Rabia A. Gilani

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.

Sensitivity to the Aromatase Inhibitor Letrozole Is Prolonged After a “Break” in Treatment

Using a hormone-dependent xenograft model, we established that loss of response to letrozole was accompanied by upregulation of the Her-2/mitogen-activated protein kinase (MAPK) pathway and downregulation of estrogen receptor α (ERα) and aromatase activity. In our previous study, we showed that stopping letrozole treatment or adding trastuzumab could reverse acquired resistance. In this study, we compared the effects of intermittent letrozole treatment and switching treatment between letrozole and trastuzumab on tumor growth in an attempt to optimize discontinuous letrozole treatment. The mice were treated with letrozole until the tumors developed resistance and then were divided into three groups: (a) letrozole, (b) trastuzumab, and (c) “off” (Δ4A supplement only); tumors were collected every week to examine changes in tumor protein expression and activity. In off group tumors, Her-2/p-MAPK activation gradually decreased and ERα and aromatase protein (and activity) increased. Within the first week of trastuzumab treatment, Her-2 and MAPK were downregulated and ERα was upregulated. When letrozole-resistant MCF-7Ca tumors were taken off treatment for 4 weeks, the second course of letrozole treatment provided a much longer duration of response (P = 0.02). However, switching treatment to trastuzumab for 4 weeks did not provide any inhibition of tumor growth. Our studies revealed that the adaptation of cells to a low-estrogen environment by upregulation of Her-2/MAPK and downregulation of ERα/aromatase was reversed on letrozole withdrawal. The tumors once again became responsive to letrozole for a significant period. These results suggest that response to letrozole can be prolonged by a short “break” in the treatment. Mol Cancer Ther; 9(1); 46–56

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.

Combination of HDACi entinostat (SNDX-275) with letrozole provides control over tumor growth in MDA-MB-231 xenograft model.

Abstract #6128 It is well established that approximately 75% of human breast cancers are ER+ and therefore treated with endocrine therapy. In a past few decades endocrine therapy has made significant advancements. However, the application of these agents is limited to ER+ cancers since ER- patients are unresponsive to endocrine therapy primarily due to lack of ER expression in the tumor. The main purpose of this project is to determine whether ER- breast cancer tumors that display poor anti-proliferative response to aromatase inhibitor (AI) letrozole, can be sensitized by co-treatment with HDACi entinostat. Based on preliminary studies, we hypothesize that by inhibiting HDAC ER is re-expressed making the cells sensitive to the anti-proliferative effects of AIs. In our previous studies we have shown that HDACi entinostat can revert the ERα repression and upregulate ERα and aromatase in vitro and in vivo. In this study we are showing that this activation of aromatase and re-expression of ERα renders ER- breast cancer tumors (xenografts of MDA-MB-231 cells) responsive to letrozole.
 MDA-MB-231 xenografts were grown in ovariectomized female nude mice. Mice were inoculated with 2.5X 10 6 cells per site subcutaneously. When the tumors reached a measurable size ∼150 mm 3 , the mice were grouped into 6 groups (n=10), such that the mean tumor volumes across the groups was not statistically different ( p=0.99 ). The mice were injected with Δ 4 A (100 μg/day), Δ 4 A plus letrozole (10 μg/day), entinostat (2.5 mg/kg/day), entinostat plus Δ 4 A, entinostat plus Δ 4 A plus letrozole or vehicle. The mice were injected 5 times a week. The tumors were measured every week with calipers and the tumor volumes were calculated using formula, 4/3 π r 1 2 r 2 . The mice in the entinostat plus Δ 4 A plus letrozole group had the least tumor growth rate (0.004+0.081), which was lower than entinostat plus Δ 4 A (0.115+0.079) and Δ 4 A plus letrozole (0.096+0.080). This data suggests a trend towards improved inhibition of tumor growth with combination of entinostat plus letrozole. The mice were sacrificed on week 9 due to large tumor volumes. The tumors and uteri were excised, cleaned, weighed and stored for additional analysis.
 In addition, ability of this combination to inhibit migration in vitro was examined by wound healing assay. The combination of entinostat plus letrozole provides superior inhibition of migration (p compared to control, entinostat and letrozole alone. This suggests that the combination of entinostat plus letrozole has potential of inhibiting metastatic spread along with tumor growth.
 These findings indicate that ERα in ER- breast cancer cells is silenced along with aromatase but can be restored with HDACi. Thus activation of silenced ER and intratumoral aromatase by HDACi could open a new avenue for management of ER- advanced breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6128.

