Letrozole-resistant Tumors Research Articles

Glyceollins Trigger Anti-Proliferative Effects in Hormone-Dependent Aromatase-Inhibitor-Resistant Breast Cancer Cells through the Induction of Apoptosis.

Aromatase inhibitors (AIs) are standard treatment for estrogen-dependent postmenopausal breast tumors; however, resistance develops leading to tumor relapse and metastasis. We previously demonstrated that glyceollin inhibits proliferation, survival, and migration of hormone-independent letrozole-resistant breast cancer. Since many AI-resistant tumors remain hormone-dependent, identifying distinctions between estrogen-receptor-positive (ER+) and ER-negative (ER-) AI-resistant tumor response to therapy is critical. We hypothesize that treating ER+ letrozole-resistant T47D breast cancer cells (T47DaromLR) with a combination of 10 μM glyceollin and 0.5 μM lapatinib (a dual EGFR/HER2 inhibitor) will decrease cell proliferation through induction of apoptosis. The T47DaromLR cells were found to overexpress HER2 and MAPK while maintaining aromatase and ER levels compared to their letrozole-sensitive (T47Darom) counterparts. In the absence of estrogen stimulation, glyceollin ± lapatinib had no effect on the proliferation of the T47Darom cells, while glyceollin treatment caused 46% reduction in the proliferation of T47DaromLR cells, which was further diminished when combined with lapatinib. While neither agent influenced cell migration, glyceollin and lapatinib reduced S and G2/M phase cell entry and exclusively induced apoptosis by 1.29-fold in the T47DaromLR cells. Taken together, these results suggest that glyceollins and lapatinib may have potential as a novel combination therapeutic approach for hormone-dependent, letrozole-resistant tumors.

ER+ Breast Cancers Resistant to Prolonged Neoadjuvant Letrozole Exhibit an E2F4 Transcriptional Program Sensitive to CDK4/6 Inhibitors.

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancers treated with prolonged neoadjuvant letrozole.Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER+ breast cancers from patients treated with preoperative letrozole (median, 7 months).Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P = 2.56E-15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER+ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER+ tumors in METABRIC.Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER+ breast cancer cells and in patients' ER+ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517-29. ©2018 AACR.

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

Abstract 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&amp;lt;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&amp;lt;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.

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 (Δ(4)A), the aromatizable substrate. When the tumors reached about 300 mm(3), 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.

Abstract 1314: HDAC inhibitor entinostat reverses letrozole resistance in MCF-7Ca xenografts through modulation of Her-2.

