Angela Brodie

Aromatase inhibitors and their application in breast cancer treatment

Estrogens are known to be important in the growth of breast cancers in both pre- and postmenopausal women. The number of breast cancer patients with hormone-dependent disease increases with age, as does the incidence of breast cancer. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. Because aromatase catalyzes the rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme have been developed in the last few years as a logical treatment strategy. Two classes of aromatase inhibitors, steroidal and nonsteroidal compounds, are now in use. Among the steroid substrate analogs, formestane and examestane have been shown to be effective in breast cancer patients with advanced disease. Highly potent and selective nonsteroidal inhibitors have recently been found to suppress plasma and urinary estrogens by more than 95% in breast cancer patients. Two of these compounds recently were approved in the United States and have been shown to be more effective than other second-line agents in terms of overall response rates and treatment failure, as well as better tolerated. Although studies of the efficacy of these agents in earlier stage disease are awaited, it is evident that aromatase inhibitors can extend the duration of treatment in breast cancer patients.

Aromatase and its inhibitors

Inhibitors of aromatase (estrogen synthetase) have been developed as treatment for postmenopausal breast cancer. Both steroidal substrate analogs, type I inhibitors, which inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now available. 4-hydroxyandrostenedione (4-OHA), the first selective aromatase inhibitor, has been shown to reduce serum estrogen concentrations and cause complete and partial responses in approximately 25% of patients with hormone responsive disease who have relapsed from previous endocrine treatment. Letrozole (CGS 20, 269) and anastrozole (ZN 1033) have been recently approved for treatment. Both suppress serum estrogen levels to the limit of assay detection. Letrozole has been shown to be significantly superior to megace in overall response rates and time to treatment failure, whereas anastrozole was found to improve survival in comparison to megace. Both were better tolerated than the latter. The potential of aromatase within the breast as a significant source of estrogen mediating tumor proliferation and which might determine the outcome of inhibitor treatment was explored. Using immunocytochemistry and in situ hybridization, aromatase and mRNAarom was detected mainly in the epithelial cells of the terminal ductal lobular units (TDLU) of the normal breast and also in breast tumor epithelial cells as well as some stromal cells. Increase in proliferation, measured by increased thymidine incorporation into DNA and by PCNA immunostaining in response to testosterone was observed in histocultures of breast cancer samples. This effect could be inhibited by 4-OHA and implies that intratumoral aromatase has functional significance. An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. This model also allows evaluation of the efficacy of aromatase inhibitors and antiestrogens in tumors of estrogen receptor positive, human breast carcinoma cells transfected with the human aromatase gene. Thus, the cells synthesized estrogen which stimulated tumor formation. Both aromatase inhibitors and antiestrogens were effective in suppressing tumor growth in this model. However, letrozole was more effective than tamoxifen. When the aromatase inhibitors were combined with tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Thus, there was no additive or synergistic effects of combining tamoxifen with aromatase inhibitors. This suggests that sequential treatment with these agents is likely to be more beneficial to the patient in terms of longer response to treatment.

Comprehensive pharmacology and clinical efficacy of aromatase inhibitors.

The goal of hormone therapy is to deprive breast tumours of estrogens, since estrogens have been implicated in the development or progression of tumours. This can be accomplished by the use of antiestrogens that block estrogen action or by inhibiting aromatase, the enzyme that catalyses the final and rate-limiting step in estrogen biosynthesis.A number of steroidal and nonsteroidal compounds have been developed as aromatase inhibitors. This review highlights the valuable role that a few of these aromatase inhibitors have played, and continue to play, in the treatment of breast cancer. Following background information regarding the biochemistry of aromatase, the rationale for its inhibition, and an outline of the test systems for evaluating and characterising aromatase inhibitors, the discussion focuses on the new generation of aromatase inhibitors that are in clinical trials or clinically available Specifically, it discusses the pharmacology and clinical efficacy of formestane, exemestane, rogletimide, fadrozole, vorozole, anastrozole and letrozole.The role of these agents as the optimal second-line agents (after tamoxifen) for the treatment of advanced breast cancer has been established; their prospects in other clinical settings and as potential breast cancer chemopreventives are warranted but are yet to be fully determined.

Aromatase inhibitor development and hormone therapy: a perspective.