Abstract 1311: Preclinical assessment of an HDAC inhibitor combined with a retinoid in AI resistant breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Treatment with aromatase inhibitors (AIs) is highly effective against breast cancer in ER positive postmenopausal women. However, some patients eventually become resistant to AIs. Tumor initiating cells (TICs) represent a subpopulation of tumor cells, which show self-renewal capacity. We are focused towards discovering strategies to reduce the growth of breast cancer TICs, which may result in resistance. We have developed a xenograft model that mimics post-menopausal hormone responsive breast cancer. In this model, aromatase transfected human hormone sensitive MCF-7 cells (MCF-7Ca) are inoculated in ovariectomized athymic nude mice and allowed to grow in presence of Δ4A (aromatizable substrate of estrogen). Using this model, we have established that although AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. In the current study, we investigated the effect of ATRA (All-trans Retinoic acid) (125μg/day, ip) and a histone deacetylase (HDAC) inhibitor entinostat (SNDX- 275) (50μg/day, po) with or without letrozole on letrozole resistant tumors in a xenograft model system. Ovariectomized athymic nude mice bearing xenografts of MCF-7Ca cells were treated with letrozole till they became resistant (15 weeks). At this time, the mice were grouped to receive ATRA, entinostat plus ATRA or the combination of ATRA plus entinostat plus letrozole till week 23. The mice treated with entinostat plus ATRA plus letrozole showed a significant decrease in tumor growth rate compared to mice treated with single agents or entinostat plus ATRA (p<0.0001, p=0.02). On week 20, two mice from each treatment group were euthanized and tumors were harvested. The tumors were digested enzymatically with collagenase and hyaluronidase and freed of debris using centrifugation and filtration. Mammosphere forming ability of TICs in the tumor tissue was measured by seeding 10,000 viable cells from each treated tumors under non-adherent conditions to access the self-renewal capacity. The combination of ATRA plus ENT plus letrozole significantly (p<0.01) reduced number of mammospheres formed compared to single agents alone. Quantitative PCR analysis of tumors cells showed a significant downregulation of the known TIC molecular markers, BCRP, ALDH, BMI-1 and Nanog compared to letrozole treated tumors. Similar results were also obtained when LTLT-Ca (long term letrozole treated MCF-7Ca) cells were treated with ATRA and entinostat in combination with letrozole and then seeded (5000 cells) in non-adherent conditions. Combination of ATRA plus entinostat plus letrozole significantly (p<0.0001) reduced mammosphere-forming ability of the LTLT-Ca cells. Overall, these studies indicate that the combination of ATRA, entinostat and letrozole is effective in reducing tumor recurrence in letrozole resistant tumors. Citation Format: Preeti Shah, Gauri J. Sabnis, Olga Goloubeva, Armina Kazi, Amanda Schech, Rabia Gilani, Yael Gau, Saranya Chumsri, Angela Brodie. Preclinical assessment of an HDAC inhibitor combined with a retinoid in AI resistant breast cancer. [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 1311. doi:10.1158/1538-7445.AM2013-1311

Abstract 787: Involvement of HER2, HIF-1, and BCRP in cancer stem cell characteristics of letrozole-resistant breast cancer cells

Studies by us and others suggest that acquired resistance to aromatase inhibitors (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 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) and CSCs have been implicated in drug resistance. Therefore, this current study aimed to 1) compare the CSC characteristics of letrozole-resistant (LTLTCa) and parental letrozole-sensitive (MCF-7Ca) breast cancer cells; 2) determine the roles of HER2, BCRP, and HIF-1 on CSC characteristics; and 3) compare the effects of HER2 inhibition and of differentiating agent all-trans retinoic acid (ATRA) on CSC population of the LTLTCa cells. LTLTCa cells were found to have higher expression of CSC characteristics compared to parental MCF-7Ca cells: 9.17%±2.0% vs. 0.02% ±0.01% (p 200 mammospheres/10,000 cells to ∼50 mammospheres/10,000 cells). Flow cytometry also demonstrated that LTLTCa cells high in both ALDH activity and CD44 positivity, were also high in HER2. Inhibition of HIF-1 (by siRNA) or BCRP (by pharmacological inhibitors or siRNA) significantly decreased mammosphere formation from 406 mammospheres/80,000 cells to 93 and 81 mammospheres/80,000 cells, respectively (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 787. doi:1538-7445.AM2012-787

Abstract 2303: The potential role of nonhypoxic HIF-1 in HER2-mediated aromatase inhibitor resistant breast cancer