Abstract Development of aromatase inhibitors (AIs) has significantly improved the treatment outcome of hormone responsive post-menopausal breast cancer. However, not all tumors respond initially and others eventually acquire resistance. Using our hormone sensitive human aromatase expressing MCF-7Ca xenograft model, we also saw that although AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. Furthermore, we observed that Her-2 protein levels within the tumors were upregulated as early as 4 weeks (as tumors were responding). Her-2 levels increased further as the tumors became resistant. A cell line was isolated from these Long-Term Letrozole Treated tumors (LTLT-Ca), which showed increased Her-2 and reduction in ERα and aromatase. The increase in Her-2 protein levels was not accompanied by gene amplification but we observed that Her-2 protein in LTLT-Ca cells was more stable and had a longer half-life than that in MCF-7Ca cells. In addition, when MCF-7Ca cells were deprived of estrogen (E2W) or treated with letrozole, stability of Her-2 protein was immediately increased, suggesting that lower estrogen levels stabilize Her-2. In order to identify novel approaches to restoring sensitivity to AIs, we treated letrozole resistant MCF-7Ca tumors with histone deacetylase (HDAC) inhibitor entinostat (ENT). Addition of ENT to either letrozole or exemestane in letrozole resistant MCF-7Ca tumors led to increased ERα expression, aromatase activity and significantly inhibited further tumor growth, along with downregulation of Her-2, p-Her-2, p-MAPK and p-Akt. Based on the observations made in letrozole resistant tumors treated with entinostat (with or without letrozole), we hypothesized that entinostat reverses letrozole resistance due to modulation of Her-2. Entinostat reduced interaction between HSP-90 and Her-2 in entinostat treated tumors, resulting in degradation of Her-2. Based on the findings that Her-2 is upregulated even when the tumors are responding, we evaluated the effect of adding entinostat to letrozole from the start of treatment in the MCF-7Ca xenografts. Tumors of MCF-7Ca cells were allowed to form in the presence of androstenedione (Δ4A), aromatizable substrate for estrogen. When the tumors reached ∼300mm3, the mice were grouped to receive Δ4A 100μg/day (control), letrozole (1μg/day), entinostat (50μg/day) or combination of entinostat plus letrozole. Mice receiving letrozole treatment had significantly lower tumor growth rate than control (p=0.0001), so did mice treated with the combination of entinostat plus letrozole (p&amp;lt;0.0001). The growth rate of tumors treated with entinostat + letrozole was similar to the growth rate of letrozole alone suggesting that in the absence of acquired or de novo resistance pathways the addition of entinostat did not provide benefit beyond the aromatase inhibitor alone. Citation Format: Gauri J. Sabnis, Armina Kazi, Olga Goloubeva, Saranya Chumsri, Angela Brodie. HDAC inhibitor entinostat reverses letrozole resistance in MCF-7Ca xenografts through modulation of Her-2. [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 1314. doi:10.1158/1538-7445.AM2013-1314

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

Abstract 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 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&amp;lt;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&amp;lt;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&amp;lt;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

Hyaluronic Acid-Bound Letrozole Nanoparticles Restore Sensitivity to Letrozole-Resistant Xenograft Tumors in Mice

Letrozole is a potent aromatase inhibitor and superior to other defined selective estrogen receptor modulators such as tamoxifen in treating hormone-responsive postmenopausal breast cancer patients. Patients who receive this drug may become insensitive to the effects of estrogen deprivation induced by letrozole. Letrozole has known side effects on bone metabolism due to systemic ablation of estrogen production. The purpose of this study was to examine the therapeutic efficacy of hyaluronic acid-bound letrozole nanoparticles (HA-Letr-NPs) in restoring sensitivity to letrozole-resistant (LTLT-Ca) cells. To target letrozole to LTLT-Ca cells, hyaluronic acid-bound letrozole nanoparticles were prepared by nanoprecipitation using biodegradable PLGA-PEG co-polymer. Binding specificity of HA to CD44 on the cell surface was analyzed in vitro using FITC-CD44 Ab and CD44 siRNA by flow cytometry. Effects on in vitro cytotoxicity and aromatase enzymatic activity of HA-Letr-NPs were performed in MCF-7 breast cancer cells, MCF-7 cells over-expressing aromatase (MCF-7/Aro), and LTLT-Ca cells resistant to letrozole. Preclinical efficacy of HA-Letr-NPs was examined in mice using LTLT-Ca xenograft tumors. HA-Letr-NPs were restricted to a maximum size of 100 nm. The in vitro drug release assay showed that the highest released concentration of letrozole occurred after 23 hours at 37 degrees C in phosphate-buffered saline. HA-Letr-NPs on MCF-7/Aro and LTLT-Ca cells showed an IC50 of 2 microM and 5 microM, respectively. HA-Letr-NPs were more efficacious in inhibiting tumor growth, reducing in vitro cellular and in vivo tumor aromatase enzyme activity more than the corresponding Letr-NPs or letrozole. HA-Letr-NPs restored and maintained a prolonged sensitivity and targeted delivery of letrozole in letrozole-resistant tumors in vivo.

