Aromatase Inhibitor-resistant Breast Cancer Research Articles

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.

Acquired resistance to everolimus in aromatase inhibitor-resistant breast cancer.

We previously reported the establishment of several types of long-term estrogen-depleted-resistant (EDR) cell lines from MCF-7 breast cancer cells. Type 1 EDR cells exhibited the best-studied mechanism of aromatase inhibitor (AI) resistance, in which estrogen receptor (ER) expression remained positive and PI3K signaling was upregulated. Type 2 EDR cells showed reduced ER activity and upregulated JNK-related signaling. The mTOR inhibitor everolimus reduced growth in cells similar to Type 1 EDR cells. The present study generated everolimus-resistant (EvR) cells from Types 1 and 2 EDR cells following long-term exposure to everolimus in vitro. These EvR cells modeled resistance to AI and everolimus combination therapies following first-line AI treatment failure. In Type 1 EvR cells, everolimus resistance was dependent on MAPK signaling; single agents were not effective, but hormonal therapy combined with a kinase inhibitor effectively reduced cell growth. In Type 2 EvR cells, ER expression remained negative and a JNK inhibitor was ineffective, but a Src inhibitor reduced cell growth. The mechanism of acquired everolimus resistance appears to vary depending on the mechanism of AI resistance. Strategies targeting resistant tumors should be tailored based on the resistance mechanisms, as these mechanisms impact therapeutic efficacy.

Abstract P4-04-13: Androstenedione initiates rapid non-genomic signalling mediated by cytoplasmic androgen receptor in aromatase inhibitor resistant breast cancer

Abstract Aromatase inhibitors (AI) are the recommended first line therapy used to treat postmenopausal breast cancer. These compounds work by inhibiting the aromatase enzyme thus preventing the conversion of circulating androgens to estrogen; as a consequence they alter the tumour intracrinology and create an unopposed highly androgenic steroid environment. We have previously reported androgen receptor (AR) protein levels to be up-regulated in an AI resistant cell line (LetR cells), and subsequently identified a mechanism by which AR drives a more aggressive phenotype in AI resistant breast cancer [1]. In this current study, LetR cells are shown to be responsive to the weak androgen androstenedione (4AD), and treatment with this steroid drives an invasive phenotype in vitro. In support of this data, clinical studies have also reported increases in the serum levels of 4AD in patients that recur on AI therapy [2]. In the canonical pathway, androgens bind to the AR which results in a conformational change to an active state. Non-canonical AR activation occurs when ligand-transformed AR interacts with molecular partners within the cytosol to induce rapid intracellular signalling cascades. These events do not depend upon AR mediated gene transcription and occur extremely quickly within a manner of minutes [3]. In vitro studies using western blot analysis and co-localisation experiments have indicated 4AD treatment potentiates a resistant phenotype through non-genomic AR actions initiated by rapid second messenger signalling within the cytoplasm. Mass spectrometry (LC–MS/MS) analysis has identified androgen-mediated, rapid cytoplasmic AR protein interactions, resulting in the identification of AR partners unique to our resistant model. Of note, evaluation of AR protein and p-ERK1/2 in a cohort of primary breast cancer patients (n=363) demonstrated that high levels of cytoplasmic AR significantly diminished survival in ER+ PR- patients (p=0.023, Fisher's exact). Elevated pERK1/2 when concomitant with increased levels of cytoplasmic AR result in a significant decrease in the period of disease free survival (p=0.018). Further investigations into these AR interactors will help elucidate mechanisms of resistance to AI therapy, and in turn these novel AR protein partners will aid the identification of patients who would benefit from anti-AR therapy.

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

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

Single CpG site methylation controls estrogen receptor gene transcription and correlates with hormone therapy resistance

Hormone therapy is the most effective treatment for patients with estrogen receptor α-positive breast cancers. However, although resistance occurs during treatment in some cases and often reflects changed estrogen receptor α status, the relationship between changes in estrogen receptor α expression and resistance to therapy are poorly understood. In this study, we identified a mechanism for altered estrogen receptor α expression during disease progression and acquired hormone therapy resistance in aromatase inhibitor-resistant breast cancer cell lines. Subsequently, we investigated promoter switching and DNA methylation status of the estrogen receptor α promoter, and found marked changes of methylation at a single CpG site (CpG4) in resistant cells. In addition, luciferase reporter assays showed reduced transcriptional activity from this methylated CpG site. This CpG region was also completely conserved among species, suggesting that it acts as a methylation-sensitive Ets-2 transcription factor binding site, as confirmed using chromatin immunoprecipitation assays. In estrogen receptor α-positive tumors, CpG4 methylation levels were inversely correlated with estrogen receptor α expression status, suggesting that single CpG site plays an important role in the regulation of estrogen receptor α transcription.

Abstract P3-04-18: The impact of circulating androgens on the androgen receptor in aromatase inhibitor resistant breast cancer

Abstract Aromatase Inhibitor (AI) therapy is the gold standard first line therapy for post-menopausal breast cancer. Following AI treatment the conversion of circulating androgens into estrone can be diminished by >99%, completely altering the tumour steroid microenvironment. Estrogens have been extensively studied in terms of the role of sex steroids as causative agents of breast cancer but interestingly, a case–control study nested within the European Prospective Investigation into Cancer and Nutrition reported that elevated levels of both serum androgens and estrogens are associated with increased breast cancer risk (E2 RR: 2.28 and androstenedione RR: 1.94). Also clinical studies have reported increases in the serum levels of androstenedione (4AD) in patients that recur on AI therapy. In order to begin to address the role of circulating androgens and the androgen receptor (AR) in AI resistant breast cancer, RNA sequencing was performed on AI resistant (MCF7-aro-LetR) cells cultured with 4AD in the presence of letrozole. Gene expression analysis was performed using the cBioPortal software found that PI3K signalling pathways were upregulated in response to 4AD treatment as well as the upregulation of pathways involved in exocytosis and hormone secretion. We found elevated levels of PI3K and AR in endocrine resistant breast cancer compared to sensitive cell lines and that our AI resistant LetR cells showed significant reduction in cell growth in response to the combined pan class PI3K inhibitor BEZ235 and anti-AR treatment. Clinically, we evaluated the potential role of PI3K signalling in AI resistance a breast cancer patient TMA (n=488) was stained immunohistochemically for phospho-AKT and AR. The protective effect conferred by high AR expression in the total patient population (p=0.02) and tamoxifen treated population (p=0.05) was lost in the AI treated population (p=0.49). For p-AKT staining, patients that were positive for p-AKT significantly associate with the more aggressive luminal B classification yet exhibit an inverse association with luminal A subtype. Kaplan Meier analysis revealed that AR is associated with a more favourable outcome in p-AKT negative patients but that this is lost in the context of elevated p-AKT. Furthermore, interrogation of the breast invasive carcinoma TCGA dataset (n=963) revealed that patients with upregulated AR mRNA and wildtype PI3K exhibited more invasive disease compared with those harbouring a PI3K mutation (p=0.02) (n=56). Further investigations into the AR interacting partners will help elucidate potential mechanisms of action and the role of androgens and AR in facilitating breast cancer tumourigenesis. Citation Format: Creevey L, Ali A, Bleach R, Hill ADK, Young L, McIlroy M. The impact of circulating androgens on the androgen receptor in aromatase inhibitor resistant breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-18.

Abstract P4-10-01: Estrogen induced apoptosis can be mimicked by targeting unfolded protein response

Abstract Estrogen signaling is considered to promote growth in estrogen receptor positive (ER+) breast cancers and its actions can be blocked by antagonists like tamoxifen or by inhibiting the synthesis of estrogen by aromatase inhibitors. Both of these classes of drug are used to treat ER+ breast cancers in the clinic. Paradoxically, before the discovery of tamoxifen and aromatase inhibitors high dose estrogen (HDE) was the choice of endocrine therapy to treat post-menopausal breast cancers. Recent clinical trials have observed 30% clinical benefit rate with high as well as low doses of estrogen-therapy in aromatase-inhibitor resistant breast cancers. Despite its clinical success, the precise underlying mechanism of estrogen-therapy by which it triggers the tumor regression remains unknown. Studies in the laboratory have indicated that unfolded protein response (UPR) and apoptotic pathways may play important role in estrogen-induced apoptosis. Using MCF7:5C cells, which can proliferate independent of estrogen and are hyper-sensitive to estrogen as evident by induction of apoptosis, we demonstrate that increased global protein translational load as the trigger for estrogen-induced apoptosis. This subsequently leads to endoplasmic reticulum (EnR) stress and activates the protein kinase-RNA-like endoplasmic reticulum kinase (PERK) pathway of UPR. Our results also suggest that sustained phosphorylation of eukaryotic initiation factor 2-alpha (eIF2-α), a downstream target of PERK activation, may be crucial in estrogen-induced apoptosis. Phosphorylation of eIF2-α attenuated global translation but preferentially allowed high expression of transcription factors, including, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). ATF4 and CHOP are known to activate apoptosis. Notably, we were able to recapitulate this phenotype by pharmacologically inhibiting the regulatory subunits of the protein phosphatase 1, GADD34 (growth arrest and DNA damage inducible protein) and CReP (constitutive repressor of eIF2α phosphorylation), that are responsible for de-phosphorylation of eIF2-α. This was evident in MCF7:5C cells as well as another estrogen-independent breast cancer cell line LCC9 that is resistant to both tamoxifen and fulvestrant but does not undergo estrogen mediated apoptosis. We further observed that the combination of 4-hydroxy-tamoxifen (4OHT) and pharmacological inhibitors of GADD34 and CReP potentiated its apoptotic action in both LCC9 and MCF7:5C cells. These results not only enhance our understanding of the apoptotic mechanism of estrogen but also provides crucial evidence that estrogen-induced apoptosis can be mimicked by manipulating the unfolded protein response even in breast cancer cells that are not susceptible to estrogen mediated apoptosis. Citation Format: Sengupta S, Sevigny C, Clarke R. Estrogen induced apoptosis can be mimicked by targeting unfolded protein response [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-10-01.

