Aromatase Inhibitor-resistant Breast Cancer Cells Research Articles

Abstract 3863: Aminoflavone confers activity against aromatase inhibitor-resistant breast cancer cells in part via miR125b2-3p restoration

Abstract Aromatase inhibitor therapy is the most crucial adjuvant treatment option for postmenopausal women with hormone receptor-positive (HR+) non-metastatic breast cancer. However, up to 30% of these patients will experience relapse due in part to aromatase inhibitor resistance. We recently identified miR125b2-3p as a putative tumor suppressor in HR+ breast cancer. We also found that small molecule Aminoflavone (AF) induces miR125b2-3p expression in HR+ breast cancer cells. In the current study, we hypothesized that AF confers anticancer actions in breast cancer cells resistant to aromatase inhibitors via miR125b2-3p reactivation. Using the Alamar Blue and colony forming assays, we discovered that AF demonstrated anticancer activity in estrogen-deprived resistant (EDR) cells designed to mimic aromatase inhibitor resistance. AntagomiR125b2-3p enhanced, while miR125b2-3p mimics diminished colony formation of EDR cells. AF-mediated suppression of colony formation in EDR cells was attenuated during co-treatment with antagomiR125b2-3p and was enhanced during co-treatment with miR125b2-3p mimics. Both miR125b2-3p mimics and AF suppressed EDR cell migration. AntagomiR125b2-3p counteracted AF-mediated suppression of cell migration. Our in silico profiling studies identified the alpha6-integrin, a mediator of breast cancer stem cell activity and metastasis, as one miR-125b2-3p target. Immunohistochemistry of patient tumor samples revealed that alpha 6-integrin protein expression intensified following relapse on the aromatase inhibitor anastrozole. MCF-7 cells transfected to overexpress the alpha 6-integrin were less responsive to anastrozole than those transfected with empty vector. These data suggest that AF restores the tumor suppressor function of miR125b2-3p to inhibit the alpha 6-integrin and confer activity against aromatase inhibitor-resistant breast cancer. Citation Format: Eileen J. Brantley, Nicole Mavingire, Salma Khan, Gayathri Nagaraj, Shawnee Angeloni, Charles Wang, Ubaldo Soto. Aminoflavone confers activity against aromatase inhibitor-resistant breast cancer cells in part via miR125b2-3p restoration. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3863.

Dissecting the Roles of PDCD4 in Breast Cancer.

The human programmed cell death 4 (PDCD4) gene was mapped at chromosome 10q24 and encodes the PDCD4 protein comprised of 469 amino acids. PDCD4 inhibits protein translation PDCD4 inhibits protein translation to suppress tumor progression, and its expression is frequently decreased in breast cancer. PDCD4 blocks translation initiation complex by binding eIF4A via MA-3 domains or by directly binding 5’ mRNA internal ribosome entry sites with an RNA binding domain to suppress breast cancer progression and proliferation. Numerous regulators and biological processes including non-coding RNAs, proteasomes, estrogen, natural compounds and inflammation control PDCD4 expression in breast cancer. Loss of PDCD4 expression is also responsible for drug resistance in breast cancer. HER2 activation downregulates PDCD4 expression by activating MAPK, AKT, and miR-21 in aromatase inhibitor-resistant breast cancer cells. Moreover, modulating the microRNA/PDCD4 axis maybe an effective strategy for overcoming chemoresistance in breast cancer. Down-regulation of PDCD4 is significantly associated with short overall survival of patients, which suggests that PDCD4 may be an independent prognostic marker for breast cancer.

Evidence for Enhanced Exosome Production in Aromatase Inhibitor-Resistant Breast Cancer Cells.

