Machine learning and molecular dynamics reveal anti-osteoporosis potential of dietary flavonoids

The androgen receptor undergoes an androgen-specific NH(2)- and COOH-terminal interaction between NH(2)-terminal motif FXXLF and activation function 2 in the ligand binding domain. We demonstrated previously that activation function 2 forms overlapping binding sites for the androgen receptor FXXLF motif and the LXXLL motifs of p160 coactivators. Here we investigate the influence of the NH(2)- and COOH-terminal interaction on androgen receptor function. Specificity and relative potency of the motif interactions were evaluated by ligand dissociation rate and the stability of chimeras of transcriptional intermediary factor 2 with full-length and truncated androgen or glucocorticoid receptor. The results indicate that the androgen receptor activation function 2 interacts specifically and with greater avidity with the single FXXLF motif than with the LXXLL motif region of p160 coactivators, whereas this region of the glucocorticoid receptor interacts preferentially with the LXXLL motifs. Expression of the LXXLL motifs as a fusion protein with the glucocorticoid receptor resulted in loss of agonist-induced receptor destabilization and increased half-time of ligand dissociation. The NH(2)- and COOH-terminal interaction inhibited binding and activation by transcriptional intermediary factor 2. We conclude that the androgen receptor NH(2)- and COOH-terminal interaction reduces the dissociation rate of bound androgen, stabilizes the receptor, and inhibits p160 coactivator recruitment by activation function 2.

Targeting Androgen Receptor in Estrogen Receptor-Negative Breast Cancer

Decision letter: Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer

Editor's evaluation: Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer

Author response: Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer

Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR as a target for radiosensitization. In addition, 70-95% of all estrogen receptor (ER) positive (ER+) breast cancers also have coexpression of AR, suggesting extended utility of AR inhibition in the radiosensitization of these AR+, ER+ tumors. Here we assessed the efficacy of AR inhibition in ER+, AR+ breast cancers to better understand the role of AR signaling across breast cancer models. Further, we also investigated the effect of ER inhibition on radiosensitization of ER+ breast cancer models. Methods: IC50 values were determined for MDV3100 (enzalutamide), ARN-509 (apalutamide), and ODM-201 (darolutamide) in TNBC cell lines (AR+ TNBC: MDA-MB-453, ACC-422, and SUM-185PE, and AR- TNBC: MDA-MB-231) and ER+ breast cancer cell lines (AR+, ER+: ZR-75-1, BT-474, CAMA-1, and AR-, ER+: MCF-7). IC50 values for tamoxifen were determined for ER+ breast cancer cell lines (MCF-7, T47D, ZR-75-1), and ER- (SUM-159) cells. Clonogenic survival assays were performed to assess radiosensitization with ER or AR inhibition with tamoxifen or second generation anti-androgens, respectively, in TNBC and ER+ breast cancer models. Results: AR inhibition with enzalutamide, apalutamide, and darolutamide showed limited single agent growth inhibition efficacy in AR+ TNBC and AR+, ER+ breast cancer cell lines (IC50 > 10 μM). AR inhibition with enzalutamide did not induce radiosensitivity in vitro. In AR+, ER+ CAMA-1 cells, AR blockade with enzalutamide had a radioprotective effect with enhancement ratios (enhR) of 0.76-0.83. No radiosensitization was observed in BT-474 (enhR: 0.92-1.01) or ZR-75-1 cells (enhR: 0.94-1.00). Radiosensitization was also assessed with anti-androgens apalutamide and darolutamide in AR+ breast cancer models. Inhibition of ER with tamoxifen, however, induced radiosensitization in MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells. No radiosensitization was observed with tamoxifen in ER- SUM-159 cells. Conclusion: Although AR is a mediator of radioresistance in AR+ TNBC, AR inhibition does not provide comparable radiosensitization in AR+, ER+ models and may actually confer a radioprotective effect. In contrast, our results demonstrate ER inhibition is an effective radiosensitizing strategy in ER+ breast cancers, independent of AR status. This work highlights the complexities of androgen and estrogen receptor signaling in AR+, ER+ breast tumors and underscores the necessity for understanding context dependent effects when translating into patients with AR+ breast cancer. Citation Format: Anna R. Michmerhuizen, Amanda Zhang, Rachel Schwartz, Andrea M. Pesch, Benjamin C. Chandler, Cassandra L. Ritter, Meilan Liu, Kari Wilder-Romans, Daniel E. Spratt, Daniel R. Wahl, Shyam Nyati, Lori J. Pierce, Corey Speers. Hormone receptor inhibition as a strategy for radiosensitization of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6271.

