Amanda Schech

The importance of HER2 signaling in the tumor-initiating cell population in aromatase inhibitor-resistant breast cancer

Aromatase inhibitors (AIs) are an effective therapy in treating estrogen receptor-positive breast cancer. Nonetheless, a significant percentage of patients either do not respond or become resistant to AIs. Decreased dependence on ER-signaling and increased dependence on growth factor receptor signaling pathways, particularly human epidermal growth factor receptor 2 (EGFR2/HER2), have been implicated in AI resistance. However, the role of growth factor signaling remains unclear. This current study investigates the possibility that signaling either through HER2 alone or through interplay between epidermal growth factor receptor 1 (EGFR/HER1) and HER2 mediates AI resistance by increasing the tumor initiating cell (TIC) subpopulation in AI-resistant cells via regulation of stem cell markers, such as breast cancer resistance protein (BCRP). TICs and BCRP are both known to be involved in drug resistance. Results from in vitro analyses of AI-resistant versus AI-sensitive cells and HER2-versus HER2+ cells, as well as from in vivo xenograft tumors, indicate that (1) AI-resistant cells overexpress both HER2 and BCRP and exhibit increased TIC characteristics compared to AI-sensitive cells; (2) inhibition of HER2 and/or BCRP decrease TIC characteristics in letrozole-resistant cells; and (3) HER2 and its dimerization partner EGFR/HER1 are involved in the regulation of BCRP. Overall, these results suggest that reducing or eliminating the TIC subpopulation with agents that target BCRP, HER2, EGFR/HER1, and/or their downstream kinase pathways could be effective in preventing and/or treating acquired AI resistance.

Histone Deacetylase Inhibitor Entinostat Inhibits Tumor-Initiating Cells in Triple-Negative Breast Cancer Cells

Mortality following breast cancer diagnosis is mainly due to the development of distant metastasis. To escape from the primary site, tumor cells undergo the epithelial-to-mesenchymal transition (EMT), which helps them acquire a more motile and invasive phenotype. In our previous study, we showed that class I selective HDAC inhibitor entinostat reverses the EMT phenotype through reversal of epigenetic repression of E-cadherin. Recent evidence suggests that a subset of cells within a breast tumor may drive the metastatic outgrowth following escape from the primary site. These cells, termed tumor-initiating cells (TIC), represent a great threat to overall prognosis. They are critical in terms of drug resistance and tumor initiation at metastatic sites. Acquisition of EMT traits has also been shown to impart TIC phenotype to the cells, making EMT a “dual-threat” for prognosis. In the current study, we show that entinostat treatment can reduce the percentage of TIC cells from triple-negative breast cancer (TNBC) cells. Entinostat treatment was able to reduce the CD44high/CD24low cell population, ALDH-1 activity, as well as protein and mRNA expression of known TIC markers such as Bmi-1, Nanog, and Oct-4. Next, we inoculated MDA-MB-231 cells transfected with firefly luciferase (231/Luc) in mammary fat pad of NSG mice. The mice were then treated with entinostat (2.5 mg/kg/d), and tumor development and formation of metastasis were assessed by bioluminescence imaging. Treatment with entinostat significantly reduced tumor formation at the primary site as well as lung metastasis. As such, entinostat may help prevent development of distant metastasis. Mol Cancer Ther; 14(8); 1848–57. ©2015 AACR.

Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer

IntroductionAlthough aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2.MethodsIn vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance.ResultsBasal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP.ConclusionsOne of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.