Aromatase inhibitors (AIs) represent the standard anti-hormonal therapy for post-menopausal estrogen receptor-positive breast cancer, but their efficacy is limited by the emergence of AI resistance (AIR). Exosomes act as vehicles to engender cancer progression and drug resistance. The goal of this work was to study exosome contribution in AIR mechanisms, using estrogen-dependent MCF-7 breast cancer cells as models and MCF-7 LTED (Long-Term Estrogen Deprived) subline, modeling AIR. We found that exosome secretion was significantly increased in MCF-7 LTED cells compared to MCF-7 cells. MCF-7 LTED cells also exhibited a higher amount of exosomal RNA and proteins than MCF-7 cells. Proteomic analysis revealed significant alterations in the cellular proteome. Indeed, we showed an enrichment of proteins frequently identified in exosomes in MCF-7 LTED cells. The most up-regulated proteins in MCF-7 LTED cells were represented by Rab GTPases, important vesicle transport-regulators in cancer, that are significantly mapped in “small GTPase-mediated signal transduction”, “protein transport” and “vesicle-mediated transport” Gene Ontology categories. Expression of selected Rab GTPases was validated by immunoblotting. Collectively, we evidence, for the first time, that AIR breast cancer cells display an increased capability to release exosomes, which may be associated with an enhanced Rab GTPase expression. These data provide the rationale for further studies directed at clarifying exosome’s role on endocrine therapy, with the aim to offer relevant markers and druggable therapeutic targets for the management of hormone-resistant breast cancers.

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.

Forskolin increases the effect of everolimus on aromatase inhibitor-resistant breast cancer cells.

Aromatase inhibitor (AI) resistance is a major obstacle in the treatment of estrogen receptor-positive breast cancer. Everolimus (EVE) ameliorates AI-resistant breast cancer and is therefore used in cancer treatment. However, some patients show resistance to EVE. Here, we used 30 clones of long-term estrogen-deprived (LTED) MCF-7 cells as a model of AI-resistant breast cancer. We examined changes in protein phosphatase type 2A (PP2A) and cancerous inhibitor of PP2A (CIP2A), a negative regulator of PP2A, in LTED cells treated with EVE. In LTED cells with high sensitivity to EVE, CIP2A expression decreased at low EVE concentrations; however, in LTED cells poorly sensitive to EVE, CIP2A and PP2A did not change upon exposure to EVE. Therefore, we hypothesized that there is a relation between expression of CIP2A and sensitivity to EVE. Knockdown of CIP2A increased the sensitivity to EVE in three clones poorly sensitive to EVE. Additionally, we found that treatment with FSK, which activates PP2A, increased the sensitivity of the cells to EVE. Our data point to CIP2A and PP2A as novel therapeutic targets for AI-resistant breast cancer.

Abstract 2918: Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) successfully treat many estrogen receptor expressing breast cancers by depleting circulating estrogen levels. Unfortunately, many patients eventually develop resistance to this treatment. In order to elucidate the mechanism(s) of AI-resistance, our lab has developed an AI-resistant breast cancer cell line MCF-7:5C which was clonally derived from parental MCF-7 cells following long-term estrogen deprivation. Unlike MCF-7 cells, the AI-resistant MCF-7:5C cell line grows robustly in the absence of estrogen and is highly aggressive. We have previously reported that interferon induced transmembrane protein 1 (IFITM1) is overexpressed in MCF-7:5C cells and in AI-resistant breast tumors. Additionally, we have shown that loss of IFITM1 leads to cell death. What remains to be addressed is how IFITM1 expression is regulated in AI-resistant breast cancer cells. IFITM1 is a type 1 interferon (IFN) stimulated gene which is not expressed in normal tissue and is only induced by the type1 IFNs (IFNα/β). In previous studies we have found that MCF-7:5C cells produce elevated levels of IFNα, which binds to the type 1 IFN receptor, IFNAR, and induces JAK/STAT signaling, resulting in the overexpression of IFITM1. In this study, we further characterized the mechanisms of IFITM1 overexpression in AI-resistant MCF-7:5C cells. We have found that mucin 1 (MUC1) associates with STAT1 and stabilizes its phosphorylated form, thereby enhancing IFITM1 expression. This interaction is disrupted by treatment with the JAK inhibitor, Ruxolitinib. MUC1 is a transmembrane O-glycosylated protein that is overexpressed in 90% of breast cancers and known to promote the survival of the epithelium through formation of mucous, interaction with transcription factors and inhibition of apoptosis. A luciferase reporter determined that MUC1 contributes directly to IFITIM1 transcription. MUC1 expression is hormonally controlled and is normally enhanced by estrogen stimulation. In this study we found that MUC1 expression is dysregulated in AI-resistant MCF-7:5C cells and is instead reduced by estrogen stimulation. In contrast with the AI-sensitive MCF-7 cells, western blot and immunofluorescent staining showed that MUC1 expression was enhanced by treatment with the pure anti-estrogen, fulvestrant. Additionally, Annexin V/PI staining and PARP cleavage indicated that loss of MUC1 expression induced cell death in MCF-7:5C cells. We found that treatment with fulvestrant protects MCF-7:5C cells from this phenomenon, indicating that loss of MUC1 induces apoptosis in an ERα dependent manner. MCF-7:5C cells are sensitive to apoptosis following exposure to estrogen, which can be enhanced by loss of IFITM1 expression. We report that loss of MUC1 expression similarly enhances estrogen-induced apoptosis, suggesting that the communication between MUC1 and STAT1 also influences estrogen signaling in AI-resistant breast cancer. Citation Format: Asona Lui, Joan Lewis-Wambi. Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2918.

