Andrew Asher Protter

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide

IntroductionThe androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ERu2009+u2009tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer.MethodsWe examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR.ResultsIn a cohort of 192 women with ERu2009+u2009breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HRu2009=u20094.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HRu2009=u20094.04, 95% CI: 1.68, 9.69; pu2009=u20090.002) and disease specific survival (HRu2009=u20092.75, 95% CI: 1.11, 6.86; pu2009=u20090.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/ARu2009+u2009breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis.ConclusionsAR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer

Enzalutamide (formerly MDV3100 and available commercially as Xtandi®), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration‐resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: binding of androgens to AR, AR nuclear translocation, and association of AR with DNA. Here, we investigate the effects of enzalutamide on AR signaling, AR‐dependent gene expression and cell apoptosis.

Combination Therapy with a Second-Generation Androgen Receptor Antagonist and a Metastasis Vaccine Improves Survival in a Spontaneous Prostate Cancer Model

Purpose: Enzalutamide, a second-generation androgen antagonist, was approved by the U.S. Food and Drug Administration (FDA) for castration-resistant prostate cancer (CRPC) treatment. Immunotherapy has been shown to be a promising strategy for prostate cancer. This study was performed to provide data to support the combination of enzalutamide and immunotherapy for CRPC treatment. Experimental Design: Male C57BL/6 or TRAMP (transgenic adenocarcinoma of the mouse prostate) prostate cancer model mice were exposed to enzalutamide and/or a therapeutic vaccine targeting Twist, an antigen involved in epithelial-to-mesenchymal transition and metastasis. The physiologic and immunologic effects of enzalutamide were characterized. The generation of Twist-specific immunity by Twist-vaccine was assessed. Finally, the combination of enzalutamide and Twist-vaccine to improve TRAMP mice overall survival was evaluated. Results: Enzalutamide mediated immunogenic modulation in TRAMP-C2 cells. In vivo, enzalutamide mediated reduced genitourinary tissue weight, enlargement of the thymus, and increased levels of T-cell excision circles. Because no changes were seen in T-cell function, as determined by CD4+ T-cell proliferation and regulatory T cell (Treg) functional assays, enzalutamide was determined to be immune inert. Enzalutamide did not diminish the ability of Twist-vaccine to generate Twist-specific immunity. Twist was confirmed as a valid tumor antigen in TRAMP mice by immunohistochemistry. The combination of enzalutamide and Twist-vaccine resulted in significantly increased overall survival of TRAMP mice compared with other treatment groups (27.5 vs. 10.3 weeks). Notably, the effectiveness of the combination therapy increased with disease stage, i.e., the greatest survival benefit was seen in mice with advanced-stage prostate tumors. Conclusions: These data support the combination of enzalutamide and immunotherapy as a promising treatment strategy for CRPC. Clin Cancer Res; 19(22); 6205–18. ©2013 AACR.

Novel p38α Mitogen‐Activated Protein Kinase Inhibitor Shows Analgesic Efficacy in Acute Postsurgical Dental Pain

SCIO‐469 is a selective p38α mitogen‐activated protein kinase (MAPK) inhibitor for preclinical models of acute pain. This prospective, double‐blind, randomized clinical study compared efficacy and safety of oral SCIO‐469, ibuprofen, and placebo in postsurgical dental pain. Subjects (n = 263) undergoing extraction of 1 or more impacted mandibular third molars received preoperative treatment with SCIO‐469 (150, 210, or 300 mg), ibuprofen (400 mg), or placebo; the 210‐mg group received 90 mg postoperatively. A 4‐point categorical scale and a 100‐mm visual analogue scale were used to measure pain intensity. The primary end point was median time from first incision to first rescue medication using the Kaplan‐Meier product limit estimator. All SCIO‐469 groups had significantly longer times to rescue medication compared with placebo; preoperative and postoperative treatment with 210 + 90 mg SCIO‐469 resulted in 8.1 hours versus 4.1 hours to rescue for placebo (P = .003). Ibuprofen also increased time to rescue medication (6.6 hours) versus placebo (P = .04). Dizziness, headache, and nausea were the most frequently reported adverse events. This is the first clinical demonstration of antinociceptive effects in acute pain with preoperative administration of a p38α MAPK inhibitor.

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.

Restricted Delivery of Talazoparib Across the Blood–Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma

Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1–3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2–M arrest in vitro. In vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp−/− versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b−/− mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood–brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735–46. ©2017 AACR.

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

Novel small molecules induced ROS accumulation and antitumor activity based on an oxidative stress gene signature.

e14575Background: Reactive oxygen species (ROS) homeostasis is crucial for cell survival. Drugs that possess anti-tumor effects by suppressing ROS metabolism in cancer cells have potential as a new...

Abstract B30: Talazoparib efficacy is enhanced by noncytotoxic doses of temozolomide-mediated DNA damage in prostate cancer cell lines