Zoledronic Acid Reverses the Epithelial–Mesenchymal Transition and Inhibits Self-Renewal of Breast Cancer Cells through Inactivation of NF-κB

Zoledronic acid, a third-generation bisphosphonate, has been shown to reduce cell migration, invasion, and metastasis. However, the effects of zoledronic acid on the epithelial–mesenchymal transition (EMT), a cellular process essential to the metastatic cascade, remain unclear. Therefore, the effects of zoledronic acid on EMT, using triple-negative breast cancer (TNBC) cells as a model system, were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers, N-cadherin, Twist, and Snail, and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability, induced cell-cycle arrest, and decreased the proliferative capacity of TNBC, suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype, the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins, BMI-1 and Oct-4, and both prevented and eliminated mammosphere formation. To understand the mechanism of these results, the effect of zoledronic acid on established EMT regulator NF-κB was investigated. Zoledronic acid inhibited phosphorylation of RelA, the active subunit of NF-κB, at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter, providing a direct link between inactivation of NF-κB signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased, correlating modulation of EMT to decreased self-renewal. On the basis of these results, it is proposed that through inactivation of NF-κB, zoledronic acid reverses EMT, which leads to a decrease in self-renewal. Mol Cancer Ther; 12(7); 1356–66. ©2013 AACR.

Advances in mechanisms of resistance to aromatase inhibitors

Clinically, there are two distinct types of aromatase inhibitor (AI) resistance, namely acquired and innate resistance. Because the underlying mechanisms of these two types of resistance may not be mutually exclusive, strategies to tackle these resistances may not be effective when used interchangeably. Activation of growth factor receptor pathways is the hallmark of acquired AI resistance. These pathways can be targeted either at the cell surface receptor level or their downstream signaling cascades. Currently, everolimus in combination with exemestane represents a new standard of care for patients progressing on non-steroidal AIs. HDAC inhibitors have also shown promising results For innate resistance, the combination of fulvestrant and AI in the front line setting represents a new treatment option, particularly for patients who present with de novo metastatic disease. A Phase III trial is currently ongoing to evaluate the benefit of CDK 4/6 inhibitor, palbociclib, in the first line setting in combination with AI.

Zoledronic Acid Inhibits Aromatase Activity and Phosphorylation: Potential Mechanism for Additive Zoledronic Acid and Letrozole Drug Interaction

Zoledronic acid (ZA), a bisphosphonate originally indicated for use in osteoporosis, has been reported to exert a direct effect on breast cancer cells, although the mechanism of this effect is currently unknown. Data from the ABCSG-12 and ZO-FAST clinical trials suggest that treatment with the combination of ZA and aromatase inhibitors (AI) result in increased disease free survival in breast cancer patients over AI alone. To determine whether the mechanism of this combination involved inhibition of aromatase, AC-1 cells (MCF-7 human breast cancer cells transfected with an aromatase construct) were treated simultaneously with combinations of ZA and AI letrozole. This combination significantly increased inhibition of aromatase activity of AC-1 cells when compared to letrozole alone. Treatment of 1 nM letrozole in combination with 1 μM or 10 μM ZA resulted in an additive drug interaction on inhibition of cell viability, as measured by MTT assay. Treatment with ZA was found to inhibit phosphorylation of aromatase on serine residues. Zoledronic acid was also shown to be more effective in inhibiting cell viability in aromatase transfected AC-1 cells when compared to inhibition of cell viability observed in non-transfected MCF-7. Estradiol was able to partially rescue the effect of 1 μM and 10 μM ZA on cell viability following treatment for 72 h, as shown by a shift to the right in the estradiol dose-response curve. In conclusion, these results indicate that the combination of ZA and letrozole results in an additive inhibition of cell viability. Furthermore, ZA alone can inhibit aromatase activity through inhibition of serine phosphorylation events important for aromatase enzymatic activity and contributes to inhibition of cell viability.