Abstract 2295: Regulation of Her-2 in long-term letrozole resistant cells by HSP-90

Abstract 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. Using our MCF-7Ca xenograft model, we also observed that although, AI letrozole provides a longer control over tumor growth than tamoxifen, tumors eventually began to grow. A cell line was isolated from these Long-Term Letrozole Treated tumors (LTLT-Ca). These cells and had lower expression of ER and aromatase compared to parental MCF-7Ca cells. On the other hand, Her-2/MAPK pathway was upregulated. Inhibition of Her-2 using trastuzumab resulted in reversal of resistance and restoration of sensitivity to estrogens, antiestrogens and AIs, Histone deacetylase (HDAC) inhibitor, entinostat also reversed AI resistance. Similarly, stopping letrozole treatment also resulted in reversal of letrozole resistance. In all these studies, we found that downregulation of Her-2 was associated with reversal of letrozole resistance. In order to determine the mechanism of regulation of Her-2 in LTLT-Ca cells, we compared parental MCF-7Ca cells to LTLT-Ca for basal levels of Her-2 protein, protein stability, mRNA and mRNA stability. Cell lysates or mRNA were then collected at various time points after trating with Actinomycin D to inhibit RNA synthesis and cycloheximide to inhibit translation. Although, our previous data shows that letrozole resistant tumors do not have amplification of the Her-2 gene (FISH). LTLT-Ca cells have higher basal level of Her-2 protein and mRNA (4.5-fold higher mRNA; p&amp;lt;0.001). We also found that Her-2 protein in LTLT-Ca cells is more stable and has a longer half-life than MCF-7Ca cells. However, Her-2 mRNA in LTLT-Ca did not have a longer half-life. In LTLT-Ca cells, more association between Her-2 and HSP-90 was observed compared to parental MCF-7Ca cells using IP. When hormone sensitive MCF-7Ca cells were placed in estrogen-deprived conditions, there was increased association of Her-2 with HSP-90. Stability of Her-2 protein was regulated by HSP-90. Inhibition of HSP-90 with HSP-90 inhibitor 17-DMAG downregulated Her-2 and also reduced the half-life of Her-2. Furthermore, 17-DMAG also effectively reduced viability of parental MCF-7Ca and LTLT-Ca cells (in 72 hours) with IC50 of 2.5μM and 1.7μM respectively. In addition, combination of letrozole with HSP-90 inhibitors such as 17-DMAG, HSP-990 and AUY9122 inhibited the growth of both LTLT-Ca and MCF-7Ca cells significantly better than single agent alone. Our results indicate that HSP-90 plays a role in regulation of Her-2 protein, which modulates resistance to letrozole. This suggests use of HSP-90 inhibitors may inhibit the upregulation of Her-2 and thereby overcome subsequent resistance to aromatase inhibitors. 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 2295. doi:10.1158/1538-7445.AM2011-2295

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

Abstract 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&amp;lt;0.05). Tumors were collected at autopsy and analyzed. ENT was able to increase ERα expression and aromatase activity in the letrozole resistant tumors. In addition, ENT also downregulated Her-2, p-Her-2, p-MAPK and p-Akt. 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.

