF1R signaling and an anti-proliferative effect in two of the three cell lines tested. In addition R1507 induced synthetic lethality in combination with acute estrogen deprivation in MCF7 cells, an effect we have recently reported for direct PI3 kinase inhibitors under the same circumstances (1). While the proapoptotic effects of R1507 were lost in the estrogen-independent growth phase of MCF7 cells, the anti-proliferative effect was not, indicating that R1507 might have efficacy in patients with endocrine therapy resistant tumors. In other cell lines the efficacy of R1507 was variable and synthetic lethality was not observed, suggesting that the development of predictive biomarkers for R1507 should be a priority. We are currently addressing the activity of R1507 in a clinical trial with two cohorts, one with acquired resistance to letrozole, where the antibody is added to continued letrozole treatment and a second group of patients with stable disease on letrozole to determine if R1507 can induce tumor regression under these circumstances.1. Crowder et al Cancer Research 69:3955-62, 2009 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3126.

Enhanced Estrogen Receptor (ER) α, ERBB2, and MAPK Signal Transduction Pathways Operate during the Adaptation of MCF-7 Cells to Long Term Estrogen Deprivation

The mechanisms involved in resistance to estrogen deprivation are of major importance for optimal patient therapy and the development of new drugs. Long term culture of MCF-7 cells in estrogen (E2)-depleted medium (long term estrogen deprivation; LTED) results in hypersensitivity to E2 coinciding with elevated levels of estrogen receptor (ER) alpha phosphorylated on Ser118 and MAPK, together with several of its downstream targets associated previously with ERalpha phosphorylation. Our data suggest elevated MAPK activity results from enhanced ERBB2 expression in the LTED cells versus the wild-type (wt), and treatment with the tyrosine kinase inhibitor ZD1839 revealed increased sensitivity in both transcription and proliferation assays. Similarly the MEK inhibitor U0126 decreased transcription and proliferation in the LTED cells and reduced their sensitivity to the proliferative effects of E2, while having no effect on the wt. However, the complete suppression of MAPK activity in the LTED cells did not inhibit ERalpha Ser118 phosphorylation suggesting that ER activity remained ligand-dependant. The LTED cells also expressed elevated levels of insulin-like growth factor-1R, and inhibition of phosphatidylinositol 3-kinase activity with LY294002 reduced basal ERalpha transactivation by 70% in the LTED cells compared with the wt. However, LY294002 had no effect on ERalpha Ser118 phosphorylation. These data suggest that although elevated levels of MAPK occur during LTED and influence the phenotype, this is unlikely to be the sole pathway operating to achieve adaptation.

Insulin-like Growth Factor Receptor Levels Are Regulated by Cell Density and by Long Term Estrogen Deprivation in MCF7 Human Breast Cancer Cells

This work describes a reciprocal relationship between cell density and levels of insulin-like growth factor receptors (IGFR) in MCF7 human breast cancer cells, which adds a new dimension to the mechanism of cross-talk between estrogen and insulin-like growth factors in the regulation of breast cancer cell growth. The reduced binding of both (125)I-IGF1 and alphaIR3 anti-IGFR antibody to whole cells showed that IGFR are lost from the surface of MCF7 cells as cell density increases, and this occurred irrespective of the presence or absence of estradiol. Western immunoblotting further confirmed loss of type I IGFR from MCF7 cells with increasing cell density. Long term estrogen deprivation was found to increase the levels of IGFR at all cell densities, such that after 96 weeks of estrogen deprivation, IGFR levels had become similar at the highest cell density in the absence of estradiol to the IGFR levels at the lowest cell density in the estrogen-maintained cells, and the levels of IGFR could be increased still further by estradiol. This overexpression of IGFR in the estrogen-deprived cells correlated with a reversal of response to exogenously added ligand, in that concentrations of insulin, IGFI, and IGFII that had stimulated growth of the estrogen-maintained cells became growth inhibitory to the estrogen-deprived cells. Blockade of the IGFIR with the alphaIR3 anti-IGFR antibody could partially inhibit the growth of the estrogen-deprived cells, suggesting that up-regulation of IGFR in these cells may contribute to the mechanism of adaptation to growth in steroid-deprived conditions which results in progression to estrogen independence of cell growth.