Cyproterone acetate (CPA) is a steroidal antiandrogen used clinically in the treatment of prostate cancer. Compared with steroidal agonists for the androgen receptor (AR) (e.g. dihydrotestosterone, R1881), CPA is bulkier in structure and therefore seemingly incompatible with the binding pockets observed in currently available x-ray crystal structures of the AR ligand-binding domain (LBD). We solved the x-ray crystal structure of the human AR LBD bound to CPA at 1.8A in the T877A variant, a mutation known to increase the agonist activity of CPA and therefore facilitate purification and crystal formation of the receptor.drug complex. The structure demonstrates that bulk from the 17alpha-acetate group of CPA induces movement of the Leu-701 side chain, which results in partial unfolding of the C-terminal end of helix 11 and displacement of the loop between helices 11 and 12 in comparison to all other AR LBD crystal structures published to date. This structural alteration leads to an expansion of the AR binding cavity to include an additional pocket bordered by Leu-701, Leu-704, Ser-778, Met-780, Phe-876, and Leu-880. Further, we found that CPA invokes transcriptional activation in the L701A AR at low nanomolar concentrations similar to the T877A mutant. Analogous mutations in the glucocorticoid receptor (GR) and progesterone receptor were constructed, and we found that CPA was also converted into a potent agonist in the M560A GR. Altogether, these data offer information for structure-based drug design, elucidate flexible regions of the AR LBD, and provide insight as to how CPA antagonizes the AR and GR.

Reply of the Authors

The transcriptional activity of androgen receptor (AR) is modulated by coregulators that have a significant influence on a number of functional properties of AR, including the ligand specificity as well as the DNA binding capacity. Because androgens and AR have pivotal roles in the development and