Aromatase Inhibitors and Breast Cancer

We have developed a breast cancer intratumoral aromatase model to simulate the postmenopausal breast cancer patient in order to compare the antitumor efficacy of aromatase inhibitors (AIs) and antiestrogens (AEs). The AI letrozole sustained growth inhibition longer than the AE tamoxifen. Nevertheless, eventually tumors began to grow despite continued treatment. Estrogen receptor-alpha (ER-alpha) levels decreased below control levels concomitant with increased phosphorylation of ER-alpha and unaltered progesterone receptor (PgR) levels. Expression of Her-2, p-Shc, Grb-2, p-Raf, p-Mekl/2, and p-MAPK was increased in the letrozole-resistant tumors. When cells isolated from letrozole-resistant tumors (LTLTCa cells) were treated with inhibitors of the Her-2 signaling pathway, such as trastuzumab (herceptin), ER-alpha was restored. Furthermore, sensitivity of LTLTCa cells to AIs and AEs was regained. These findings suggest cross-talk between ER and Her-2 signaling. To prevent activation of the Her-2 pathway and resistance to AIs, mice were treated with a combination of an AI anastrozole and the ER downregulator fulvestrant. This resulted in no increase in Her-2 and p-MAPK levels, and tumor growth was significantly inhibited. Thus, blocking both ER and Her-2 signaling delayed development of resistance to AIs. This hypothesis was supported by the finding that growth of letrozole-resistant tumors was reduced when xenografts were treated with trastuzumab combined with letrozole. In addition, resistance to letrozole could be reversed by discontinuing letrozole. Our findings indicate that after letrozole treatment is stopped, the antitumor effect of letrozole can be restored when the AI treatment is resumed.

Preclinical modeling of endocrine response: a combination therapy approach with the ERβ agonist, diarryl propionitrile and letrozole restores sensitivity to letrozole-resistant breast cancer cells.

Abstract Abstract #17 The majority of breast cancer patients are postmenopausal women with estrogen receptor-positive (ER+) tumors. Approximately 66% of breast carcinomas contain aromatase. Aromatase inhibitors are proving to be more effective than tamoxifen in the treatment of postmenopausal women with ER+ breast cancer. However, the development of resistance to treatment is a concern. The transition of the ER+ tumors from a responsive to unresponsive state is associated with different molecular pathways. We have therefore investigated the properties of resistance that develops in response to the aromatase inhibitor letrozole in the preclinical MCF-7-aromatase xenograft animal model following long-term treatment with letrozole by using the derived letrozole-resistant MCF-7-aromatase LTLTca breast cancer cells. The combination treatment of letrozole and the ERβ agonist diarryl propionitrile (DPN) restored sensitivity to letrozole in the letrozole-resistant LTLTca cells. Studies from our group using this postmenopausal breast cancer preclinical model clearly showed the therapeutic advantage of the ERβ agonist in overcoming unresponsiveness to letrozole. A 67% reduction in tumour size was observed in the group of animals bearing the letrozole-resistant LTLTca tumors that received the combination of letrozole and DPN. A novel delivery method using letrozole-DPN nanoparticles showed enhanced sensitivity (∼80%) to the combination nanoparticles. Letrozole was found to be relatively ineffective in these animals with only a 36% reduction in tumor size with tumors having a mean tumor weight of 0.95g by the end of four weeks of treatment. Biochemical analysis showed a reduction in the tumor aromatase enzyme levels in mice treated with letrozole + DPN when compared to letrozole alone. QRT-PCR as well as protein analysis studies of the letrozole-resistant LTLTca tumors showed a decrease in P27 levels and elevated expression levels of HER-2. Expression levels of cyclin D1 and the ERα to ERb ratio was higher in the untreated letrozole-resistant tumors compared to tumors that responded to the combination therapy. These findings provide further support for the development and testing of novel therapeutic approaches for selective regulation of ER-dependent (ERα and β) actions. Combination treatments involving agents that modulate ERβ activity may provide therapeutic advantage in the treatment, for overcoming resistance to estrogen-ablation and prevention of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 17.