A nude mouse model of obesity to study the mechanisms of resistance to aromatase inhibitors

Obesity is a risk factor for breast cancer progression. Breast cancer patients who are overweight or obese or have excess abdominal fat have an increased risk of local or distant recurrence and cancer-related death. Hormone depletion therapies can also cause weight gain, exacerbating the risk for these patients. To understand the effect of obesity on hormone-dependent human breast cancer tumors, we fed ovariectomized athymic nude mice a diet containing 45% kcal fat and 17% kcal sucrose (high fat sucrose diet (HFSD)), 10% kcal fat (low fat diet (LFD)), or a standard chow diet (chow). The mice fed the HFSD developed metabolic abnormalities consistent with the development of obesity such as weight gain, high fasting blood glucose, and impaired glucose tolerance. These mice also developed hyperinsulinemia and insulin resistance. The obese mice also had a higher tumor growth rate compared to the lean mice. Furthermore, the obese mice showed a significantly reduced responsiveness to letrozole. To understand the role of obesity in this reduced responsiveness, we examined the effect of insulin on the growth of MCF-7Ca cells in response to estrogen or letrozole. The presence of insulin rendered MCF-7Ca cells less responsive to estrogen and letrozole. Exogenous insulin treatment of MCF-7Ca cells also resulted in increased p-Akt as well as ligand-independent phosphorylation of ERα. These findings suggest that diet-induced obesity may result in reduced responsiveness of tumors to letrozole due to the development of hyperinsulinemia. We conclude that obesity influences the response and resistance of breast cancer tumors to aromatase inhibitor treatment.

Abstract 1471: HER2 regulated miRNA expression in letrozole resistant breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MicroRNAs (miRNAs) are short non-coding regulatory RNA molecules that modulate the expression of specific proteins by binding to target messenger RNAs (mRNAs) and causing either degradation of the mRNAs or inhibition of their translation to protein. Thus, miRNAs play an important role in a variety of normal cellular processes (e.g., differentiation, cell growth, cell death, etc.), and in diseases, such as cancer. MiRNAs have been implicated in breast cancer, but very little is known about their role in aromatase-inhibitor (AI)-resistant breast cancer. Studies by us and others suggest that acquired resistance to AIs (e.g., letrozole, let), which inhibit the aromatase enzyme from converting androgens (e.g., androstenedione) to estrogen, involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. Recent work in our lab has further indicated that HER2s effects on resistance are mediated by hypoxia inducible factor 1 (HIF-1) and cancer stem cells. The aim of this current study was to determine if miRNAs were also involved in this mechanism. Initial miRNA microarray results by Zhou et al. identified a number of miRNAs that were either upregulated or down regulated in ER-/HER2+, let-resistant LTLTCa cells vs. ER+/HER2- let-sensitive MCF-7Ca cells. Of particular interest, was data indicating increased expression of miRNA181a and miRNA222 in LTLTCa cells. MiRNA181a has been linked to HIF-1 and cancer stem cells. MiRNA222 has been linked to drug resistance in breast cancer cells. In vitro miRNA isolation and RT-PCR analyses confirmed the microarray results: miRNA181a and miRNA22 levels were at least 13-fold and 10-fold higher in LTLTCa cells vs. MCF-7Ca cells, respectively. MiRNA expression was also analyzed in mouse tumor xenografts. Compared to control (androstenedione-supplemented) tumors, let-treated tumors had increased miRNA181a and 222 expression, starting at 2 weeks of let treatment, when HER2 has previously been shown to be expressed, and further increasing through 16 weeks of letrozole treatment, when tumors are letrozole-resistant. Since HER2 is associated with let resistance, the effect of inhibiting HER2 expression and/or activity on miRNA expression was determined. Lapatinib and trastuzumab either alone or in combination, significantly decreased miRNA 181a expression (0.1-fold vs. vehicle). MiRNA 181a and 222 expression was also analyzed in another ER-/HER2+ breast cancer cell line, SKBR3. Compared to MCF-7Ca cells, miRNA 181a, but not miRNA 222, was significantly upregulated 16-fold in SKBR3 cells. Lastly, preliminary studies were conducted to look at miRNA181a expression in patient tumor samples. Significant and comparable levels of miRNA181a were detected in both ER+/HER2+, ER-/HER2+ samples Overall, these results suggest that miRNAs 181a and 222 are regulated by HER2 and may mediate its effects on AI-resistance. Note: This abstract was not presented at the meeting. Citation Format: Armina A. Kazi, Gauri Sabnis, Qun Zhou, Saranya Chumsri, Amanda Schech, Preeti Shah, Angela Brodie. HER2 regulated miRNA expression in letrozole resistant breast cancer. [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 1471. doi:10.1158/1538-7445.AM2014-1471