Abstract 717: Overexpression of interferon-stimulated genes is critical for the survival of aromatase inhibitor-resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) successfully treat many estrogen receptor expressing breast cancers by depleting circulating estrogen levels. Unfortunately, the majority of patients treated with AIs eventually develop resistance to this treatment. In order to elucidate the mechanism(s) of AI-resistance our lab has developed an AI-resistant breast cancer cell line MCF-7:5C which was clonally derived from parental MCF-7 cells following long-term estrogen deprivation. The AI-resistant MCF-7:5C cells grow robustly in the absence of estrogen, are highly aggressive, and constitutively overexpress a panel of type 1 interferon (IFN) stimulated genes (ISGs) including IFITM1, IFIT1, IFI27, PLSCR1, STAT1/2, OAS1 and MX1 as compared to the AI-sensitive MCF-7 cells. Human tissue microarray analysis also indicates that IFITM1 and PLSCR1 are overexpressed in AI-resistant/recurrence breast tumors as compared to primary tumors. ISGs are not expressed basally in normal tissue and are only induced by type1 IFNs (IFNα and β) to protect the host from viral infections. IFNα signals through a specific receptor, IFNAR1/2, which uses JAK/STAT signaling to produce the ISGs. The significance of constitutive overexpression of ISGs in AI-resistant breast cancer is not known. In this study, we investigated the functional importance of ISGs in the AI-resistant breast cancer cell line, MCF-7:5C, and the mechanism(s) that drive their overexpression in these cells. Using ELISA, we detected a 3-fold higher level of IFNα in AI-resistant MCF-7:5C cells than in the parental MCF-7 cells and FACS analysis revealed that the IFNα was produced by all cells in culture, not a distinct subset of the population. Blockade of IFNα signaling using a neutralizing antibody to IFNAR, as well as transient knockdown of IFNα and its transcription factor IRF7 dramatically reduced ISG expression. Most notably, loss of IFNα and suppression of type 1 IFN signaling resulted in the death of the AI-resistant MCF-7:5C cells, while having no effect on the parental MCF-7 cells, thus confirming its pro-survival function. Apart from IFNα signaling, we also found evidence of post-transcriptional regulation of ISGs in the AI-resistant cells. Specifically, several of the ISGs had a significantly longer mRNA half-life in the MCF-7:5C cells than in the parental MCF-7 cells. Additionally, knockdown of the RNA binding protein Human Antigen R (HuR) significantly reduced the mRNA stability and expression of the ISGs. Electron microscopy confirmed that AI-resistant MCF-7:5C cells exhibit classic morphological signs of anti-viral response and ISG expression, including dilated ER, and enhanced cell-cell adhesion as compared with the AI-sensitive MCF-7 cell line. Overall, we have found that AI-resistant breast cancer cells maintain elevated levels of IFNα in order to drive constitutive overexpression of ISGs which sustains their survival in estrogen deprived conditions. Citation Format: Asona Lui, Hye-Joung Choi, Joan Lewis-Wambi. Overexpression of interferon-stimulated genes is critical for the survival of aromatase inhibitor-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 717. doi:10.1158/1538-7445.AM2015-717

