Armina A. Kazi

Estrogen Rapidly Activates the PI3K/AKT Pathway and Hypoxia-Inducible Factor 1 and Induces Vascular Endothelial Growth Factor A Expression in Luminal Epithelial Cells of the Rat Uterus

Abstract We have previously shown that 17beta-estradiol (E2) increases vascular endothelial growth factor A (Vegfa) gene expression in the rat uterus, resulting in increased microvascular permeability, and that this involves the simultaneous recruitment of hypoxia-inducible factor 1 (HIF1) and estrogen receptor alpha (ESR1) to the Vegfa gene promoter. Both events require the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. However, those studies were carried out using whole uterine tissue, and while most evidence indicates that the likely site of E2-induced Vegfa expression is luminal epithelial (LE) cells, other studies have identified stromal cells as the site of that expression. To address this question, the pathway regulating Vegfa expression was reexamined using LE cells rapidly isolated after E2 treatment. In addition, we further characterized the nature of the receptor through which E2 triggers the signaling events that lead to Vegfa expression using the specific ESR1 antagonist ICI 182,780. In agreement with previous results in the whole uterus, E2 stimulated Vegfa mRNA expression in LE cells, peaking at 1 h (4- to 14-fold) and returning to basal levels by 4 h. Treatment with E2 also increased phosphorylation of AKT in LE cells, as well as of the downstream mediators FRAP1 (mTOR), GSK3B, and MDM2. The alpha subunit of HIF1 (HIF1A) was present in LE cells before E2 treatment, was unchanged 1 h after E2, but was >2-fold higher by 4 h. Chromatin immunoprecipitation analysis showed that HIF1A was recruited to the Vegfa promoter by 1 h and was absent again by 4 h. The E2 activation of the PI3K/AKT pathway, HIF1A recruitment to the Vegfa promoter, and Vegfa expression were all blocked by ICI 182,780. In summary, the rapid E2-induced signaling events that lead to the expression of Vegfa observed previously using the whole uterus occur in LE cells and appear to be initiated via a membrane form of ESR1.

New Insight into the Transcriptional Regulation of Vascular Endothelial Growth Factor Expression in the Endometrium by Estrogen and Relaxin

Abstract: Increased uterine capillary permeability, which can be induced by both estrogen and relaxin, is required for endometrial growth and implantation. This effect is mediated in both cases by estrogen receptors (ERs), via stimulation of vascular endothelial growth factor (VEGF) expression. The sites on the VEGF promoter through which induction occurs, however, are completely unclear. We have used the technique of chromatin immunoprecipitation in vivo to localize the site of ER action and identify other transcription factors that are involved. We have found that ERa associates with Sp1/Sp3 at a GC‐rich region of the promoter. More interesting, however, is the observation that estrogen also induces rapid, transient binding of hypoxia‐inducible factor 1 (HIF‐1), which mediates VEGF transcription in response to hypoxia, to the promoter. The estrogen‐induced HIF‐1 binding closely matches the estrogen‐induced pattern of VEGF expression in the uterus, suggesting that HIF‐1 is involved in that induction, and probably that of many other genes as well (HIF‐1 is now known to regulate the expression of more than 40 genes). It is likely that studies now under way will also link relaxin‐induced VEGF expression to HIF‐1. This is based on the similarities in the effects of the two hormones on VEGF expression and on their shared ability to activate the PI3K and MAPK pathways, both of which can activate HIF‐1.

HDAC inhibitor entinostat restores responsiveness of letrozole-resistant MCF-7Ca xenografts to aromatase inhibitors through modulation of Her-2.

We previously showed that in innately resistant tumors, silencing of the estrogen receptor (ER) could be reversed by treatment with a histone deacetylase (HDAC) inhibitor, entinostat. Tumors were then responsive to aromatase inhibitor (AI) letrozole. Here, we investigated whether ER in the acquired letrozole-resistant tumors could be restored with entinostat. Ovariectomized athymic mice were inoculated with MCF-7Ca cells, supplemented with androstenedione (Δ4A), the aromatizable substrate. When the tumors reached about 300 mm3, the mice were treated with letrozole. After initial response to letrozole, the tumors eventually became resistant (doubled their initial volume). The mice then were grouped to receive letrozole, exemestane (250 μg/d), entinostat (50 μg/d), or the combination of entinostat with letrozole or exemestane for 26 weeks. The growth rates of tumors of mice treated with the combination of entinostat with letrozole or exemestane were significantly slower than with the single agent (P < 0.05). Analysis of the letrozole-resistant tumors showed entinostat increased ERα expression and aromatase activity but downregulated Her-2, p-Her-2, p-MAPK, and p-Akt. However, the mechanism of action of entinostat in reversing acquired resistance did not involve epigenetic silencing but rather included posttranslational as well as transcriptional modulation of Her-2. Entinostat treatment reduced the association of the Her-2 protein with HSP-90, possibly by reducing the stability of Her-2 protein. In addition, entinostat also reduced Her-2 mRNA levels and its stability. Our results suggest that the HDAC inhibitor may reverse letrozole resistance in cells and tumors by modulating Her-2 expression and activity. Mol Cancer Ther; 12(12); 2804–16. ©2013 AACR.

Inhibition of Oxygen-Induced Hypoxia-Inducible Factor-1α Degradation Unmasks Estradiol Induction of Vascular Endothelial Growth Factor Expression in ECC-1 Cancer Cells in Vitro

Estradiol (E(2)) rapidly and strongly induces vascular endothelial growth factor (VEGF) transcription in uterine endometrial epithelial cells in vivo. We have shown that this is mediated by both the estrogen receptor-alpha and hypoxia-inducible factor (HIF)-1alpha. By contrast, E(2) induces little or no VEGF expression in cultured breast or endometrial cancer cells, which lack HIF-1alpha due to the abnormally high concentration of oxygen ( approximately 20%) to which they are exposed. To test the hypothesis that restoring HIF-1alpha in cultured cells would restore the ability of E(2) to induce VEGF expression, we treated human endometrial cancer cells (ECC-1) with cobalt chloride (CoCl(2);100 microm), which prevents oxygen-induced HIF-1alpha degradation. HIF-1alpha was absent in untreated ECC-1 cells but detectable by 4 h after treatment with CoCl(2) alone, as was a significant increase in VEGF mRNA. E(2) plus CoCl(2) induced detectable HIF-1alpha expression at 2 h and an even higher level than that induced by CoCl(2) alone at 4 h; this HIF-1alpha was localized in the nuclei. This was accompanied by increasing VEGF expression, with the increase at 4 h severalfold higher than that induced by CoCl(2) alone and was concurrent with recruitment of both HIF-1alpha and estrogen receptor-alpha to the VEGF promoter. These results confirm that HIF-1alpha plays an essential role in E(2)-induced expression of VEGF. Through the induction of increased microvascular permeability and the consequent exudation of plasma growth factors, VEGF in turn may play an essential role in cancer cell proliferation in vivo.

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.

Seribantumab, an anti-ERBB3 antibody, delays the onset of resistance and restores sensitivity to letrozole in an estrogen receptor-positive breast cancer model

Heregulin-driven ERBB3 signaling has been implicated as a mechanism of resistance to cytotoxic and antiendocrine therapies in preclinical breast cancer models. In this study, we evaluated the effects of seribantumab (MM-121), a heregulin-blocking anti-ERBB3 monoclonal antibody, alone and in combination with the aromatase inhibitor letrozole, on cell signaling and tumor growth in a preclinical model of postmenopausal estrogen receptor–positive (ER+) breast cancer. In vitro, heregulin treatment induced estrogen receptor phosphorylation in MCF-7Ca cells, and long-term letrozole-treated (LTLT-Ca) cells had increased expression and activation levels of EGFR, HER2, and ERBB3. Treatment with seribantumab, but not letrozole, inhibited basal and heregulin-mediated ERBB receptor phosphorylation and downstream effector activation in letrozole-sensitive (MCF-7Ca) and -refractory (LTLT-Ca) cells. Notably, in MCF-7Ca–derived xenograft tumors, cotreatment with seribantumab and letrozole had increased antitumor activity compared with letrozole alone, which was accompanied by downregulated PI3K/MTOR signaling both prior to and after the development of resistance to letrozole. Moreover, the addition of an MTOR inhibitor to this treatment regimen did not improve antitumor activity and was not well tolerated. Our results demonstrate that heregulin-driven ERBB3 signaling mediates resistance to letrozole in a preclinical model of ER+ breast cancer, suggesting that heregulin-expressing ER+ breast cancer patients may benefit from the addition of seribantumab to antiendocrine therapy. Mol Cancer Ther; 14(11); 2642–52. ©2015 AACR.