Estrogen and NMDA receptor antagonism: effects upon reference and working memory

Since both estrogen and NMDA receptor antagonists act on the hippocampus CA1 region and behaviorally affect hippocampal memory tasks, we examined how estrogen depletion (ovariectomy) and NMDA receptor antagonism interact upon spatial memory of the mouse. After ovariectomy or sham operation, mice were given a 2-week recovery before behavioral tests began under the influence of vehicle or (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP 2, 5 and 10 mg/kg) intraperitoneal injections. CPP is a competitive, full NMDA receptor antagonist. Spatial reference memory was tested by the water maze, spatial working memory was tested by the radial arm maze, while overall locomotive activity was monitored by the Y-maze. Results from the water maze and the Y-maze did not show any spatial reference memory or activity differences between sham-operated and ovariectomized mice. The radial arm maze, however, highlighted some working memory differences between intact and ovariectomized mice. CPP treatment impaired dose dependently –the performance of ovariectomy and sham-operated mice equally on both water maze and radial arm maze, while the drug had no effect on Y-maze performance. These results suggest that short term estrogen deprivation has no effect upon spatial –reference memory, while it impairs spatial working memory. This effect is probably not mediated by NMDA receptors.

Effects of Age and Long Term Ovariectomy on Prolactin Secretion, as Assessed by the Reverse Hemolytic Plaque Assay*

PRL secretion from pituitary lactotrophs was assessed using the reverse hemolytic plaque assay in young (2- to 3-month-old), middle-aged (10- to 12-month-old), and middle-aged long term ovariectomized (LT-OVX) rats to investigate whether 1) a change in the percentage of pituitary cells secreting PRL is detectable in middle-aged animals, 2) the amount of PRL secreted per cell changes with age, 3) aging involves a change in responsiveness to TRH and/or dopamine (DA), and 4) LT-OVX can prevent any of these changes. Young and middle-aged rats were OVX for 1 week. LT-OVX rats were OVX at 2-3 months of age and used when they were 10-12 months old. All animals were implanted with Silastic capsules containing estradiol (E2) in sesame oil and killed 3 or 8 days later. Anterior pituitaries were collected, and cells were dispersed and prepared for the reverse hemolytic plaque assay. Three days after E2 was implanted, the percentage of anterior pituitary cells that secrete PRL was higher in middle-aged compared to young rats. LT-OVX prevented this increase; the percentage of cells secreting PRL was significantly lower in LT-OVX than in both young and middle-aged rats. Basal secretion of PRL per cell was not different in young compared to middle-aged rats and was significantly lower in LT-OVX than in either young or middle-aged rats. TRH induced similar increases in plaque size in young and middle-aged rats, but had no effect in LT-OVX rats. DA (10(-7) M) inhibited plaque size only in LT-OVX rats; however, higher concentrations of DA were equally effective in the three experimental groups. Eight days after E2 was implanted, the percentage of cells that secrete PRL increased in LT-OVX rats, but was still significantly lower than that in middle-aged animals. After 8 days of E2 treatment, PRL release was similar in the three experimental groups under basal conditions. In LT-OVX rats TRH produced a small increase in PRL secretion (30-40%); DA suppressed PRL release in a similar manner in the three groups. These data demonstrate that middle-aged rats exhibit an increase in the percentage of cells secreting PRL without a concomitant detectable change in the amount of PRL released by single cells and/or a change in responsiveness to TRH or DA. Long term estrogen deprivation prevents this age-related change, suppresses responsiveness to TRH, and enhances sensitivity to DA.