The introduction of aromatase inhibitors as a new class of agents represents a further step in improving breast cancer treatment and possibly in preventing this disease. Although these agents are now used in the first- and second-line treatment of postmenopausal breast cancer, the heterogeneity of patients enrolled in clinical trials prevents a thorough assessment of the effectiveness of potential sequential and combination therapies. Such investigations are more easily performed in the laboratory, and to this end, a tumor model in nude mice was established to simulate several aspects of the postmenopausal breast cancer patient. This model showed that aromatase inhibitors are more efficient than tamoxifen at reducing tumor volume. Additionally, the combination of an aromatase inhibitor plus tamoxifen does not improve the antiproliferative results obtained with the aromatase inhibitor alone, a finding corroborated in the Arimidex, Tamoxifen Alone or in Combination adjuvant clinical trial. To investigate the effect that potential sequences of treatment have on tumor growth, letrozole was administered in sequence with tamoxifen to nude mice bearing human xenografts. Tumor growth was significantly reduced with the sequence compared with tamoxifen alone. Additionally, when agents were alternated every 4 weeks, mice started on letrozole fared better than those started on tamoxifen. Finally, letrozole alone provided the best and most sustained reduction in tumor growth. These experiments suggest the means to evaluate therapeutic combinations in the laboratory to guide potential trial designs and provide the best chance of success to the patients who enter these clinical trials.

Complete IGF Signaling Blockade by the Dual-Kinase Inhibitor, BMS-754807, Is Sufficient To Overcome Tamoxifen and Letrozole Resistance In Vitro and In Vivo.

Resistance to hormonal therapy is a clinically unmet need in breast cancer. IGF signaling has been identified as a major mechanism of resistance to hormonal therapy in breast cancer. As components of the IGF signaling pathway are expressed in most breast cancers, the development of IGF-1R monoclonal antibody (mAb) and tyrosine kinase inhibitors (TKI) are active areas of clinical investigations. A key distinction between the mAb and TKIs are their differences in their ability to inhibit the Insulin Receptor (InsR). While targeting the InsR with TKIs may have a theoretical liability of hyperglycemia, targeting only the IGF-1R may have the theoretical liability of incompletely blocking IGF signaling. As InsR isoform A expression, which can transduce IGF-II-mediated proliferation, is higher in breast cancers compared to normal breast tissue, we investigated whether IGF-1R or IGF-1R/InsR inhibition was sufficient for overcoming resistance to hormonal therapy. To determine the optimal combination strategies for clinical investigations, we tested the hypothesis that IGF signaling inhibition could overcome primary (or de novo/intrinsic) and secondary (or acquired/selected) resistance to hormonal therapy. For these studies, we used either hormone therapy-naive or hormone therapy-resistant variants of the breast cancer model, MCF-7/AC-1, which has been engineered to stably express full-length human aromatase. We employed and compared a novel, potent dual kinase inhibitor of the IGF-1R and InsR, BMS-754807, which is currently in early clinical investigations, with the IGF-1R antibody mAb391. BMS-754807 has been shown to induce apoptosis more potently than mAb391 in Rh41 human rhabdomyosarcoma cells. In vitro, BMS-754807 demonstrated profound synergy in combination with tamoxifen and letrozole (median effect combination index In vivo , BMS-754807 enhanced the anti-tumor activity of tamoxifen and letrozole in hormone-naive tumors and induced regression of tumors resistant to tamoxifen or letrozole when combined with letrozole. This activity was not observed with mAb therapy, which resulted in greater up-regulation of InsR-A and erbB receptor expression and activation. This suggested a greater susceptibility to resistance pathways with mAb therapy. Dual IGF-1R/InsR blockade alone or in combination was tolerated by the animals and has no significant change in glucose homeostasis. Gene expression profiling experiments to compare the difference between the effects of tamoxifen in combination with BMS-754807 and with mAb revealed alternative pathway signaling is one of the potential mechanisms of resistance.In summary, combined hormonal therapy with BMS-754807 overcomes primary and secondary resistance to tamoxifen and letrozole and was well tolerated. IGF-1R blockade with a mAb alone is insufficient to overcome resistance and induces InsR over-expression. Thus, IGF signaling through either InsR or IGF-1R may be a major mechanism of resistance to hormonal therapy. These data suggest that blockade of IGF-1 and IGF-II from activation of IGF-1R and InsR, with agents such as BMS-754807 have promise in extending the benefits of hormonal therapy in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 402.

