The role of androgen receptor (AR) activation and expression is well understood in prostate cancer. In breast cancer, expression and activation of AR is increasingly recognized for its role in cancer development and its importance in promoting cell growth in the presence or absence of estrogen. As both prostate and breast cancers often share a reliance on nuclear hormone signaling, there is increasing appreciation of the overlap between activated cellular pathways in these cancers in response to androgen signaling. Targeting of the androgen receptor as a monotherapy or in combination with other conventional therapies has proven to be an effective clinical strategy for the treatment of patients with prostate cancer, and these therapeutic strategies are increasingly being investigated in breast cancer. This overlap suggests that targeting androgens and AR signaling in other cancer types may also be effective. This manuscript will review the role of AR in various cellular processes that promote tumorigenesis and metastasis, first in prostate cancer and then in breast cancer, as well as discuss ongoing efforts to target AR for the more effective treatment and prevention of cancer, especially breast cancer. In order to develop effective treatment strategies for patients with each of these cancer types, it is important to understand how AR is functioning similarly and differently to drive tumor growth.Androgens are expressed at different levels in men and women, and while they are important for proper development, they can also drive tumor growth. Most notably, the role of the androgen receptor (AR) in prostate cancer has been extensively studied. Recent data suggest that AR signaling may also be important in breast cancer, glioblastoma, and additional tumor types with AR expression2. As is characteristic of this type of receptor, inactive forms of AR are located in the cytoplasm, bound to heat shock proteins (HSPs)3. The HSPs are responsible for proper protein folding, prevention of misfolding and maintaining 3D protein structure during events of cellular stress5. AR, like other receptors in this family, is activated by the binding of androgen molecules to its ligand binding domain (LBD). Androgen binding results in AR homodimerization and translocation into the nucleus, where AR binds to androgen response elements (AREs) resulting in activation and transcription of a variety of downstream genes. Binding of AR results in the activation of diverse signaling pathways, including multiple signaling pathways that have been implicated in cancer, including the PI3K/AKT pathway6. AR also contributes to cell growth and proliferation differently in the context or absence of ER, and AR has an influence on cell cycle and DNA damage repair. Further, AR has non-genomic functions that can influence cell growth, migration, metastasis, and apoptosis. Due to its many downstream effects, antiandrogen therapies have long been of therapeutic interest along with combining AR antagonism with conventional chemotherapy or radiation therapy.AR belongs to the Type I class of nuclear hormone transcription factors along with the estrogen receptor (ER), progesterone receptor (PR), and glucocorticoid receptor (GR)7, and others develop mutations in the LBD of AR in response to antiandrogen treatment8. These mutations can render cells refractory to androgen deprivation as there is constitutive AR activation, even in the absence of androgens. This results in activation of AR including AR binding to AREs and constitutive AR-regulated gene expression. More recently, a role for AR in the progression of breast cancer has been described. While AR’s function has not been fully characterized in breast cancer, work done in prostate cancer informs the potential function of AR in breast cancer.The AR has been well characterized as a key driver for the growth of prostate cancer in men. In this context, castration or androgen deprivation therapy (ADT) is a first line of therapy for men with metastatic prostate cancer. Despite the efficacy of ADT, resistance is near-universal. In some men, resistance can be mediated by AR amplification9. Breast cancers that express the ER (ER+) grow in response to the presence of estrogen and are more responsive to endocrine ablation11. This understanding led to some of the first “molecularly targeted therapies” that established the use of aromatase inhibitors (AIs) or selective estrogen receptor modulators (SERMs), which block the production and signaling of estrogen12. AIs and SERMs have been used as effective therapies for women with tumors that express ER14. Despite having identified the presence of AR expression in breast cancer many years ago15, little is known about the role of androgen signaling in breast cancer, though its importance as a potentially effective therapeutic target is increasingly appreciated and will be discussed herein. We begin with a review of the various processes known to be mediated by AR signaling, as recent studies have shed light on the role of AR with other pathways known to be abrogated in cancer.Similar to the role that AR plays in prostate cancer development and progression, the ER has been recognized for the integral role that it plays in driving the development of the majority of breast cancers16. More recently, three distinct classes of alterations in FOXA1 have been described in prostate cancer, each with unique structural and phenotypic consequences17. The Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP and are found in the wing-2 region of the DNA-binding forkhead domain. Functionally these mutations allow for enhanced chromatin mobility and binding frequency and strongly transactivate a luminal AR program. The second class of activating mutations are found in metastatic prostate cancer and are characterized by a truncated C-terminal domain. These mutations increase FOXA1 DNA affinity and promote metastasis by