Abstract 3993: MicroRNA analysis of aromatase inhibitor-resistant breast cancer cells reveals upregulation of a unique miRNA cluster on chromosome 14

Abstract Estrogen signaling has been shown to play a large role in the development and progression of breast tumors that express the estrogen receptor (ERα). Thus, endocrine therapies that either inhibit ERα action (tamoxifen or fulvestrant) or inhibit estrogen synthesis (aromatase inhibitors) have been increasingly valuable for the management of ER+ breast cancer. A frequent obstacle, however, is acquired resistance to endocrine therapies, often characterized by aberrant ERα expression and heightened growth factor signaling. Another aspect of acquired endocrine resistance that is now being examined is the role of microRNA (miRNA). miRNAs are 18-22 base pair RNA molecules whose primary role is to inhibit expression of mRNA targets by inhibition of translation or by degradation of the mRNA. Here, we have conducted next generation sequencing of the miRNA from three breast cancer cell lines - one ER+ line that is estrogen-dependent and AI-sensitive (MCF-7), and two ER+ long-term estrogen deprived (LTED) models of AI resistance (MCF-7:5C and MCF-7:2A). We found that in LTED/AI-resistant MCF-7:5C and MCF-7:2A cells there were over 330 and 240 miRNAs differentially expressed, respectively, compared to AI-sensitive MCF-7 breast cancer cells. Initially, we used qPCR to validate the expression of miRNAs that that were differentially expressed in both LTED cell lines vs. MCF-7. Notably, LTED cell lines had upregulated miR-181a (> 4 to 8-fold) and downregulated miR-221/222 expression compared to MCF-7. Elevated miR-181a has been shown to be associated with more aggressive breast cancers whereas miR-221/222 play a role in tamoxifen resistance through targeting ERα. Further bioinformatic analysis also revealed that the LTED cells had very unique expression profiles compared to one another, indicating that they have adapted to an estrogen-depleted environment through potentially different mechanisms. In MCF-7:5C cells, which have lost expression of the progesterone receptor (PR), a miRNA cluster on chromosome 14 is upregulated. This upregulated cluster, which encodes miR-127, miR-654, miR-889 and others, has been shown to be associated with Luminal B (ER+/PR-) breast cancer, consistent with the hormone receptor status of the MCF-7:5C cells. The oncogenic, estrogen-responsive miR-17/92 cluster on chromosome 13 is upregulated in the MCF-7:2A cells and these miRNAs target proteins including PTEN, p21, and Bim. Overall, these findings reveal altered expression of miRNAs in AI-resistant breast cancer cells and they suggest that targeting these miRNAs might be an effective strategy to reverse AI resistance in breast cancer patients. Citation Format: Erin L. Hayes, Jennifer Knapp, Joan Lewis-Wambi. MicroRNA analysis of aromatase inhibitor-resistant breast cancer cells reveals upregulation of a unique miRNA cluster on chromosome 14. [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 3993. doi:10.1158/1538-7445.AM2015-3993

Abstract OT2-1-08: A phase II open label study of everolimus in combination with anti-estrogen therapy in hormone receptor-positive HER2-negative advanced breast cancer