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 2053: Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer

Estrogen receptor-positive (HR+) breast cancer is the most common form of breast cancer that is accountable for the majority of breast cancer mortality. Currently, the cyclin dependent kinase inhibitor palbociclib in combination with endocrine therapy represents the new standard first and second line therapy for patients with metastatic HR+ breast cancer. While palbociclib has been shown to significantly improved progression free survival in combination with aromatase inhibitor (AI) and fulvestrant, resistance will inevitably occur in patients with metastatic breast cancer. However, the mechanisms of resistance to the combination of palbociclib and AI remain largely unknown. It is also unclear whether resistance mechanisms would be the same if palbociclib was given as first line treatment in combination with letrozole vs. if palbociclib was given as second line treatment after letrozole resistance had already occurred. To address this, previously well established and well characterizied letrozole-sensitive MCF7Ca cells and letrozole-resistant LTLTCa cells were subjected to continuous, long term treatment with increasing doses of letrozole and palbociclib until resistance to both drugs was achieved (MCF7Calet+palb and LTLTCapalb, respectively). Preliminary MTT cell viability assays indicate palbociclib IC50s of 750 nM in MCF7Ca and LTLTCa cells vs. 21 uM in palbociclib and letrozole-resistant cells. First line and second line palbociclib resistance correlated with changes in morphology, protein expression, and cancer stem cell characteristics. Under phase contrast microscopy, first line and second line palbociclib-resistant MCF7Ca and LTLTCa cells were larger in size, more irregular in shape, and tended not to grow in epithelial cell-like groups compared to palbociclib-sensitive MCF7Ca and letrozole cells, with second-line palbociclib resistant cells exhibiting these characteristics the most. Western blot showed that ER protein expression in ER+/HER2- MCF7Ca cells decreased with first line palbociclib and letrozole resistance, and that both ER and HER2 protein expression were decreased with second line palbociclib resistance in ERlow/HER2+ LTLTCa cells. Lastly, mammosphere assays demonstrated increasing percentage of cancer stem cells with letrozole resistance alone (4 per 1000 cells plated MCF7Ca vs. 58 per 1000 planted LTLTCa) and with palbociclib and palbociclib resistance (317 per 1000 cells plated MCF7Ca and 202 per 1000 cells plated LTLTCa cells). Overall, these results indicate that 1) resistance to palbociclib, whether as first line or second line treatment has significant effects on breast cancer cells that may be relevant to patient diagnosis and treatment. Citation Format: Armina A. Kazi, Antony Sare, Saranya Chumsri, Angela Brodie. Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer [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 2053. doi:10.1158/1538-7445.AM2017-2053

Abstract 1062: Overexpression of miRNAs 181a and 222 play a role in triple negative breast cancer, and are targeted by entinostat

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer in women. Clinically, this subtype is characterized by high recurrence rates, poor prognosis, and lack of targeted therapies. Therefore, there is considerable need to identify TNBC-specific biomarkers that can serve as a diagnositc markers and therapeutic targets. MIcroRNAs (miRNAs), may be such biomarkers. 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, they 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 there is not consistent agreement as to which miRNAs are involved in TNBCs, nor have molecular targeting drugs been identified that could treat TNBCs by affecting miRNAs. Previous studies by our lab and others indicate that miRNAs 181a and 222 are involved in estrogen-receptor independence, cancer stem cells, and drug resistance (ex. letrozole resistance). Thus, in this study, the expression of miRNAs 181 and 222 in TNBCs, the effect of inhibiting each miRNA on cell viability and cancer stem cells, and the effect of histone deactylase inhibitor entinostat on miRNA expression are explored. RT-PCR analysis of miRNA expression in both HS578T and BT547 TNBCs and MCF-7 cells (represents the least aggressive subtype), and in representative breast cancer patient biopsy samples indicates that both miRNA 181a and 222 are upregulated by at least 15-fold in TNBCs compared to MCF-7 (least aggressive subtype). Specific inhibition of miRNA 181a via siRNA/miRNA inhibitor in HS578T cells significantly decreased cell viability by at least 80%, as determined by MTT assay, and cancer stem cells by 50%, as determined by mammosphere assays. In addition, inhibition of miRNA 181a produced morphological changes in HS578T cells, in which cells lost their protrusions, became more round, and grew in colonies. Lastly, treatment of HS578T cells or of patient derived xenografts with entinostat, which is currently being explored as a TNBC-targeting drug, decreased miRNA 181a and 222 expression and produced similar results as miRNA inhibition on cell viability, cancer stem cells, and morphology. Overall, these results suggest that miRNAs 181a and 222 are overexpressed in TNBCs and may play a role in regulation of cell viability and cancer stem cells. They also indicate that HDAC inhibitor, entinostat, may be an effective treatment for TNBCs through their action on miRNAs 181a and 222. Citation Format: Armina A. Kazi, Alexa Giammarino, Nicholas Musacchio, Gauri Sabnis, Amanda Schech, Angela Brodie. Overexpression of miRNAs 181a and 222 play a role in triple negative breast cancer, and are targeted by entinostat. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1062.