Preclinical modeling of endocrine response and resistance

The authors developed a breast cancer intratumoral aromatase model system to compare the antitumor efficacy of several aromatase inhibitors (AIs) and antiestrogens (AEs). Although the AI letrozole caused sustained growth inhibition, tumors eventually began to grow, even when treatment was maintained. For the current study, the mechanisms of resistance to letrozole during the course of treatment were investigated. Estrogen receptor alpha (ER-alpha) levels decreased below control levels in letrozole-resistant tumors. The decrease was simultaneous to an increase in phosphorylation of ER-alpha and an unaltered expression of progesterone receptor (PgR). Expression levels of HER-2, activated (phosphorylated) SHC-adaptor protein (p-Shc), growth factor receptor-bound protein 2 (Grb-2), p-Raf, phosphorylated mitogen-activated protein kinase kinase 1/2 (p-Mekl/2), and phosphorylated mitogen-activated protein kinase (p-MAPK) were increased. When cells isolated from letrozole-resistant tumors (LTLTCa cells) were treated with inhibitors of the HER-2 signaling pathway, ER-alpha expression and estradiol-stimulated transactivation was restored. The HER-2 blocker trastuzumab also restored the sensitivity of LTLTCa cells to AIs and AEs. These findings suggested that there is crosstalk between ER and HER-2 signaling. To prevent activation of the HER-2 pathway and resistance to AIs, mice were treated with a combination of AIs and the ER down-regulator fulvestrant. There was no increase in HER-2 or p-MAPK expression, and tumor growth was inhibited significantly. When trastuzumab was added to unresponsive tumors under letrozole treatment, it significantly inhibited tumors growth compared with switching to trastuzumab alone. However, the trastuzumab plus letrozole combination was more effective than letrozole alone only in refractory breast tumors. These results suggested that blocking both ER and HER-2 signaling may delay the development of resistance to AIs in patients with recurrent breast cancer.

Predicting response and resistance to endocrine therapy

Selection for endocrine therapy requires the identification of markers that accurately predict response/resistance. In this report, the authors review their published work and abstract results from an unpublished study to illustrate the potential of RNA microarrays from sequential tumor biopsies from patients who were offered neoadjuvant endocrine therapy treatment to identify the molecular signatures associated with tumor sensitivity/resistance. Clinical response was assessed by serial ultrasound measurements in postmenopausal women with large, primary, estrogen receptor-rich breast cancers who received neoadjuvant treatment with letrozole for 3 months. Tumor RNA from biopsies that were taken before and after 14 days of treatment was hybridized on Affymetrix U133A chips to determine expression profiles. Classic estrogen-dependent genes and markers of proliferation were changed with treatment in most tumors but were poorly associated with clinical response (they frequently were changed in letrozole-resistant tumors). Differential expression patterns could be used to identify heterogeneity in clinically resistant tumors. The results indicated that molecular profiling of early changes with letrozole treatment offers the opportunity to distinguish between clinically responsive and nonresponsive tumors and provides important information about the heterogeneity of endocrine resistance.

Xenograft models for aromatase inhibitor studies

As several aromatase inhibitors are now available for treating breast cancer, we developed a model system to compare their antitumor efficacy and to explore strategies for their optimal use. Tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca) and can therefore synthesize as well as respond to estrogen. Results from this model have been predictive of clinical outcome. Thus, inhibiting estrogen action and estrogen synthesis by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen in combination did not result in synergy. Moreover, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in response to sequential letrozole treatment. However, our findings indicate that letrozole alone was better than all other treatments. Although letrozole resulted in long sustained growth inhibition, tumors eventually grew despite continued treatment. Mechanisms of resistance to letrozole were investigated during the course of treatment. ER was initially upregulated in responding tumors, but subsequently decreased below control levels in tumors no longer responsive to letrozole. Her-2 as well as adapter proteins (p-Shc and Grb-2) and signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), were all increased in letrozole resistant tumors. In LTLT cells, isolated from the letrozole resistant tumors and treated with inhibitors of the MAPKinase pathway, MAPK activity was decreased and ER expression restored to control levels. Inhibitors of EGFR/Her-2 also restored the sensitivity of LTLT cells to letrozole. These results suggest that crosstalk occurs between ER and tyrosine kinase receptor signaling. Therefore, to investigate whether down-regulating ER would prevent activation of MAPK and resistance to letrozole, xenografts were treated with letrozole and faslodex in combination. Her-2 and MAPK were not increased and tumor growth was inhibited throughout 29 weeks of treatment. These results suggest that blocking both ER and growth factor mediated transcription may delay development of resistance to letrozole and maintain its growth inhibition of breast cancer.