Abstract Background: Hormone receptor positive (HR+) breast cancer, defined as estrogen receptor and/or progesterone receptor positive, accounts for 70% of invasive breast cancers. While most HR+ tumors initially respond to anti-estrogen therapy, resistance, linked to crosstalk between cell signaling and signal transduction pathways, subsequently develops in almost 100%. Preclinical data has shown that the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway is activated in long-term estrogen-deprived and aromatase inhibitor-resistant breast cancer cells. It is hypothesized that in this group of endocrine-resistant patients (pts), resistance to anti-estrogen therapy is driven by the PI3K/Akt/mTOR pathway and hence the inhibition of this pathway may reverse resistance. This study will evaluate the efficacy of adding everolimus (an inhibitor of mTOR) to anti-estrogen therapy in pts with HR+ metastatic breast cancer (MBC) who have progressed on anti-estrogen therapy. Study Objectives: This multi-center, open-label Phase II trial is designed to determine the efficacy (primary endpoint is PFS), safety and tolerability of everolimus in combination with anti-estrogen therapy in pts with HR+, HER2-negative MBC with disease progression on prior anti-estrogen therapy. An exploratory objective of this study is to evaluate the prognostic value of the VeriStrat assay in this population. VeriStrat testing has been used to predict treatment efficacy in clinical trials of non small cell lung cancer, head and neck squamous carcinoma, and MBC. Eligibility: Pts ≥18 years with HR+, HER2-negative breast cancer that is either unresectable, locally recurrent, or metastatic are eligible. Pts must have progressed on anti-estrogen therapy (recurrence while on/within 12 months of treatment for early stage breast cancer, or progressed while on/within 1 month of treatment for advanced/MBC), but anti-estrogen therapy is not required to be the last therapy prior to enrollment. No washout for anti-estrogen therapy is required, and ≤1 previous chemotherapy regimen is allowed. Post menopausal or pre-/ peri-menopausal women on tamoxifen are eligible and ovarian function suppression is permitted. Prior treatment with a mTOR inhibitor is not allowed. Additional eligibility requirements include: measurable or evaluable disease, ECOG ≤2, adequate bone marrow and organ function and an adequate lipid profile (fasting serum cholesterol ≤300 mg/dL or ≤7.75 mmol/L and fasting triglyceride ≤2.5 x ULN). Trial Design: Everolimus will be administered at a dose of 10 mg PO daily in combination with the FDA prescribed doses of the most recent anti-estrogen therapy on which the pt progressed. Treatment cycles will be 4 weeks, with response evaluations by CT scans every 2 cycles. Pts will be treated until disease progression or unacceptable toxicity occurs. With an alpha=0.5, a sample size of 42 will provide 80% power to detect improvement in median PFS. Blood samples for the VeriStrat assay will be collected at baseline, on Cycle 3 Day 1, at disease progression and at the first follow-up visit post disease progression. Archival tumor tissue samples will be collected for comprehensive genomic profiling. This trial has a total accrual goal of 46 pts. Citation Format: Denise A Yardley, Mythili Shastry, Laura M DeBusk, Howard A Burris III, John D Hainsworth. A phase II open label study of everolimus in combination with anti-estrogen therapy in hormone receptor-positive HER2-negative advanced breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT2-1-08.

Cross-talk between ER and HER2 regulates c-MYC-mediated glutamine metabolism in aromatase inhibitor resistant breast cancer cells

Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant clinical problem for a considerable number of patients. The oncogenic transcription factor c-MYC (hereafter referred to as MYC), which regulates glutamine metabolism in cancer cells, has been linked to endocrine resistance. We were interested in whether MYC-mediated glutamine metabolism is also associated with aromatase inhibitor (AI) resistant breast cancer. We studied the expression and regulation of MYC and the effects of inhibition of MYC expression in both AI sensitive and resistant breast cancer cells. Considering the role of MYC in glutamine metabolism, we evaluated the contribution of glutamine to the proliferation of AI sensitive and resistant cells, and performed RNA-sequencing to investigate mechanisms of MYC-mediated glutamine utilization in AI resistance. We found that glutamine metabolism was independent of estrogen but still required estrogen receptor (ER) in AI resistant breast cancer cells. The expression of MYC oncogene was up-regulated through the cross-talk between ER and human epidermal growth factor receptor 2 (HER2) in AI resistant breast cancer cells. Moreover, the glutamine transporter solute carrier family (SLC) 1A5 was significantly up-regulated in AI resistant breast cancer cells. ER down-regulator fulvestrant inhibited MYC, SLC1A5, glutaminase (GLS) and glutamine consumption in AI resistant breast cancer cells. Inhibition of MYC, SLC1A5 and GLS decreased AI resistant breast cancer cell proliferation. Our study has uncovered that MYC expression is up-regulated by the cross-talk between ER and HER2 in AI resistant breast cancer cells. MYC-mediated glutamine metabolism is associated with AI resistance of breast cancer.