Purpose: To investigate the effects of talazoparib alone and in combination with temozolomide (TMZ) on prostate cancer cells Introduction: Talazoparib is a potent, orally bioavailable, small molecule inhibitor of PARP (poly-ADP ribose polymerase) enzyme activity that also traps PARP protein onto DNA at sites of DNA damage; both activities result in cancer cell death (Murai et al. Mol Cancer Ther. 2014;13:433-43). Talazoparib is currently under investigation for clinical activity in patients who have locally advanced and/or metastatic breast cancer with germline BRCA mutations (EMBRACA; Clinical Trials.gov; NCT01945775). In the current nonclinical study, talazoparib demonstrated potent antitumor effects on androgen receptor (AR)-positive prostate cancer cells expressing both full length AR (LNCaP cells) as well as the androgen-independent splice variant, AR-V7 (22RV1 cells), when used either as a single agent or in combination with low, noncytotoxic concentrations of TMZ, a DNA alkylating therapeutic. Methods: Viability of prostate cancer cells (LNCaP and 22RV1 cells) after 7 days of treatment was measured by Cell Titer Glo (Promega) after treatment with vehicle, talazoparib, TMZ or a combination of the two. DNA damage was determined by immunofluorescence against gH2AX (EMD Millipore), and the amount of PARP trapped on DNA, determined by Western Blot analysis of chromatin probed with antibodies specific for PARP. Cellular homeostasis was determined by flow cytometry analysis for cell cycle stage, and apoptosis indicated by Annexin V staining (BD Pharmingen) or caspase activation with Caspase Glo (Promega) assays. Nucleotide adducts, N7 methyl-29-deoxyguanosine (N7-MedG) and O6-Methyl-29-deoxyguanosine (O6-MedG), from 1-, 2- and 4-hour TMZ-treated calf thymus DNA and genomic DNA from LNCaP cells treated with TMZ were detected after DNA hydrolysis and quantitated by LC-mass spectrometry in multiple reaction monitoring mode. Results: Talazoparib treatment resulted in a concentration-dependent inhibition of cell viability in LNCaP (AR wildtype) and 22RV1 (AR-V7) cells. Talazoparib9s antitumor effects were greatly enhanced when combined with noncytotoxic TMZ concentrations. In a cell-free study using calf thymus DNA, TMZ generated both N7-MedG and O6-MedG DNA adducts, with the N7-MedG being more abundant than the O6-MedG. Analysis of DNA from LNCaP cells treated with TMZ generated detectable N7-MedG DNA adducts. Low, noncytotoxic concentrations of TMZ potentiated the effects of talazoparib on prostate cancer cell viability independent of AR variant status. Treatment with talazoparib alone and in combination with TMZ was associated with increased gH2AX staining (DNA damage) and enhanced PARP-DNA trapping complexes. The combination treatment also induced an increase in apoptotic cells at early time points. Conclusion: Talazoparib is cytotoxic to prostate cancer cells as a single agent in nonclinical cell based models. Herein, we demonstrate that combining noncytotoxic doses of TMZ with talazoparib improves efficacy in killing of LNCaP and 22RV1 cells. The increased efficacy corresponds with increased DNA-damage and increased trapped PARP-DNA complexes. Low, noncytotoxic doses of TMZ are shown to generate DNA damage sites in vitro and in treated LNCaP cells, providing a mechanistic basis for the combination effect of talazoparib and TMZ. Together, these nonclinical data provide scientific rationale for a clinical trial strategy of combining talazoparib with low, noncytotoxic doses of TMZ to enhance efficacy in prostate cancer. Citation Format: Jeffrey N. Lindquist, Vernon T. Phan, Eduardo Riquelme, Kakoli Mukherjee, Olivia Farias, Ashu Gupta, Mohd Raja, Roopa Rai, Hirdesh Uppal, Andrew A. Protter. Talazoparib efficacy is enhanced by noncytotoxic doses of temozolomide-mediated DNA damage in prostate cancer cell lines [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B30.

Abstract LB-109: MDV3100, an androgen receptor signaling inhibitor, inhibits tumor growth in breast cancer preclinical models regardless of estrogen receptor status

Background: The androgen receptor (AR) is detected by immunohistochemistry in approximately 75% of all invasive breast cancer, with ∼88% of estrogen receptor (ER) positive (ER+) tumors also expressing AR and ∼20 to 30% of ER negative (ER-) tumors also retaining AR expression. Potent inhibition of AR activity could be a therapeutic strategy in AR+ ER+ breast cancer. MDV3100 is an androgen receptor signaling inhibitor (ARSI), which inhibits AR activity via three mechanisms: 1) inhibition of androgen binding to AR, 2) inhibition of AR nuclear translocation, and 3) inhibition of nuclear AR-DNA binding; and has demonstrated an overall survival benefit in men with post-docetaxel prostate cancer. Methods: Two ER+/AR+ breast cancer cell lines, MCF7and BCK4 (recently derived from a pleurocentesis), were used to assess the proliferative effect of dihydrotestosterone (DHT) and estradiol (E2) in ovariectomized mice. MDV3100 was compared to bicalutamide and tamoxifen. Growth effects of MDV3100 on ER-/AR+ cells in tissue culture and in xenografts were also examined. Results: Both bicalutamide and MDV3100 inhibited DHT-mediated proliferation of ER+/AR+ cell lines. Although MDV3100 binds AR very effectively and does not bind ER, it inhibited E2-mediated proliferation. MDV3100 also blocked E2 mediated upregulation of AR, PR, and SDF-1. MDV3100 inhibited E2-stimulated tumor growth of MCF7 mammary xenografts as effectively as tamoxifen. With ER-/AR+ cells (MDA-MB-453), MDV3100 reduced DHT-induced nuclear translocation, cell growth in tissue culture and tumor growth in mouse orthotopic xenografts. Conclusions: MDV3100 blocked both DHT- and E2-mediated growth of breast cancer cells, whereas bicalutamide enhanced E2-mediated proliferation. MDV3100 may have unique therapeutic utility in patients with AR+ breast cancer, regardless of ER status. Funding: DOD Breast Cancer Program Idea Award BC074403, Avon Foundation for Women, and University of Colorado Cancer Center pilot project funds to JKR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-109. doi:1538-7445.AM2012-LB-109