Although aromatase inhibitors (AIs; i.e., letrozole) 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 resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. The mechanism by which HER2 is involved in AI resistance remains unclear. It is, therefore, important to elucidate the HER2-mediated pathway that contributes to AI resistance, and to identify other relevant factors involved that can be used as biomarkers of AI resistance and targets for therapy. One such factor may be HIF-1, a heterodimeric transcription factor made up of an inducible alpha (α) subunit and a constitutively expressed beta (β) subunit. HIF-1 regulates genes important for cell survival, metabolic adaptation, and angiogenesis. Oxygen (O 2 ) tension is a well-known regulator of HIF-1α, but other factors independent of O 2 can also regulate it. Previous studies have shown that transfecting HER2 into cells and activation of kinase pathways increase HIF-1α expression and/or activity. Unlike the well-studied role of hypoxia-regulated HIF-1α in a variety of cancers, nonhypoxic regulation of HIF-1α and its role in cancer remains largely unclear. Here we have investigated HIF1α in AI resistance. Preliminary results indicate that basal HIF-1α protein expression is 3-fold (p 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 2303. doi:10.1158/1538-7445.AM2011-2303

Abstract 4611: Growth inhibitory effects of lapatinib on letrozole resistant breast cancer cells via HER2-AIB1-ERα crosstalk

Aromatase inhibitors (AI), such as letrozole, provide effective endocrine therapy for estrogen receptor positive (ER+) breast cancers, but not all patients respond to AIs and those that do may become resistant. Thus, it is clinically important to determine the factors and signaling mechanisms involved in AI resistance and provide potential therapeutic strategies to overcome and/or prevent it. Previous work from this laboratory comparing long-term letrozole treated MCF-7Ca breast cancer (LTLTCa) cells resistant to growth inhibition by letrozole with their parental letrozole-sensitive MCF-7Ca cells, indicated that AI resistance involves a switch from estrogen (E 2 )-ERα dependent to HER2/MAPK pathway-dependent growth and that inhibition of HER2/MAPK pathway signaling via trastuzumab can restore ERα levels and letrozole sensitivity. We observed that the upregulation of HER2 is through protein stabilization and not gene amplification. This current study further investigates the factors and mechanism(s) involved in letrozole resistance in LTLTCa cells, specifically steroid receptor coactivator Amplified in Breast Cancer-1 (AIB1/SRC-3). AIB1 is often overexpressed in breast cancer cells, is associated with poor prognosis, and has been linked with both E 2 -ERα and HER2 signaling. Indeed, western blot analyses showed that LTLTCa cells have 2.8-fold higher AIB1 protein levels than MCF-7Ca cells (p 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 4611.

Expression of ERα and Aromatase in MDA-MB-231 Tumors by HDAC Inhibitor Entinostat Leads to Growth Inhibition by Aromatase Inhibitor Letrozole.

The treatment for hormone receptor-positive breast cancer has improved significantly since the development of aromatase inhibitors (AIs). Nevertheless, AIs are ineffective in estrogen receptor-negative (ER-) tumors, which comprise of approximately 25% of breast cancers and tend to be more aggressive. Studies have shown that repression of ER in these hormone receptor-negative tumors may be due to epigenetic modifications. The discovery of recruitment of histone deacetylase enzymes in gene silencing provides a rationale for inhibition of HDAC activity to release transcriptional repression as a potential therapeutic strategy. The objective of the present study was to express ERα and aromatase with HDACI treatment and thereby sensitize tumors to growth inhibition with aromatase inhibitors. In this study we used ER negative, hormone refractory MDA-MB-231 human breast cancer cells. Treatment with HDAC inhibitor entinostat led to upregulation of ERα, aromatase and its activity in a dose dependent manner in cells and xenografts. MDA-MB-231 xenografts were grown in ovariectomized female nude mice. Mice were inoculated with 2.5 X 106 cells per site subcutaneously. When the tumors reached 150 mm3, the mice were grouped into 4 groups (n=10), so that the mean tumor volume was not statistically different across groups (p=0.88). Tumor volumes were measured twice weekly. The mice in the letrozole group had a mean tumor growth rate (β = 0.023 ± 0.014) that was not statistically different (p=0.76) from that of the control group (β = 0.038 ± 0.007). Also, the growth rate of entinostat group (β = 0.034 ± 0.011) was not significantly lower than that of the control (p=0.33). However, the growth rate of entinostat plus letrozole group ((β = -0.003 ± 0.013) was significantly lower than that of the control (p=0.01), entinostat (p=0.03) and letrozole (p=0.049) groups. The combined treatment of entinostat plus letrozole was significantly more effective than either agent alone. In addition, the ability of this combination to inhibit migration in vitro was examined by wound healing assay. The combination of entinostat plus letrozole provides superior inhibition of migration (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 401.