Aurora kinase A and B as new treatment targets in aromatase inhibitor-resistant breast cancer cells.

Aromatase inhibitors (AIs) are used for treatment of estrogen receptor α (ER)-positive breast cancer; however, resistance is a major obstacle for optimal outcome. This preclinical study aimed at identifying potential new treatment targets in AI-resistant breast cancer cells. Parental MCF-7 breast cancer cells and four newly established cell lines, resistant to the AIs exemestane or letrozole, were used for a functional kinase inhibitor screen. A library comprising 195 different compounds was tested for preferential growth inhibition of AI-resistant cell lines. Selected targets were validated by analysis of cell growth, cell cycle phase distribution, protein expression, and subcellular localization. We identified 24 compounds, including several inhibitors of Aurora kinases e.g., JNJ-7706621 and barasertib. Protein expression of Aurora kinase A and B was found upregulated in AI-resistant cells compared with MCF-7, and knockdown studies showed that Aurora kinase A was essential for AI-resistant cell growth. In AI-resistant cell lines, the clinically relevant Aurora kinase inhibitors alisertib and danusertib blocked cell cycle progression at the G2/M phase, interfered with chromosome alignment and spindle pole formation, and resulted in preferential growth inhibition compared with parental MCF-7 cells. Even further growth inhibition was obtained when combining the Aurora kinase inhibitors with the antiestrogen fulvestrant. Our study is the first to demonstrate that Aurora kinase A and B may be treatment targets in AI-resistant cells, and our data suggest that therapy targeting both ER and Aurora kinases may be a potent treatment strategy for overcoming AI resistance in breast cancer.

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

Inhibition of the proliferation of acquired aromatase inhibitor-resistant breast cancer cells by histone deacetylase inhibitor LBH589 (panobinostat)

Aromatase inhibitors (AIs) are important drugs for treating postmenopausal patients with hormone receptor-positive breast cancer. However, acquired resistance to AI therapies is a significant problem. Our study has revealed that the histone deacetylase inhibitor LBH589 treatment abrogated growth of AI-resistant cells in vitro and in vivo, causing cell cycle G2/M arrest and induced apoptosis. LBH589 treatment also reduced the level of NF-κB1 which is overexpressed when AI resistance develops. Analyzing paired tumor specimens from 12 patients, we found that NF-κB1 expression was increased in recurrent AI-resistant tumors as compared to the paired primary tumors before AI treatment. This finding was consistent with up-regulated NF-κB1 expression seen in a collection of well-established AI-resistant cell lines. Furthermore, knockdown of NF-κB1 expression significantly suppressed the proliferation of AI-resistant cells. Treatment of AI-resistant cell lines with LBH589 suppressed NF-κB1 mRNA and protein expression. In addition, LBH589 treatment abrogated growth of AI-resistant tumors in mice, and was associated with significantly decreased levels of NF-κB1 in tumors. In all, our findings strongly support further investigation of LBH589 as a novel therapeutic strategy for patients with AI-resistant breast cancer, in part by suppressing the NF-κB1 pathway.

AIB1:ERα Transcriptional Activity Is Selectively Enhanced in Aromatase Inhibitor–Resistant Breast Cancer Cells

The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy. Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole. Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH(2)-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter. This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype.