Aromatase Inhibitor-resistant Cells Research Articles

Abstract 3183: Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone

Abstract Postmenopausal women with early-stage metastatic estrogen-dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., letrozole). However, acquired resistance remains a major clinical obstacle. Previously, our group revealed a global proteomic signature of a letrozole-resistant cell line (LTLT-Ca) associated with hormone independence, enhanced cell motility and epithelial to mesenchymal (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC), we chose to utilize a two-dimensional (2D) vs three-dimensional (3D) culture system to compare the proteome of LTLT-Ca cells, as 3D culture not only enriches for CSC, but more accurately recapitulates the tumor microenvironment, morphology, function and response to therapy compared to conventional 2D culture. We hypothesize utilizing a novel systems biology approach may reveal previously unconsidered molecular changes that could aid in understanding complex signaling networks and be exploited as therapeutic targets. To address this hypothesis ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with LTLT-Ca or letrozole sensitive cells (AC-1) and intratumoral putative CSC marker expression was assessed by immunohistochemistry. Results indicate LTLT-Ca tumors were CD44high/CD24low while AC-1 tumors were CD44-/CD24low. Mammosphere formation assays were conducted and LTLT-Ca cells formed mammospheres at a 3.4-fold higher index than AC-1 cells. A quantitative proteomic analysis of whole cell lysates from LTLT-Ca (2D adherent cells) versus LTLT-Ca (3D mammospheres) was conducted. Results identified significant protein expression changes within a panel of 1010 proteins; 173 were upregulated and 186 downregulated (p<0.05, fold change >1.20). Additionally, functional enrichment analyses were performed and 19 gene ontology (GO) terms and one KEGG pathway (hsa03010:Ribosome) were over-represented (BH adjusted p-value < 0.01) by the cognate genes. Notably, there was a 35.04-fold increase in midasin (MDN1), a nuclear chaperone protein required for maturation and nuclear export of pre-60S ribosome subunit. Increased MDN1 expression was strongly correlated with highly tumorigenic breast cancer spheres. Additionally, Kaplan-Meier survival plots demonstrate that increased MDN1 levels were positively correlated with decreased relapse free survival in estrogen receptor negative breast cancer, and is constitutively expressed as breast tumors progress from atypical ductal hyperplasia to ductal carcinoma in situ to invasive, metastatic breast cancer. The TGCA database was interrogated and MDN1 was frequently amplified or mutated in breast tumors. Taken together our study for the first time implicates a role for ribosomal assembly in AI resistant cells enriched for CSCs and underscores a potential for MDN1 in the progression and responsiveness to therapy. Citation Format: Syreeta L. Tilghman, Jamal Pratt, Shawn D. Llopis, A. Michael Davidson, Rashidra R. Walker, Patrick Carriere, Ian R. Davenport, Wensheng Zhang, Karen Zhang. Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2017-3183

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.

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 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.

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.

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.

AKT-aro and HER2-aro, models for de novo resistance to aromatase inhibitors; molecular characterization and inhibitor response studies

Aromatase inhibitors (AI) are currently the first line therapy for estrogen receptor (ER)-positive postmenopausal women. De novo AI resistance is when a patient intrinsically does not respond to an AI therapy as well as other targeted endocrine therapy. To characterize this type of resistance and to examine potential therapies for treatment, we have generated two cell models for de novo resistance. These models derive from MCF-7 cells that stably overexpress aromatase and Akt (AKT-aro) or HER2 (HER2-aro). Evaluation of these cell lines revealed that the activities of aromatase and ER were inhibited by AI and ICI 187280 (ICI) treatment, respectively; however, cell growth was resistant to therapy. Proliferation in the presence of the pure anti-estrogen ICI, indicates that these cells do not require ER for cell growth and distinguishes these cells from the acquired AI resistant cells. We further determined that the HSP90 inhibitor 17-DMAG suppressed the growth of the AI-resistant cell lines studied. Our analysis revealed 17-DMAG-mediated decreased expression of growth promoting signaling proteins. It was found that de novo AI resistant AKT-aro and HER2-aro cells could not be resensitized to letrozole or ICI by treatment with 17-DMAG. In summary, we have generated two cell lines which display the characteristics of de novo AI resistance. Together, these data indicate the possibility that HSP90 inhibitors may be a viable therapy for endocrine therapy resistance although additional clinical evaluation is needed.

PD01-05: Histone Deacetylase Inhibitor LBH589 (Panobinostat) Suppresses the Activated-NFκB Pathway in Acquired Aromatase Inhibitor Resistant Breast Cancer Cells.

Abstract Background: Aromatase inhibitors (AIs) are effective in treating postmenopausal patients with estrogen receptor (ER)-positive breast cancer. However, resistance to endocrine therapies remains a major problem in the management of ER-positive breast cancer. Recently, histone deacetylase inhibitors (HDACis) show promise as cancer therapeutics, but the full scope of their utility remains unknown. Experimental Design: In order to search for critical genes in developing resistance to AI, a microarray study was performed on AI resistant cells derived from MCF-7aro cells that overexpress aromatase and on AI resistant tumors from our mouse experiments. Moreover, using MCF-7aro cells that are resistant to anastrozole (Ana-R), letrozole (Let-R), and exemestane (Exe-R), as well as LTEDaro, we evaluated the significance of NFKB1 and several regulatory genes in AI resistance, and the anti-NFκB1 activity of HDACi LBH589 in vitro and in vivo. Results: Significant changes (Fold change cutoff > 1.5, false discovery rate < 0.05) in the expression levels of 22 genes among three networks were identified from microarray analysis of our in vitro and in vivo models of acquired AI resistance. NFKB1 is a key player in one of three networks, including 11 genes, and is functionally up-regulated in all AI resistant cells. NFκB1 knockdown suppresses the proliferation of AI resistant cells more than that of MCF-7aro cells. Moreover, AI resistant cell lines are highly sensitive to LBH589 treatment and apoptosis is induced in these cell lines. LBH589 suppressed NFκB1 as demonstrated at both the mRNA and protein levels in LTEDaro and other AI resistant cell lines. Furthermore, the LBH589 treatment abrogated tumor growth in mice, and was associated with significant decreased levels of NFKB1 in tumors. Conclusions: Our findings provide new insights into how the NFκB1-mediated networks play roles in AI resistance, and strongly support that LBH589 offers a novel therapeutic strategy for patients with AI resistant breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-05.

Abstract 1472: MRP4 and PGT, members of the COX-2 pathway, may contribute to breast cancer progression

Abstract Prostaglandin E2 (PGE2) is the major product of the cyclooxygenase 2 (COX-2) enzyme. PGE2 is a substrate of multiple drug resistance-associated protein 4 (MRP4) which exports it from the cell. PGE2 can then signal through any of its four GPCR-family receptors EP1-4. Each receptor is connected to a different cell function including adenylate cyclase activation, calcium flux, and inositol trisphosphate mobilization. Alternatively, extracellular PGE2 can be imported through prostaglandin transporter (PGT) and inactivated by 15-hydroxy prostaglandin dehydrogenase (15-PGDH). PGE2 plays a role in tumor progression as evidenced by the anti-tumor activity of COX-2 inhibitors. Adverse effects of these inhibitors have forced a search to identify other potential therapeutic targets in the COX-2 pathway (such as the receptors, transporters, and associated metabolic enzymes of PGE2). We hypothesize that disregulation of the COX-2 pathway can lead to increased metastatic potential in breast cancer. High extracellular PGE2 in the tumor microenvironment can stem from high COX-2 expression or elevated eicosanoid export activity. Continued prostaglandin signaling is thought to occur due to the interplay between increased expression of the exporting protein (MRP4) shuttling PGE2 into the pericellular space and decreased expression of the importing protein (PGT) removing PGE2 from the pericellular space. Higher concentrations of PGE2 in the pericellular space allow for increased signaling through PGE2 receptors EP1-4. Using a panel of breast cancer cell lines, we demonstrate that highly metastatic tumor cells can express low levels of COX-2, but still actively signal through PGE2 receptors. MRP4 mRNA and protein expression is increased in tumor cell lines with high metastatic potential while expression of PGT mRNA and protein is decreased in these cells when compared to cells with lower metastatic potential. This should lead to higher concentrations of extracellular PGE2 in the tumor microenvironment. Additionally, we have examined MCF7 cells stably transfected with aromatase and an anastrozole-resistant derivative of this cell line for expression of COX-2, EP4, EP1, MRP4 and PGT. At the mRNA level, COX-2, EP1, MRP4 and PGT decrease in the aromatase inhibitor-resistant cells. EP4 mRNA levels increase in the resistant cells. Examining the relative expression levels of all key members in the COX-2 pathway as they relate to tumor progression could highlight new therapeutic targets under this classification of breast cancer. 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 1472. doi:10.1158/1538-7445.AM2011-1472

Abstract PD05-07: Use of Natural ER Beta Modulators in Treating Hormonal Therapy Resistant Breast Tumors

Abstract Although systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) such as letrozole or block actions of estrogen/ER actions by anti-estrogens (AEs) like tamoxifen are well tolerated, unfortunately, essentially all breast cancers in women with advanced breast cancer develop resistance to these two classes of agents, and the benefit of adjuvant therapy (where micrometastatic disease is present but cannot be directly observed) is also limited by the development of resistance. In addition, side effects associated with the systemic inhibition of aromatase with current compounds limits their long-term utility of them as chemopreventative agents. We have investigated whether treatment of letrozole resistant breast cancer cells with selective plant-derived ER beta agonists (Liquiritigenin; LIQ or Nyasol; NYA) alone or in combination with letrozole affects the growth of AI resistant breast cancer cells, thereby restores sensitivity to AI. To test whether combination therapy restores sensitivity to letrozole, we have treated letrozole resistant breast cancer cells [LTLTca, derived from MCF-7-aromatase tumors resistant to letrozole] with either letrozole alone or with a combination of letrozole and LIQ/NYA. In addition, effects of these compounds on cell growth was also tested using different breast cancer cells including MCF-7, MCF-7-aromatase, ZR-75-1, ZR-75-aromatase, MDA-MB-231 along with tamoxifen resistant MCF-7 or MCF-7- PELP/ZR-75-PELP1. Compared to single agents, combination treatment not only restored sensitivity to letrozole but also resulted in decreased cell proliferation and increased apoptosis as well as increased ERα and decreased ERα levels in resistant cells. Combination of LIQ or NYA not only diminished cell growth in MCF-7-HER2, MCF-7-PELP1, and SKBR-3 breast cancer cells (that express endogenous aromatase) but also affected the expression of aromatase. Detailed investigations indicated that LIQ/NYA affects the induction of aromatase by specifically inhibiting breast cancer specific aromatase promoter 1.3 as well as its activity through inflammatory cytokine-mediated actions. These studies suggest the therapeutic benefit of LIQ or NYA ERα agonist to resensitize breast cancer cells that are resistant to AEs or AI and their ability to selectively inhibit tumor induction of aromatase. Our study suggests a novel role of natural ER beta agonists as valuable therapeutic modulators to treat endocrine sensitive as well as resistant tumors. (Supported by V-Foundation & P30 CA 54174, RRT) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-07.

Abstract 606: Heat shock protein 90 inhibitors: New mode of therapy to overcome aromatase inhibitor resistance

Abstract Results from several major trials have revealed that aromatase inhibitors (AIs) are superior to tamoxifen with regard to disease progression, incidences of locoregional and distant relapses, and contralateral breast cancers. Although AIs are highly effective drugs (with outstanding specificity and potency), unfortunately, a significant number of AI-treated breast cancer patients will eventually develop disease progression due to acquired resistance. Hormone refractory breast cancers result from complex molecular changes and are challenging to cure. Recently, we examined whether the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) can inhibit the growth of AI-resistant breast cancers and the mechanisms by which 17-DMAG affects proliferation. AI-responsive MCF-7aro and AI-resistant LTEDaro breast epithelial cells were used in this study. We observed that 17-DMAG inhibited proliferation in both MCF-7aro and LTEDaro cells in a dose-dependent manner. 17-DMAG induced apoptosis and G2 cell cycle arrest in both cell lines. Although inhibition of HSP90 decreased the levels of ERα, the ERα transcriptional activity was not affected when cells were treated with 17-DMAG together with estradiol. Moreover, detailed mechanistic studies suggested that 17-DMAG inhibits cell growth via degradation of HSP90 client proteins AKT and HER2. Furthermore, our experiments using AI responsive MCF-7aro cells have revealed that HSP90 inhibitors such as 17-DMAG and AIs such as letrozole can suppress the proliferation of hormone-dependent breast cancer cells in a synergistic manner. To study de novo AI-resistance, our lab has generated MCF-7 cell models to overexpress both aromatase and either Akt (MCF-7Aktaro) or HER2 (MCF-7aroHER2). We have found that HSP90 inhibitor 17-DMAG is also effective on suppressing the proliferation of these de novo AI resistant cells. Collectively, results from this study provide data to support the theory that HSP90 inhibitors may be an effective therapy to delay and treat AI-resistant breast cancers (This research was supported by the NIH grant CA44735). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 606.