Aromatase and breast cancer

Several aromatase inhibitors and also new antiestrogens are now available for treating breast cancer. We have developed a model to compare the antitumor efficacy of these agents and to explore strategies for their optimal use. Results from the model have been predictive of clinical outcome. In this model, tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca). The possibility that blockade of estrogen action and estrogen synthesis may be synergistic was explored by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen alone and in combination. The results indicated that letrozole alone was better than all other treatments. In addition, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in mice switched to letrozole treatment. However, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors eventually adapt and grow during letrozole treatment, we determined the expression of signaling proteins in tumors during the course of letrozole treatment compared to the tumors of control mice. Tumors initially up-regulated the ER while responding to treatment, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Also, Her-2 and adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), but not in the Pl3/Akt pathway, were increased in tumors no longer responsive to letrozole. To investigate whether sensitivity to letrozole could be regained, cells were isolated from the letrozole resistant tumors (LTLT) and treated with inhibitors of the MAPKinase pathway (PD98059 and UO126). These compounds reduced MAPK activity and increased ER expression. EGFR/Her-2 inhibitors, gefitinib and AEE78S although not effective in the parental MCF-70a cells, restored the sensitivity of LTLT cells to letrozole. In xenografts, beginning treatment with letrozole and faslodex to down regulate the ER prevented increases in Her-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results suggest that blocking both ER- and growth factor-mediated transcription may delay development of resistance and maintain growth inhibition of ER+ breast cancer.

The intratumoral aromatase model: studies with aromatase inhibitors and antiestrogens

Aromatase inhibitors have now been approved as first-line treatment options for hormone-dependent advanced breast cancer. When compared to tamoxifen, these aromatase inhibitors provide significant survival and tolerability advantages. However, the optimal use of an aromatase inhibitor and tamoxifen remains to be established. To date, the intratumoral aromatase xenograft model has proved accurate in predicting the outcome of clinical trials. Utilizing this model, we performed long-term studies with tamoxifen and letrozole to determine time to disease progression with each of the treatment regimens. Aromatase-transfected MCF-7Ca human breast cancer cells were grown as tumor xenografts in female ovariectomized athymic nude mice in which androstenedione was converted to estrogen and stimulated tumor growth. When tumor volumes were approximately 300 mm 3, the animals were grouped for continued supplementation with androstenedione only (control) or for treatment with letrozole 10 μg per day (long-term), tamoxifen 100 μg per day (long-term), letrozole alternating to tamoxifen (4-week rotation), tamoxifen alternating to letrozole (4-week rotation), or a combination of the two drugs. Tumors of control mice had doubled in volume in 3–4 weeks. In mice treated with tamoxifen and the combination, tumor doubling time was significantly shorter (16 and 18 weeks, respectively) than with letrozole (34 weeks). Furthermore, alternating letrozole and tamoxifen treatment every 4 weeks was less effective than letrozole alone. Tumors doubled in 17–18 weeks when the starting treatment was tamoxifen and in 22 weeks when the starting treatment was letrozole. Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole (10 μg per day). However, when mice with letrozole-resistant tumors were switched to antiestrogen treatment, tumors did not respond to tamoxifen (100 μg per day) or faslodex (1 mg per day). This suggests that advanced breast cancers treated with letrozole may be insensitive to subsequent second-line hormonal agents. Thus, although letrozole was determined to be an effective second-line treatment option for tumors progressing on tamoxifen, antiestrogen therapy does not appear to be effective for tumors progressing on letrozole. However, response to second-line treatment was observed in a model where tumors that had progressed on letrozole were transplanted to new mice. These tumors had been allowed to grow in the presence of supplemented androstenedione but absence of letrozole. This suggests that resistance to letrozole may be reversible, allowing tumors to respond to subsequent antiestrogens and letrozole.