Nicholas C. D'Amato

Multiple Molecular Subtypes of Triple-Negative Breast Cancer Critically Rely on Androgen Receptor and Respond to Enzalutamide In Vivo

Triple-negative breast cancer (TNBC) has the lowest 5-year survival rate of invasive breast carcinomas, and currently there are no approved targeted therapies for this aggressive form of the disease. The androgen receptor (AR) is expressed in up to one third of TNBC and we find that all AR+ TNBC primary tumors tested display nuclear localization of AR, indicative of transcriptionally active receptors. While AR is most abundant in the “luminal AR (LAR)” molecular subtype of TNBC, here, for the first time, we use both the new-generation anti-androgen enzalutamide and AR knockdown to demonstrate that the other non-LAR molecular subtypes of TNBC are critically dependent on AR protein. Indeed, AR inhibition significantly reduces baseline proliferation, anchorage-independent growth, migration, and invasion and increases apoptosis in four TNBC lines (SUM159PT, HCC1806, BT549, and MDA-MB-231), representing three non-LAR TNBC molecular subtypes (mesenchymal-like, mesenchymal stem–like, and basal-like 2). In vivo, enzalutamide significantly decreases viability of SUM159PT and HCC1806 xenografts. Furthermore, mechanistic analysis reveals that AR activation upregulates secretion of the EGFR ligand amphiregulin (AREG), an effect abrogated by enzalutamide in vitro and in vivo. Exogenous AREG partially rescues the effects of AR knockdown on proliferation, migration, and invasion, demonstrating that upregulation of AREG is one mechanism by which AR influences tumorigenicity. Together, our findings indicate that non-LAR subtypes of TNBC are AR dependent and, moreover, that enzalutamide is a promising targeted therapy for multiple molecular subtypes of AR+ TNBC. Mol Cancer Ther; 14(3); 769–78. ©2015 AACR.

A TDO2-AhR Signaling Axis Facilitates Anoikis Resistance and Metastasis in Triple-Negative Breast Cancer

The ability of a cancer cell to develop resistance to anoikis, a programmed cell death process triggered by substratum detachment, is a critical step in the metastatic cascade. Triple-negative breast cancers (TNBC) exhibit higher rates of metastasis after diagnosis, relative to estrogen-positive breast cancers, but while TNBC cells are relatively more resistant to anoikis, the mechanisms involved are unclear. Through gene expression and metabolomic profiling of TNBC cells in forced suspension culture, we identified a molecular pathway critical for anchorage-independent cell survival. TNBC cells in suspension upregulated multiple genes in the kynurenine pathway of tryptophan catabolism, including the enzyme tryptophan 2,3-dioxygenase (TDO2), in an NF-κB-dependent manner. Kynurenine production mediated by TDO2 in TNBC cells was sufficient to activate aryl hydrocarbon receptor (AhR), an endogenous kynurenine receptor. Notably, pharmacologic inhibition or genetic attenuation of TDO2 or AhR increased cellular sensitivity to anoikis, and also reduced proliferation, migration, and invasion of TNBC cells. In vivo, TDO2 inhibitor-treated TNBC cells inhibited colonization of the lung, suggesting that TDO2 enhanced metastatic capacity. In clinical specimens of TNBC, elevated expression of TDO2 was associated with increased disease grade, estrogen receptor-negative status, and shorter overall survival. Our results define an NF-κB-regulated signaling axis that promotes anoikis resistance, suggest functional connections with inflammatory modulation by the kynurenine pathway, and highlight TDO2 as an attractive target for treatment of this aggressive breast cancer subtype.

Cooperative Dynamics of AR and ER Activity in Breast Cancer

Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha–positive (ER+) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared with dihydrotestosterone (DHT). Estradiol-induced AR-binding sites were enriched for estrogen response elements and had significant overlap with ER-binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER+/AR+ breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER+/AR+ patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Finally, in a comparison of ER+/AR+ primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide preclinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single-agent efficacy may be possible in tumors resistant to traditional endocrine therapy, as clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER. Implications: This study suggests that AR plays a previously unrecognized role in supporting E2-mediated ER activity in ER+/AR+ breast cancer cells, and that enzalutamide may be an effective therapeutic in ER+/AR+ breast cancers. Mol Cancer Res; 14(11); 1054–67. ©2016 AACR.

Androgen Receptor Supports an Anchorage-Independent, Cancer Stem Cell-like Population in Triple-Negative Breast Cancer

Preclinical and early clinical trials indicate that up to 50% of triple-negative breast cancers (TNBC) express androgen receptor (AR) and are potentially responsive to antiandrogens. However, the function of AR in TNBC and the mechanisms by which AR-targeted therapy reduces tumor burden are largely unknown. We hypothesized that AR maintains a cancer stem cell (CSC)-like tumor-initiating population and serves as an antiapoptotic factor, facilitating anchorage independence and metastasis. AR levels increased in TNBC cells grown in forced suspension culture compared with those in attached conditions, and cells that expressed AR resisted detachment-induced apoptosis. Culturing TNBC cells in suspension increased the CSC-like population, an effect reversed by AR inhibition. Pretreatment with enzalutamide (Enza) decreased the tumor-initiating capacity of TNBC cells and reduced tumor volume and viability when administered simultaneously or subsequent to the chemotherapeutic paclitaxel; simultaneous treatment more effectively suppressed tumor recurrence. Overall, our findings suggest that AR-targeted therapies may enhance the efficacy of chemotherapy even in TNBCs with low AR expression by targeting a CSC-like cell population with anchorage independence and invasive potential. Cancer Res; 77(13); 3455-66. ©2017 AACR.

Synergy between Androgen Receptor Antagonism and Inhibition of mTOR and HER2 in Breast Cancer

The androgen receptor (AR) is widely expressed in breast cancer, and evidence suggests dependence on AR signaling for growth and survival. AR antagonists such as enzalutamide and seviteronel have shown success in preclinical models and clinical trials of prostate cancer and are currently being evaluated in breast cancer. Reciprocal regulation between AR and the HER2/PI3K/mTOR pathway may contribute to resistance to HER2- and mTOR-targeted therapies; thus, dual inhibition of these pathways may synergistically inhibit breast cancer growth. HER2+ and triple-negative breast cancer cell lines were treated with AR antagonist plus anti-HER2 mAb trastuzumab or mTOR inhibitor everolimus. Apoptosis, cell proliferation, and drug synergy were measured in vitro. Pathway component genes and proteins were measured by qRT-PCR, Western blot, and reverse phase protein array. In vivo, HER2+ breast cancer xenografts were treated with enzalutamide, everolimus, trastuzumab, and combinations of these drugs. AR antagonists inhibited proliferation of both HER2+ and TNBC cell lines. Combining AR antagonist and either everolimus or trastuzumab resulted in synergistic inhibition of proliferation. Dihydrotestosterone caused increased phosphorylation of HER2 and/or HER3 that was attenuated by AR inhibition. Everolimus caused an increase in total AR, phosphorylation of HER2 and/or HER3, and these effects were abrogated by enzalutamide. Growth of trastuzumab-resistant HER2+ xenograft tumors was inhibited by enzalutamide, and combining enzalutamide with everolimus decreased tumor viability more than either single agent. AR antagonists synergize with FDA-approved breast cancer therapies such as everolimus and trastuzumab through distinct mechanisms. Treatment combinations are effective in trastuzumab-resistant HER2+ breast cancer cells in vivo. Mol Cancer Ther; 16(7); 1389–400. ©2017 AACR.

Abstract P2-14-02: Preclinical Evaluation of Enzalutamide in Breast Cancer Models

Background: The vast majority of breast cancers express the androgen receptor (AR) with ∼80% of ER+ and 30% to50% of ER− tumors being positive for AR by immunohistochemistry (IHC). Approximately 30% of breast cancer patients with estrogen receptor positive (ER+) tumors do not respond to tamoxifen or aromatase inhibitors and AR signaling has been implicated as a possible mechanism of this insensitivity. AR overexpression has also been associated with resistance to either tamoxifen or aromatase inhibitors in cell line models. Hypothesis: ER+ breast cancers can switch from estrogen to androgen-dependent growth as they become resistant to traditional endocrine therapies and AR can serve as a therapeutic target in AR+ breast cancers, irrespective of ER status. Methods: The proliferative effect of dihydrotestosterone (DHT) and estradiol (E2) in the presence enzalutamide (formerly MDV3100) was examined in vitro and in xenograft studies using models of both ER+ and ER− breast cancer that express AR. Furthermore, AR and ER were examined by IHC in clinical specimens from breast cancer patients treated with neoadjuvant exemestane and adjuvant tamoxifen. Tumor tissue was compared to adjacent normal breast epithelium. Results: In ER+ models, bicalutamide and enzalutamide both inhibited DHT-mediated proliferation, while enzalutamide uniquely inhibited E2-mediated proliferation. In vivo, enzalutamide significantly reduced estrogen- and androgen-mediated growth of MCF7 (ER+/AR+) xenograft tumors. Remarkably, enzalutamide demonstrated an anti-proliferative effect comparable to tamoxifen. in vitro, enzalutamide inhibits E2-stimulated tumor cell proliferation and E2 bound ER activation of genes such as PR and SDF-1, despite no observed binding of enzalutamide to ER alpha or beta. In ER−/AR+ MDA-MB-453 xenografts, enzalutamide treatment resulted in decreased nuclear AR localization, increased apoptosis and tumor growth inhibition. Conclusions: Enzalutamide demonstrated significant anti-tumor activity in preclinical models of breast cancers that express AR, regardless of ER status. When enzalutamide opposes DHT, it functions by excluding AR from the nucleus, thereby reversing the anti-apoptotic effect of AR. In contrast, when opposing E2, it causes a decrease in tumor cell proliferation. Profiling of ER+ xenografts is underway to further elucidate the mechanism by which this occurs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-14-02.

Abstract 4756: Elucidating the role of AR in breast cancer .

Background: In breast cancers, the androgen receptor (AR) is more widely expressed than estrogen receptor alpha (ER) or progesterone receptor (PR), and AR has recently emerged as a useful marker for refinement of breast cancer subtype classification. Approximately 77% of invasive breast cancer tumors are positive for AR and among the subtypes, 88% of ER+, 59% of HER2+, and 32% of triple negative breast cancers (ER-/PR-/HER2-) are positive for AR protein expression by IHC. AR expression has been associated with resistance to current endocrine therapies (tamoxifen and aromatase inhibitors) in cell line and preclinical models, and clinical studies. Hypothesis: We hypothesized that ER+ breast cancers that become resistant to traditional endocrine therapies might do so by switching from estrogen to androgen-dependence and that AR holds potential as a therapeutic target in AR+ breast cancers, whether ER+ or ER-. Methods: We quantified the percentage of cells positive for nuclear AR compared to the percentage of cells positive for ER in a cohort of tamoxifen treated patients with clinical outcome data. We also examined the effect of dihydrotestosterone (DHT) and estradiol (E2) in the presence of enzalutamide (formerly MDV3100), a novel AR inhibitor, in in vitro and in vivo models of ER+, ER-, and Her2+ breast cancer that retain AR. Results: Our clinical data indicate that a high ratio of AR to ER protein is indicative of a shorter time to relapse in patients treated with tamoxifen and a lack of response to neo-adjuvant AI treatment. In ER+ models, bicalutamide and enzalutamide both inhibit DHT-mediated proliferation, while enzalutamide uniquely inhibited E2-mediated proliferation. In xenograft tumor studies, enzalutamide significantly reduced estrogen- and androgen-mediated growth of ER+/AR+ xenograft tumors. Remarkably, enzalutamide demonstrated an anti-proliferative effect comparable to tamoxifen on tumors in E2 treated mice and inhibited the expression of classic E2-regulated genes such as SDF-1. Enzalutamide opposed DHT-stimulated proliferation of ER-/AR+ MDA-MB-453 tumors in vivo and caused decreased nuclear AR localization and increased tumor cell apoptosis. In Her2+ breast cancer cell lines, enzalutamide enhanced the response to trastuzumab and decreased the amount of both phosphorylated and total Her3. Conclusions: Enzalutamide demonstrated significant anti-tumor activity in preclinical models of breast cancers that express AR, regardless of ER status. In Her2+ breast cancer, inhibition of AR with enzalutamide may enhance response to Her2-directed therapy or overcome resistance to such agents by reducing levels of Her3. The AR:ER ratio may be a new predictor of response to traditional E2/ER directed endocrine therapy and may indicate that patients who relapse while on tamoxifen or AIs might be good candidates for AR-directed therapy. Citation Format: Nicholas C. D9Amato, Haihua Gu, Dawn R. Cochrane, Sebastian Bernales, Britta M. Jacobsen, Paul Jedlicka, Kathleen C. Torkko, Susan M. Edgerton, Ann D. Thor, Anthony D. Elias, Andrew A. Protter, Jennifer K. Richer. Elucidating the role of AR in 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 4756. doi:10.1158/1538-7445.AM2013-4756

Abstract P1-07-12: Defining the role of the kynurenine pathway in mediating anoikis resistance in triple negative breast cancer