Background: Androgen receptor (AR) is expressed in 60%-90% of breast cancers. The role of AR in breast cancer largely depends on estrogen receptor (ER) status and remains controversial. In ER+ cancers, AR expression is associated with improved prognosis. Enzalutamide (ENZ), an AR antagonist, impairs AR signaling, inhibits ER+/AR+ breast cancer cell proliferation and has been shown to mitigate resistance to anti-estrogen therapies. A recent study identified that RAD140, an AR agonist, suppressed the growth of ER+/AR+ breast cancer via stimulating AR signaling, resulting in down regulation of ERα. Both AR agonists and antagonists are effective in treating ER+ breast cancers. Identifying the subgroup of patients who most likely will benefit from an individual drug is important in clinical practice. Here, we sought to characterize the relative AR to ER levels and their relationships to drug response in ER+ breast cancers.Methods: We evaluated AR and ER expression levels among 68 samples with ER+ breast cancers. On each tumor, IHC staining for AR and ER were performed and results were scored by multiplying the percentage of positive cells by the intensity. TCGA-RPPA (reverse phase protein array) dataset was used to verify AR protein distribution in ER+ cancers. We created MCF7 and T47D cells with doxycycline-inducible AR and ER expression system to manipulate the relative AR to ER ratios and determined the antitumor activity of ENZ and RAD140 by cell proliferation assays. The levels of AR and ER in cells were measured by western blot. Linear regression was used to test the association between dose-response area under curve (AUC) and AR, ER levels. Drug preference was modeled against AR/ER expression levels (AR/ER ratios) using logistic regression.Results: Among 68 ER+ breast cancers, 69.12% were AR-positive. More than 2/3 cases (48 of 68) had more ER than AR expressed. The AR to ER ratios varied from 0 to 6. In the TCGA cohort, 84.15% of 347 ER+ patients had AR/ER ratios less than 1.The range of AR to ER ratios in TCGA dataset were comparable with our data (0.004 to 4.210). In our cell line models, the AR/ER ratios were controlled between 0.19 to 4.05. We found that the AUC of RAD140 was negatively associated with AR protein levels (P=0.008) and AR/ER ratios (P=0.013), and not significantly associated with ER expression levels. On the other hand, the AUC of ENZ was significantly negatively associated with ER protein (P=0.0016) but postiviely associated with AR/ER ratio (p=0.037). Preferred treatment comparing efficacy of the two drug can be best determined at extremes of AR/ER ratios. Using clinically relevant dosages, our model predicted that ENZ (10µM) would be a preferred treatment choice and have a better treatment efficacy compared with RAD140 when the AR/ER ratio was ≤ 0.42, whereas RAD140 (1µM) would be the preferred choice with a better treatment efficacy compared with ENZ when AR/ER ratios were ≥ 3.1 The efficacy preference of the two drugs are equivocal for AR/ER ratios between 0.42 and 3.10. Conclusion: We developed preclinical models using AR and ER expression levels to predict AR-targeting drug response. The results support the use of RAD140 in AR high patients and those with an AR/ER ratio >3.10, and enzalutamide in AR low patients and those with an AR/ER ratio <0.42. Equipoise on choice of drug was found for AR/ER ratios of 0.42-3.10. RAD140 and enzalutamide are compelling candidates for monotherapy or combination with anti-estrogen therapies in ER+/AR+ breast cancer. Future clinical validation of the models and therapeutic effect is warranted. Citation Format: Lixuan Wei, Huanyao Gao, Jia Yu, Duan Liu, Huan Zhang, Thanh Nguyen, Marie R Passow, Jodi M Carter, Richard M Weinshilboum, James N Ingle, Liewei Wang. Computational modeling of androgen receptor (AR) and estrogen receptor as predictive biomarkers of response to AR agonists and antagonists [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-36.

