Michael A. Gordon

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 Biology in Triple Negative Breast Cancer: a Case for Classification as AR+ or Quadruple Negative Disease

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) amplification. Due to the absence of these receptors, TNBC does not respond to traditional endocrine or HER2-targeted therapies that improve patient prognosis in other breast cancer subtypes. TNBC has a poor prognosis, and currently, there are no effective targeted therapies. Some TNBC tumors express androgen receptor (AR) and may benefit from AR-targeted therapies. Here, we review the literature on AR in TNBC and propose that TNBC be further sub-classified as either AR+ TNBC or quadruple negative breast cancer since targeting AR may represent a viable therapeutic option for a subset of TNBC.

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.

Constitutive expression of microRNA-150 in mammary epithelium suppresses secretory activation and impairs de novo lipogenesis

Profiling of RNA from mouse mammary epithelial cells (MECs) isolated on pregnancy day (P)14 and lactation day (L)2 revealed that the majority of differentially expressed microRNA declined precipitously between late pregnancy and lactation. The decline in miR-150, which exhibited the greatest fold-decrease, was verified quantitatively and qualitatively. To test the hypothesis that the decline in miR-150 is crucial for lactation, MEC-specific constitutive miR-150 was achieved by crossing ROSA26-lox-STOP-lox-miR-150 mice with WAP-driven Cre recombinase mice. Both biological and foster pups nursed by bitransgenic dams exhibited a dramatic decrease in survival compared with offspring nursed by littermate control dams. Protein products of predicted miR-150 targets Fasn, Olah, Acaca, and Stat5B were significantly suppressed in MECs of bitransgenic mice with constitutive miR-150 expression as compared with control mice at L2. Lipid profiling revealed a significant reduction in fatty acids synthesized by the de novo pathway in L2 MECs of bitransgenic versus control mice. Collectively, these data support the hypothesis that a synchronized decrease in miRNAs, such as miR-150, at late pregnancy serves to allow translation of targets crucial for lactation. Summary: A decline in miR-150 is critical for lactation, as pups nursed by dams in which this decline is prevented exhibit a dramatic decrease in survival compared with littermate controls.

Anti-androgen therapy in triple-negative breast cancer

Inhibition of the androgen receptor (AR) represents the most important therapeutic target in prostate cancer. Although AR is expressed in 77% of all breast cancers (BCs), even more than estrogen receptors (ERs), its role in BC growth and progression remains indefinite [Guedj et al. 2012]. AR expression is associated with somewhat more indolent BC [Lehmann et al. 2011; Liedtke et al. 2008; Cochrane et al. 2014]. The drug development pipeline of AR-targeted therapeutics in prostate cancer is facilitating the evaluation of AR signaling inhibition in triple-negative breast cancer (TNBC): including bicalutamide, a nonsteroidal partial agonist; enzalutamide, an inhibitor of nuclear localization of AR; and VT-464, a dual inhibitor of CYP17 and AR. Given the controversy in the role of AR, other ongoing or completed trials are testing dehydroepiandrosterone (DHEA) or 4-OH testosterone (see Table 1). n n n nTable 1. n nClinical trials of AR therapies in breast cancer. n n n n n nFigure 1. n nAR signaling integration in TNBC. n n n n nPreclinical justification for anti-androgen therapies in breast cancer nGene expression profiling of BC suggests a significant functional role for AR in multiple subtypes of BC [Guedj et al. 2012; Lehmann et al. 2011]. While AR is expressed to varying degrees across all BC subtypes, preclinical modeling suggests that its functional role in disease progression is subtype-specific. Gene expression profiling of TNBC has revealed a number of potential subtypes within TNBC, including basal-like 1, basal-like 2, immunomodulatory, mesenchymal-like, mesenchymal stem-like, and luminal AR (LAR) [Lehmann et al. 2011], although these subtypes do not yet dictate individualized treatment with specific targeted agents to date. Although ER expression is absent, the LAR subtype is characterized by AR signaling with a gene expression pattern similar to luminal BC. Patients with LAR tumors are more slowly growing when metastatic, however they have decreased relapse-free survival in the adjuvant setting relative to other TNBC subtypes [Cochrane et al. 2014], perhaps due to lower chemotherapy sensitivity. LAR cell line models are sensitive to the AR partial antagonist bicalutamide [Lehmann et al. 2011], and are even more sensitive to the next-generation AR inhibitor enzalutamide [Cochrane et al. 2014]. n nAR is expressed in 12–55% of cases of TNBC [Barton et al. 2015; Collins et al. 2011; Gucalp et al. 2013; Thike et al. 2014; Traina et al. 2015]. Some of the variability in frequency of expression between studies is due to different anti-AR antibodies used and to different assay cutoffs (1% versus 10%). Preclinically, BC expressing as little as 1% AR may respond to enzalutamide, although higher levels may be associated with greater response [Barton et al. 2015]. Optimal assay for response to AR inhibitors in clinic is as yet unknown. Although the LAR subtype of TNBC is AR enriched, other TNBC subtypes also express AR, and have responded to AR inhibition using preclinical models [Barton et al. 2015]. In TNBC models, AR appears to regulate amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, which when secreted could potentially support even AR negative tumor cells [Barton et al. 2015]. n nPhosphoinositide 3-kinase (PI3K3) activation through loss of phosphatase and tensin homolog (PTEN) or mutation of PIK3CA is common in TNBC [Shah et al. 2012; Kriegsmann et al. 2014], and is associated with increased AR levels in BC [Gonzalez-Angulo et al. 2009]. The combination of bicalutamide and the PI3K inhibitors pictilisib and apitolisib showed additive efficacy in PI3K-mutant TNBC cells in vitro and in vivo [Lehmann et al. 2014]. Enzalutamide plus everolimus appeared to be synergistic in multiple in vitro preclinical models of BC, including TNBC [Gordon et al. 2014].