Profiles of miRNAs matched to biology in aromatase inhibitor resistant breast cancer.

Aromatase inhibitor (AI) resistance during breast cancer treatment is mimicked by MCF-7:5C (5C) and MCF-7:2A (2A) cell lines that grow spontaneously. Survival signaling is reconfigured but cells are vulnerable to estradiol (E2)-inducible apoptosis. These model systems have alterations of stress related pathways including the accumulation of endoplasmic reticulum, oxidative, and inflammatory stress that occur prior to E2-induced apoptosis. We investigated miRNA expression profiles of 5C and 2A to characterize their AI resistance phenotypes. Affymetrix GeneChip miRNA2.0 arrays identified 184 miRNAs differentially expressed in 2A and 5C compared to E2-free wild-type MCF-7:WS8. In 5C, 34 miRNAs of the DLK1-DIO3 locus and miR-31 were overexpressed, whereas miR-222 was low. TCGA data revealed poor and favorable overall survival for low miR-31 and miR-222 levels, respectively (HR=3.0, 95% CI:1.9-4.8; HR=0.3, 95% CI:0.1-0.6). Targets of deregulated miRNAs were identified using CLIP-confirmed TargetScan predictions. KEGG enrichment analyses for 5C- and 2A-specific target gene sets revealed pathways associated with cell proliferation including insulin, mTOR, and ErbB signaling as well as immune response and metabolism. Key genes overrepresented in 5C- and 2A-specific pathway interaction networks including EGFR, IGF1R and PIK3R1 had lower protein levels in 5C compared to 2A and were found to be differentially modulated by respective miRNA sets. Distinct up-regulated miRNAs from the DLK1-DIO3 locus may cause these attenuative effects as they are predicted to interact with corresponding 3′ untranslated regions. These new miRNA profiles become an important regulatory database to explore E2-induced apoptotic mechanisms of clinical relevance for the treatment of 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

Histone deacetylase inhibitor entinostat in combination with a retinoid downregulates HER2 and reduces the tumor initiating cell population in aromatase inhibitor-resistant breast cancer.

Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.

Aromatase Inhibitor-Mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer.

Aromatase inhibitors are effective drugs that reduce or eliminate hormone-sensitive breast cancer. However, despite their efficacy, resistance to these drugs can occur in some patients. The INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiation-induced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress, Nrf2 dissociates from INrf2, is stabilized, translocates to the nucleus, and coordinately induces a battery of cytoprotective gene expression. Current studies investigated the role of Nrf2 in aromatase inhibitor resistance. RT-PCR and immunoblot assays showed that aromatase inhibitor-resistant breast cancer LTLTCa and AnaR cells express lower INrf2 and higher Nrf2 protein levels, as compared with drug-sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/degradation of Nrf2 in aromatase inhibitor-resistant cells. Higher Nrf2-mediated levels of biotransformation enzymes, drug transporters, and antiapoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance. shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells reduced resistance and sensitized cells to aromatase inhibitor exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed reduced levels of aldehyde dehydrogenase, a marker of tumor-initiating cells and significantly decreased mammosphere formation, as compared with LTLTCa-Vector control cells. The results together suggest that persistent aromatase inhibitor treatment downregulated INrf2 leading to higher expression of Nrf2 and Nrf2-regulated cytoprotective proteins that resulted in increased aromatase inhibitor drug resistance. These findings provide a rationale for the development of Nrf2 inhibitors to overcome resistance and increase efficacy of aromatase inhibitors.

Short report: Monitoring ESR1 mutations by circulating tumor DNA in aromatase inhibitor resistant metastatic breast cancer.