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor

<div>Abstract<p><b>Purpose:</b> To evaluate whether tumor androgen receptor (AR) expression was prognostic and/or predictive for endocrine treatment alone or in combination with estrogen receptor (ER). The AR has been hypothesized to have differential prognostic roles in breast cancer depending on tumor ER status, and to influence endocrine treatment response.</p><p><b>Experimental Design:</b> A population-based prospective cohort of 1,026 patients diagnosed with primary invasive breast cancer in Lund, Sweden, between 2002 and 2012 was followed until June 2014. Associations between immunohistochemical AR expression in tumor tissue microarrays, patient and tumor characteristics, and AR genotypes were analyzed. Disease-free survival (DFS) by AR status, and combined ER/AR status was assessed in various treatment groups.</p><p><b>Results:</b> AR expression was assessable in 913 tumors. AR<sup>+</sup> tumors (85.0%) were associated with higher age (<i>P</i> = 0.036) and favorable tumor characteristics. The AR<sup>+</sup> status was a prognostic marker for DFS (LogRank <i>P</i> = 0.025). There was an interaction between AR and ER expression with respect to prognosis (adjusted <i>P</i><sub>interaction</sub> ≤ 0.024). Tumors with discordant hormone receptor expressions (ER<sup>+</sup>AR<sup>−</sup> or ER<sup>−</sup>AR<sup>+</sup>) demonstrated worse prognosis compared with concordant tumor expressions (ER<sup>+</sup>AR<sup>+</sup> or ER<sup>−</sup>AR<sup>−</sup>) in multivariable models [adjusted HRs (95% confidence intervals); ≥1.99 (1.28–3.10), <i>P</i> ≤ 0.002]. ER<sup>+</sup>AR<sup>−</sup> indicated early treatment failure with aromatase inhibitors (AI) among chemonaïve patients aged 50 or older.</p><p><b>Conclusions:</b> Prediction of breast cancer prognosis and treatment response was improved by combining AR and ER status. AR negativity predicted early treatment failure with AI but not tamoxifen, a finding that warrants confirmation in a randomized setting. Patients may benefit from anti-androgens or selective AR modulators. <i>Clin Cancer Res; 21(16); 3640–50. ©2015 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> To evaluate whether tumor androgen receptor (AR) expression was prognostic and/or predictive for endocrine treatment alone or in combination with estrogen receptor (ER). The AR has been hypothesized to have differential prognostic roles in breast cancer depending on tumor ER status, and to influence endocrine treatment response.</p><p><b>Experimental Design:</b> A population-based prospective cohort of 1,026 patients diagnosed with primary invasive breast cancer in Lund, Sweden, between 2002 and 2012 was followed until June 2014. Associations between immunohistochemical AR expression in tumor tissue microarrays, patient and tumor characteristics, and AR genotypes were analyzed. Disease-free survival (DFS) by AR status, and combined ER/AR status was assessed in various treatment groups.</p><p><b>Results:</b> AR expression was assessable in 913 tumors. AR<sup>+</sup> tumors (85.0%) were associated with higher age (<i>P</i> = 0.036) and favorable tumor characteristics. The AR<sup>+</sup> status was a prognostic marker for DFS (LogRank <i>P</i> = 0.025). There was an interaction between AR and ER expression with respect to prognosis (adjusted <i>P</i><sub>interaction</sub> ≤ 0.024). Tumors with discordant hormone receptor expressions (ER<sup>+</sup>AR<sup>−</sup> or ER<sup>−</sup>AR<sup>+</sup>) demonstrated worse prognosis compared with concordant tumor expressions (ER<sup>+</sup>AR<sup>+</sup> or ER<sup>−</sup>AR<sup>−</sup>) in multivariable models [adjusted HRs (95% confidence intervals); ≥1.99 (1.28–3.10), <i>P</i> ≤ 0.002]. ER<sup>+</sup>AR<sup>−</sup> indicated early treatment failure with aromatase inhibitors (AI) among chemonaïve patients aged 50 or older.</p><p><b>Conclusions:</b> Prediction of breast cancer prognosis and treatment response was improved by combining AR and ER status. AR negativity predicted early treatment failure with AI but not tamoxifen, a finding that warrants confirmation in a randomized setting. Patients may benefit from anti-androgens or selective AR modulators. <i>Clin Cancer Res; 21(16); 3640–50. ©2015 AACR</i>.</p></div>

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 D'Amato, 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.

OBJECTIVE:Androgen (AR), Estrogen (ER) and Progesterone (PR) hormones play an important role in the prenatal and postnatal development of urogenital tract and especially the penis. The expressions of AR, ER and PR receptors in penile tissues in children with hypospadiases had also been shown previously. In this leading study, to demonstrate of the sex hormone receptor expression in cases with different types of hypospadias were aimed.METHODS:This study was designed in children operated due to hypospadiases without DSD. Biopsy samples of 3 mm’s were obtained from three different sytes as the lateral parameatal tissue and the anterior corner of the prepuce, and inner layer of posterior prepuce. The presence of AR, ER and PR receptors was investigated immunehistochemically.RESULTS:Mean age was 5.4 years in 18 children with hypospadiases; in totally 33 specimens were taken in 5 subcoronal as 5 specimens, and 7 penile as 15 specimens, and 6 penoscrotal as 13 specimens. According to sytes of samples; 13 samples were from lateral para-meatal tissues, and 13 were from anterior corners of prepuces, and 7 were from inner layers of posterior prepuces. In regard to receptor expression; ER and AR receptors were positive in 29 (87.8%) and 12 (36.4%) respectively; PR receptors were negative.CONCLUSION:This study emphasized the dominant expression of estrogen receptors in penile tissues of children with hypospadias. Although there was not a manifest correlation of androgen receptors absence in regard to the severity of hypospadias patients, there was a marked estrogen receptors presence in penile tissues. These findings suggest that the disrupted androgen and estrogen receptor interaction and/or balance could play a role during the development of external genitalia in hypospadias patients. Progesterone receptor was not present and therefore the active role in the postnatal development of hypospadias is still debatable.