In Vivo Models of AI Resistance

The goal of endocrine therapy is to deprive the breast tumor of estrogens, which are key to the growth and progression of the tumor. This can be accomplished by blocking the receptor action via antiestrogens or blocking the biosynthesis of estrogen using aromatase inhibitors. Model systems have been devised to study the effectiveness of hormonal therapy on breast tumor growth. These models proved essential in bringing endocrine agents to the forefront of breast cancer therapy. Now, these models are being exploited to develop strategies to overcome resistance to endocrine agents. Cell lines or tumor xenografts that are deprived of estrogen or treated extensively with aromatase inhibitors serve as models of breast cancers of patients that are receiving AIs in the clinical setting.

Abstract P1-07-01: Histone deacetylase inhibitor entinostat reverses epithelial to mesenchymal transition of breast cancer cells by reversing the repression of E-cadherin

Loss of ERa in breast cancer correlates with poor prognosis, increased recurrence rates and higher incidence of metastasis. In our previous studies, we have shown that histone deacetylase (HDAC) inhibitor entinostat (ENT) can upregulate ERα and aromatase in ER-negative cells and tumors, making them sensitive to aromatase inhibitors (AIs). In the current study, we are showing that ENT can also reverse epithelial to mesenchymal transition (EMT), which is considered to be a first step in the process of metastases formation. EMT is characterized by loss of intracellular adhesion (loss of E-cadherin); loss of epithelial markers such as cytokeratins and upregulation of mesenchymal markers such as vimentin; acquisition of fibroblast-like spindle morphology and increased motility. Various carcinomas undergo varying degrees of EMT and capacity to undergo EMT correlates inversely with levels of E-Cadherin. It is widely accepted that loss of E-cadherin is associated with more invasive phenotype. Epigenetic silencing of E-cadherin has been implicated in metastatic cell lines and invasive breast cancers. Triple negative breast cancer cells such as MDA-MB-231 and Hs578T show a basal phenotype characterized by loss of E-cadherin expression and higher expression of mesenchymal markers such as N-cadherin, vimentin along with transcriptional repressors such as twist and snail. In this study, we measured the effect of entinostat on the EMT. When MDA-MB-231 and Hs578T cells were treated with ENT, E-cadherin transcription was increased along with reduction in N-cadherin mRNA expression. Similar results were also seen in tumors of MDA-MB-231 and Hs578T xenografts treated with ENT (for 5 weeks and 2 weeks respectively). A dose dependent increase in E-cadherin was seen along with a dose dependent decrease in N-cadherin mRNA. Although, we did not observe any reduction in vimentin protein, phosphorylation of vimentin was increased and vimentin remodeling was changed as seen by immunofluorescence. We performed chromatin immunoprecipitation (ChIP) assay to measure the activation of E-cadherin promoter. Treatment of MDA-MB-231 and Hs578T cells increased the activation of E-cadherin promoter as seen by increased acetyl histones at the promoter region of E-cadherin. Twist and snail are known repressors of E-cadherin gene and we saw that ENT treatment reduced the association of twist and snail with the E-cadherin promoter. ENT was also able to downregulate twist, which may be responsible for reduced twist association with the E-cadherin promoter. In summary, these findings suggest that HDAC inhibitor ENT can reverse EMT and may help reduce the formation of metastasis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-01.