Acquired estrogen receptor gene (ESR1) mutations have been recently reported as a marker of resistance to aromatase inhibitors in hormone receptor positive metastatic breast cancer. We retrospectively considered seven patients treated for metastatic breast cancer with available samples from the primary tumor before any treatment, cryopreserved metastasis removed during progression and concomitant plasmas. All these seven patients were in disease progression after previous exposure to aromatase inhibitors for at least 6 months, and were assessed for ESR1 mutations detection in tumor and circulating DNA. For these patients, Sanger sequencing identified four metastases with clear ESR1 mutation and one possible, whereas digital PCR identified six mutated metastases. Then, under blind conditions and using digital PCR, corresponding circulating ESR1 mutations were successfully detected in four of these six metastatic breast cancer patients. Moreover, in two patients with serial blood samples following treatments exposure, the monitoring of circulating ESR1 mutations clearly predicted disease evolution. In the context of high interest for ESR1 mutations, our results highlight that these acquired recurrent mutations may be tracked in circulating tumor DNA and may be of clinical relevance for metastatic breast cancer patient monitoring.

Abstract P3-05-22: PI3K pathway as a novel target in androgen-driven aromatase inhibitor resistant breast cancer

Abstract Breast cancer has the highest incidence and second highest mortality rate among all cancers in women worldwide (1). The gold standard treatment for post-menopausal breast cancer is aromatase inhibitor (AI) therapy. Unfortunately, resistance is inevitable in about 30-60% of these patients (2). Research from our lab has previously identified a transcription factor, HOXC11, to be associated with breast cancer resistance and metastases (3). Prosaposin (PSAP) was then identified as a putative HOXC11 target gene in the AI resistant setting. PSAP is a secreted protein that also plays a role in metastatic prostate cancer in both a ligand dependent and independent fashion (4,5). We have previously shown PSAP to be an androgen responsive gene that can upregulate androgen receptor (AR) expression in a ligand dependent manner in AI resistance. An AR antagonist was effective at slowing down cell proliferation in AI resistant cells in vitro . However, since PSAP can also potentially act in a ligand independent fashion, this current study is focused on evaluating the role of PSAP in PI3K pathway activation in the development of AI resistance. Treatment of cells with recombinant PSAP protein (rhPSAP) resulted in increasing expression of p-AKT in a dose-dependent manner. To target this pathway of resistance, we used a pan-class PI3K/mTOR inhibitor BEZ235 on our AI resistant cells. Functional studies with BEZ235 treatment showed significant reduction in both cell motility as well as cell proliferation. Treatment of AI resistant cells with BEZ235 resulted in decreased expression of p-AKT, with little or no effect on AR expression. These findings suggest that to prevent resistance to AI therapy, combination treatment regimens including AR antagonists plus a PI3K inhibitor may ensure sustained response to therapy.

Abstract P3-05-21: Investigation into the oncogenic potential of the androgen receptor in aromatase inhibitor resistant breast cancer

Abstract The heterogenous nature of breast cancer, exemplified by a wide range of subtype classifications, has been well documented. The identification of the novel molecular apocrine breast tumours (ER-, PR-ve, HER2-ve, AR+), with a gene expression profile reflective of ER+ luminal breast cancer, has led to the proposition of an alternative role for the androgen receptor as a pseudo ER. This proposition was borne out of global expression profiling and AR recruitment of molecular apocrine cells, demonstrating that more than 50% of AR binding to the genome mirrors the pattern of ER binding suggesting that in the absence of ER, AR can mimic ER in its DNA binding capabilities. Aromatase Inhibitor (AI) therapy is the gold standard first line therapy for postmenopausal breast cancer. AIs function by abrogating the activity of the enzyme Cyp 19 (aromatase), which is responsible for converting circulating androgens to estrogen. By preventing this conversion step, this eradicates the main steroid driving breast cancer growth and in turn results in a more androgenic environment. We hypothesise that AR may also act as a pseudo ER in our AI resistant (MCF7-letrozole resistant) cells. Preliminary data demonstrates that AR is recruited to some but not all classical ER target genes as well as some AR target genes in MCF7-LetR cells, therefore highlighting the potential of AR to act as an oncogene in this setting. Analysis of AR protein expression also demonstrates higher levels of AR in AI resistant cells versus sensitive cells. In the evaluation of the responsiveness of a range of breast cancer cell lines to the AR antagonist Bicalutamide, it was noted that our AI resistant cells demonstrated responsiveness to Bicalutamide treatment. RNA sequencing experiments following AR knockdown in AI resistant cells also provide crucial information as to the key genes differentially regulated by AR. Given that resistance to AI therapy is an emerging clinical issue, further elucidation of the potential mechanisms of AI resistance and potential AR targets is highly warranted. Citation Format: Leonie Young, Laura Creevey, Azlena Ali, Arnold DK Hill, Marie McIlroy. Investigation into the oncogenic potential of the androgen receptor in aromatase inhibitor resistant 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 P3-05-21.

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.

New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.