Background: Anoikis resistance is thought to be a critical trait of metastatic cancer cells, enabling them to leave the primary tumor and travel through extracellular matrix, intravasate, and survive in the vasculature or lymphatics in transit to a metastatic site. This is particularly important for the triple-negative breast cancer (TNBC) subtype, which has a peak risk of recurrence within the first three years post-diagnosis and an increased mortality rate in the first five years as compared to other subtypes. We performed global profiling of TNBC cells in attached versus forced suspension culture conditions (using poly-HEMA coated plates) for 24 hours. These data revealed that TNBC cells surviving in suspension upregulate multiple genes involved in tryptophan catabolism, also known as the kynurenine pathway (KP), including the rate limiting enzyme tryptophan 2,3,-dioxygenase (TDO) and kynureninase (KYNU). A key metabolite of this pathway has been found to activate the aryl hydrocarbon receptor (AhR), which was also up-regulated in suspended cells. Hypothesis: We hypothesize that the ability to upregulate the kynurenine pathway (KP) facilitates TNBC cell survival in suspension and mediates the migratory/invasive potential of TNBC. Methods: We assessed mRNA and protein levels of TDO2, KYNU and AhR by RT-qPCR and western blot. AhR luciferase reporter activity, as well as known AhR regulated genes, were measured in suspension compared to adherent conditions. TNBC cells were treated with small molecule inhibitors of AhR and TDO2. Additionally, secretion of endogenous kynurenine was measured by high performance liquid chromatography (HPLC). Purified kynurenine was added to rescue AhR activity following TDO2 inhibition. Finally, anoikis sensitivity and migratory potential were measured following pharmacological inhibition of TDO2 and AhR. Results: Relative mRNA levels of TDO2, KYNU, and AhR increase 9, 7, and 2 fold respectively in suspended TNBC cells compared to adherent conditions (P Conclusions: Collectively, these results suggest that the kynurenine pathway may play a critical role in metastatic TNBC. Further mechanistic studies will focus on how the kynurenine pathway is mediating these tumorigenic properties either through the de novo synthesis of NAD+ and/or activation of AhR by kynurenine. Targeting the kynurenine pathway in the clinic may provide a therapeutic strategy to reduce TNBC mortality rates. Citation Format: Thomas Rogers, Nicholas D9Amato, Travis Nemkov, Michael Gordon, Kirk Hansen, Jennifer Richer. Defining the role of the kynurenine pathway in mediating anoikis resistance in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-12.

Abstract P3-04-06: Inhibiting androgen receptor nuclear localization decreases estrogen receptor (ER) activity and tumor growth in ER+ breast cancer

Background: Androgen receptor (AR) is widely expressed in breast tumors, but the role of AR in estrogen receptor (ER)+ tumors is controversial. In the absence of estradiol (E2), dihydrotestosterone (DHT) increases growth of ER+ breast cancer cells in vitro and in vivo. Anti-androgens such as bicalutamide (Bic) and enzalutamide (ENZ) inhibit this DHT-mediated proliferation. Surprisingly we have found that ENZ, which impairs nuclear entry of liganded AR, also inhibits E2-mediated proliferation of ER+ breast cancer cells, while Bic does not. Hypothesis: We hypothesize that nuclear localization of AR is necessary for maximal E2-mediated proliferation in ER+/AR+ breast cancer cells, and targeting AR with ENZ or other agents that impede AR nuclear entry or cause AR degradation will inhibit growth of ER+/AR+ human breast cancer cell lines and decrease tumor burden in preclinical models. Methods: ER+/AR+ MCF7, BCK4, and ZR-75-1 cells were treated with E2 plus or minus ER and AR antagonists and proliferation was measured by crystal violet staining or Incucyte live cell imaging. Nuclear AR was assessed by immunocytochemistry or nuclear/cytoplasmic fractionation. For in vivo experiments, 1x10^6 luciferase-expressing MCF7 cells were injected into the mammary fat pad of nu/nu mice and tumor growth monitored by caliper and IVIS imaging. Results: ENZ blocked E2-induced proliferation and showed synergistic activity with the ER antagonists 4-hydroxy-Tamoxifen (OH-Tam) and Fulvestrant (Fulv) in vitro. E2-induced expression of ER target genes including PR and SDF1 was also inhibited by ENZ, but not by Bic. Similarly, AR knockdown decreased baseline and E2-induced proliferation of MCF7 cells and E2-induced ER target gene expression. Both DHT and E2 treatment induced nuclear translocation of AR, which was decreased by ENZ. Nuclear translocation of AR in response to E2 occurs only in ER+ cell lines, further supporting a role for nuclear AR in E2-induced ER activity. In vivo, ENZ inhibited E2-induced growth of MCF7 tumors as effectively as Tamoxifen (Tam), and the combination of ENZ plus Tam was more effective than either drug alone. ENZ also inhibited growth of Tam-resistant MCF7 cells in vitro. Conclusions: Our results suggest that nuclear localization of AR plays a previously-unrecognized role in E2-mediated ER activity in ER+/AR+ breast cancer cells. Because of its ability to inhibit nuclear entry of liganded AR, ENZ may serve as an effective therapeutic in ER+/AR+ breast cancers. Importantly, ENZ may be particularly useful in combination with current anti-estrogen therapies (Tam or Fulv) since it affects ER, but via an indirect mechanism acting through AR. ENZ may also be effective in tumors resistant to ER-directed therapy based on in vitro data and published clinical data indicating that many such tumors express more AR protein than ER. Funded by: DOD BCRP Clinical Translational Award BC120183 to JKR, American Cancer Society Postdoctoral Fellowship PF-13-314-01 – CDD to NCD. Citation Format: Nicholas C D9Amato, Britta M Jacobsen, Dawn R Cochrane, Nicole S Spoelstra, Beatrice L Babbs, Anthony Elias, Jennifer K Richer. Inhibiting androgen receptor nuclear localization decreases estrogen receptor (ER) activity and tumor growth in ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-06.