Dicer expression in estrogen receptor-positive versus triple-negative breast cancer: an antibody comparison.

Dicer is an RNase III enzyme responsible for cleaving double-stranded RNAs into small interfering RNAs and microRNAs, which either target messenger RNA transcripts for degradation or inhibit translation. Dicer protein levels have been examined in breast cancer with contradictory results. Our goal was to resolve whether Dicer levels differ in breast cancer versus normal breast epithelium and between estrogen receptor-α-positive (ER+) or estrogen receptor-α-negative (ER-) primary breast cancers. We compared 3 different Dicer antibodies: Abcam 4A6, Abcam ab5818, and Sigma HPA000694, using immunohistochemistry and Western blot analyses. All 3 Dicer antibodies detected higher levels of Dicer in ER+ breast cancer cell lines versus ER-, and all 3 recognized exogenous overexpressed Dicer. In clinical specimens, all 3 antibodies detected higher Dicer in ER+ breast cancers versus triple-negative breast cancer (TNBC) but had very different staining patterns by immunohistochemistry on the same tumor samples. Using the optimal antibody, ab5818, selected for its sensitivity and specificity, Dicer protein expression was significantly higher in ER+ versus TNBC clinical specimens of primary tumor (P<.0001, unpaired t test). Dicer was also significantly higher in adjacent normal breast epithelium versus TNBC (P<.0001, paired t test; n=18 pairs). Differences in antibody performance may explain contrasting results observed in the literature regarding Dicer protein in breast cancer. If Dicer becomes more clinically relevant as a prognostic indicator, further antibody optimization and standardization will be critical.

The long non-coding RNA MALAT1 promotes ovarian cancer progression by regulating RBFOX2-mediated alternative splicing: MALAT1 promotes ovarian cancer progression

Ovarian cancer metastasizes via direct seeding, whereby cancer cells shed from the primary site, resist cell death in the peritoneal cavity, then metastasize to peritoneal organs. We sought to identify molecular mechanisms that facilitate ovarian cancer cell anchorage independent survival. Gene expression profiling was performed on ovarian cancer cells grown in attached or forced suspension culture and confirmed by RT‐qPCR. Anoikis was measured by Caspase 3/7 assay. Since the long non‐coding RNA Metastasis Associated Lung Adenocarcinoma transcript 1 (MALAT1) was among the transcripts most highly increased in forced suspension culture, modified anti‐sense oligonucleotides (ASO) were used to inhibit its expression. Knockdown of RBFOX2 and KIF1B was performed using shRNAs. Publically available datasets were analyzed for association of MALAT1 gene expression with clinicopathological variables. In multiple anoikis‐resistant ovarian cancer cell lines MALAT1 expression increased after 24 and 48u2009h in forced suspension culture compared to attached culture. High MALAT1 is associated with increased stage, recurrence, and reduced survival in ovarian cancer, and in a small percentage of ovarian cancers MALAT1 is amplified. MALAT1 knockdown resulted in decreased proliferation, invasion, anchorage‐independent growth, and increased anoikis. Suppression of MALAT1 also resulted in decreased expression of RBFOX2, and alternative processing of the pro‐apoptotic tumor suppressor gene KIF1B. RBFOX2 suppression resulted in preferential splicing of the pro‐apoptotic isoform of KIF1B (KIFB1B‐beta) and increased anoikis. The lncRNA MALAT1 facilitates a pro‐metastatic phenotype in ovarian cancer by promoting alternative RNA processing and differential expression of anti‐apoptosis and epithelial to mesenchymal transition (EMT)‐related genes.