Abstract 95: Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Although aromatase inhibitors (AIs) have been shown to be highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Studies suggest that acquired resistance to AIs (i.e., letrozole) involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. Recent work in our lab has also linked hypoxia inducible factor 1 (HIF-1) and its target gene breast cancer resistance protein (BCRP, a stem cell marker) to HER2 and AI resistance. Other studies have associated each of these factors with cancer stem cells (CSCs),, which have been implicated in drug resistance. Thus, the purpose of this study is to 1) determine whether HIF-1 expression is enriched in the CSC subpopulation of letrozole-resistant breast cancer cells; and 2) determine the effects of inhibiting HIF-1 on CSC characteristics. The in vitro model used for acquired letrozole resistant breast cancer is the LTLTCa cell line, which was obtained through long-term letrozole treatment of MCF-7Ca xenograft tumors. The CSC subpopulation in LTLTCa cells was isolated by flow cytometry based on their aldehyde dehydrogenase (ALDH) expression, and the CSC characteristics studied include mammosphere formation, side population percentage, and CD44+/CD24+ expression. Inhibition of HIF-1 expression was accomplished using either commercially available HIF-1α siRNA or EZN-2968, a specific RNA antagonist against HIF-1α currently in clinical trials. HIF-1α is the inducible subunit of HIF-1. RT-PCR indicates that mRNA expression of HIF-1α and stem cell markers, such as BCRP, BMI-1, and Nanog, were significantly higher (p<0.05) in ALDHhigh (CSC) vs. ALDHlow (non-CSC) cells. HIF-1 siRNA and EZN-2968 decreased HIF-1α mRNA and protein expression in LTLTCa cells within 48h (0.4 to 0.01-fold vs. negative control siRNA/RNA antagonist). This correlated with decreases in mRNA expression of stem cell markers BCRP and BMI-1, and TWIST, an EMT marker and regulator of BMI-1. HIF-1α inhibition also reduced mammosphere formation by 33% (p<0.05). Interestingly, preliminary results indicate that while EZN-2968 decreased the ratio of CD44+/24+ expression (28% vs. 52% in negative control RNA antagonist) and side population percentage (8% vs. 16% in negative control RNA antagonist) in LTLTCa cells, the commercially available HIF-1α siRNA had no effect. These results suggest that nonhypoxic HIF-1 is involved in regulating cancer stem cell characteristics in letrozole-resistant breast cancer cells, and that EZN-2968 should be further explored in the prevention and/or treatment of AI-resistant breast cancer. EZN-2968 kindly provided by Enzon Pharmaceuticals. This work is funded by DOD Breast Cancer Research Program Postdoctoral Award (BC1039031). Citation Format: Armina A. Kazi, Preeti Shah, Amanda Schech, Gauri Sabnis, Saranya Chumsri, Angela Brodie. Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2013-95

Aromatase Inhibitors for Breast Cancer Prevention

Aromatase inhibitors (AIs) offer a new treatment option for breast cancer prevention without increased risks of venous thromboembolism and endometrial cancer. Compared to placebo, both exemestane and anastrozole significantly reduced the risk of not only invasive breast cancer but also non-invasive lesions. AIs are associated with unique side effects, particularly musculoskeletal symptoms, vasomotor symptoms, and bone loss. However, these side effects are manageable. There appeared to be no difference in the incidence of cardiovascular disease and the difference in quality of life is numerically small.