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

Abstract PD05-03: HDAC Inhibitor Entinostat Restores Responsiveness of Letrozole Resistant MCF-7Ca Xenografts to AIs through Modulation of Her-2

Development of aromatase inhibitors (AIs) has significantly improved the treatment outcome of hormone responsive post-menopausal breast cancer. However, not all tumors respond and some eventually acquire resistance. We have developed a xenograft model that mimics post-menopausal hormone responsive breast cancer (MCF-7Ca). Results obtained using this model have been confirmed by numerous clinical trials. Using this model, we have established that single agent AI is better than tamoxifen in controlling tumor growth. We also observed that although, AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. A cell line was isolated from these Long-Term Letrozole Treated tumors and designated as LTLT-Ca. These cells and the tumors had lower expression of ER and aromatase compared to parental MCF-7Ca cells. On the other hand, growth factor receptor Her-2 and downstream kinases such as MAPK, Akt were upregulated. Inhibition of Her-2 using trastuzumab resulted in reversal of resistance and restoration of sensitivity to estrogens, antiestrogens and AIs. In order to restore sensitivity to AIs, we treated letrozole resistant MCF-7Ca tumors to HDAC inhibitor entinostat (ENT). In studies performed with ER negative MDA-MB-231 cells, ENT has upregulated ERα and restored sensitivity to AIs. To examine the effect of this combination on tumors that have acquired resistance to letrozole, we utilized MCF-7Ca xenografts model. We inoculated ovariectomized athymic nude mice MCF-7Ca cells and then tumors were allowed to form in the presence of androstenedione (≥4A), aromatizable substrate for estrogen. When the tumors reached ∼300mm3, the mice were randomized such that the mean tumor volume was not significantly different across the groups (p=0.13). 50 mice were treated with letrozole for total 16 weeks, during this time the tumors regressed, but eventually began to grow. When the tumors had reached double the initial volume, they were randomized again into 5 groups; letrozole continued, exemestane (250µg/day), ENT (50µg/day) or the combination of ENT with letrozole or exemestane. The mice were treated till week 26. The growth rates of tumors of mice treated with the combination of ENT with letrozole or exemestane was significantly better than single agent alone (P We have shown that upregulation of Her-2 in LTLT-Ca cells is a result of longer half-life of Her-2 protein and not gene amplification. To determine the mechanism of effect of ENT on Her-2 expression, we examined the interaction of Her-2 with HSP-90 in LTLT-Ca cells and effect of ENT on this interaction. We observed that ENT reduced the association of Her-2 protein with HSP-90, possibly reducing the stability of Her-2 protein the LTLT-Ca cells. This reduced interaction was also seen the tumors treated with the combination of ENT plus letrozole or exemestane. Our results suggest that HDAC inhibitor entinostat may modulate Her-2 and result in reverse the acquired resistance of letrozole resistant cells and tumors. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-03.

Abstract PD5-2: Preclinical assessment of HDAC inhibitor entinostat combined with all trans retinoic acid (ATRA) in aromatase inhibitor resistant breast cancer

Treatment with aromatase inhibitors (AI’s) is highly effective against breast cancer in ER positive postmenopausal women. However, some patients eventually become resistant to AIs. Tumor initiating cells (TIC’s) 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 D4A (aromatizable substrate of estrogen). Results obtained using this model have been confirmed by numerous clinical trials. Using this model, we have established that single agent AI is better than tamoxifen in controlling tumor growth. We also observed 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 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 weeks, 2 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. 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 treated with ATRA and entinostat in combination with letrozole and then seeded in non-adherent conditions. The combination of ATRA plus ENT plus letrozole significantly (p<0.01) reduced number of mammospheres formed compared to single agents alone. We have shown previously, that LTLT-Ca cells have higher percentage of side population (cells expressing higher level of efflux pumps such as BCRP) compared to MCF-7Ca cells. The treatment of LTLT-Ca cells with the combination of ENT and ATRA with letrozole drastically reduced the percentage of side population. Overall, these studies indicate that the combination of ATRA, entinostat and letrozole is effective in reducing tumor recurrence in letrozole resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD5-2.