Abstract P5-10-06: A functional role for miR-150 in breast cancer

Background: During mammary gland development, massive coordinated changes in protein expression govern the progression through pregnancy to lactation and involution. These dramatic changes are likely regulated in part at the translational level by changes in microRNAs (miRNAs). We profiled miRNA expression in mammary epithelial cells (MECs) isolated from mice at pregnancy day 14 (P14) or lactation day 2 (L2) and found that miR-150 is the most significantly downregulated miRNA between pregnancy and lactation. Interestingly, miR-150 was recently discovered to be decreased in mouse mammary tumors compared to normal mammary tissue in numerous transgenic models. However, a causal role for miR-150 has yet to be studied in human breast cancer and little is known about its functional role and relevant targets in the normal breast or breast cancer. Hypothesis: We hypothesized that miR-150 may be a tumor suppressor whose loss in breast cancer cells is an important event that allows for expression of multiple pro-tumorigenic genes. Methods: miR-150 levels in human breast cancers were evaluated in specimens of both ER+ and triple-negative breast cancer (TNBC) as compared to adjacent, non-involved normal breast epithelium by in situ hybridization. miR-150 levels were also measured by qRT-PCR in cell lines representative of multiple breast cancer subtypes. We stably restored miR-150 in TNBC cell lines by lentiviral infection and evaluated its effects on clonogenicity, growth in 3D culture, migration/invasion, and tumorigenicity. Expression of predicted miR-150 targets was assessed by immunoblot. Crossing of miR-150 fl/fl mice with BLG-Cre or MMTV/NIC transgenic mice will be utilized to determine the effects of MEC-specific loss of this miRNA on mammary tumorigenesis. Results: In clinical samples, in situ hybridization reveals that miR-150 levels are lower in both ER+ and TNBC tumor specimens compared to adjacent normal epithelium, with triple-negative tumors having the lowest expression. All breast cancer cell lines tested also have low miR-150 expression as compared to normal mammary epithelial tissue, with TNBC cell lines expressing the lowest levels. Exogenous expression of miR-150 in breast cancer cell lines caused a dramatic decrease in migration and invasion in vitro , and we are testing predicted miR-150 targets for their role in this phenotype. Experiments with transgenic models will determine if loss of miR-150 in mammary epithelial cells in the MMTV/NIC mouse model results in decreased latency or increased tumor formation and metastasis, or if miR-150 loss during pregnancy results in alterations in lactation, hyperplasia, or tumor formation. Conclusions: TNBC specimens and cell lines have the lowest expression of miR-150, though decreased miR-150 expression compared to normal mammary epithelium is a common feature of breast cancers regardless of subtype. miR-150 expression dramatically inhibits breast cancer cell migration and invasion in vitro , and ongoing experiments will determine the relevant target genes. Supported by Susan G. Komen Grant KG090415 and NIH NICHD P01 PAR-10-245 (Project 3) to JKR Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-06.