activating the Wnt pathway through TLE3 inactivation. The final class of FOXA1 genomic rearrangements are characterized by duplications and translocations within the FOXA1 locus that reconfigure regulatory elements (FOXA1 mastermind elements) to drive overexpression of FOXA1. This third class of alterations is found primarily in metastatic prostate cancer and further underscores the interaction and significance of AR and FOXA1 protein interactions17. Similar classes of alterations also been observed in breast cancer1. In prostate cancer, FOXA1 also influences the ability of AR to bind DNA and control cell cycle progression. FOXA1 binds to genes necessary for growth of castration-resistant prostate cancer (CRPC), suggesting that FOXA1 is responsible for driving cell cycle progression in CRPC both from G1 to S and G2 to M18. FOXA1 also facilitates cell cycle progression from G2 to M by acting as a cofactor for AR18. Unsurprisingly, there is also significant overlap between genomic binding sites occupied by AR and FOXA119. While AR binds to many DNA regions independent of FOXA1, DNA-binding sites often require the presence of FOXA1 for AR recruitment19. Therefore, loss of FOXA1 results in the inability of AR to bind many DNA loci19. Using H3K4me2 ChIP analyses, Sahu et al. found that there were H3K4me2 marks at ~70% of sites shared by AR and FOXA119. Furthermore, staining of FOXA1 has been shown to correlate with disease outcomes in prostate cancer patients, where even with high AR staining, low FOXA1 is associated with good prognoses, and strong FOXA1 staining correlates with poor prognoses19, indicating that FOXA1 may have an important effect on AR signaling and tumor progression. Levels of FOXA1 are also elevated in prostate tumors and metastases, and overexpression of FOXA1 in prostate cancer cell lines results in increased AR binding at novel sites that have high chromatin accessibility20. These results suggest that increased levels of FOXA1 enhance AR binding to novel sites in order to facilitate cancer cell growth20 and implicate the importance of FOXA1 on AR function and tumor progression.FOXA1 is a transcription factor which plays an important role in aiding the binding of hormone receptors to their target DNA21. Similar to prostate cancer, ChIP-seq studies have shown that there is extensive overlap between locations of AR and FOXA1 binding in breast cancer cells22. The function of AR in breast cancer is also dependent upon FOXA1, as silencing of FOXA1 inhibits AR binding of target DNA as well as cell growth22. In addition, FOXA1 functions as a transcription factor, playing an important role in aiding binding of hormone receptors, including ER and AR, to their target DNA23. When expressed with AR, FOXA1 may direct AR binding at sites of ER binding in luminal tumors24. Notably, co-expression of AR and FOXA1 was observed by immunohistochemistry (IHC) in ~15% of triple-negative breast cancer (TNBC) patients24, and AR-positive (AR+), FOXA1-positive (FOXA1+) patients had a significant decrease in recurrence-free survival and overall survival compared to TNBC patients25. These findings suggest that when co-expressed in TNBC, AR, and FOXA1 may be mediating an estrogen-like gene signature similar to those expressed in luminal breast cancers. FOXA1 has been studied extensively in the context of ER chromatin binding, and ER binding is dependent on FOXA1 in the presence or absence of ligand23. Further, similar to findings in prostate cancer, 1.8% of breast cancers harbor mutations in FOXA1, and amplifications of the FOXA1 gene locus have been observed in breast and prostate cancers26. Notably most identified mutations are in the forkhead domain of FOXA1, and tumors in this study were exclusively ER+. The implications of these mutations, however, is still under investigation in breast cancer. Interestingly, differences exist between the function of FOXA1 in directing AR binding in breast versus prostate cancers, and future studies may investigate the varied roles of FOXA1 in directing AR binding in TNBC and prostate cancer, in addition to investigating the role of AR when co-expressed with ER. Current literature suggests, however, that regardless of tumor type, FOXA1 is an important cofactor for directing the transcriptional activity of AR.FOXA1 is also essential for the growth of ER+ breast cancer cell lines27. In prostate tumors, with loss of PTEN, there are decreased levels of AR signaling28. Inhibition of PI3K in these tumors results in increased levels of AR signaling through loss of human epidermal growth factor receptor 2 (HER2)-mediated feedback inhibition of AR28. A direct physical interaction between AR and PTEN in low passage LNCaP cells has been shown to inhibit nuclear translocation of AR resulting in an increase in degradation of AR protein29. A pilot study suggested that high expression of both AR and PTEN in patients with advanced prostate cancer was associated with a higher risk of relapse at 30 months after surgery (85.7% of high AR and PTEN expressing patients verses 16.6% in patients with low AR and PTEN expression)30. Further, combination therapy with both antiandrogen  and PTEN induction was shown to reduce prostate-specific antigen (PSA) promoter activity compared to PTEN alone31. Sequencing of metastatic-CRPC (mCRPC) patients revealed that AR and PTEN are among the most commonly aberrant genes, along with the ETS family and TP5332. Therefore, these data suggest that both AR and PTEN may influence prostate tumor growth and progression.Expression of AR with PTEN has also been investigated in prostate cancer. In prostate cancer patients, high AR expression with low PTEN expression is associated with poor clinical outcomesPTEN promoter, and there is a positive correlation between AR and PTEN transcript levels27. In addition, high expression of AR and PTEN is correlated with better clinical outcomes for breast cancer patients27. Interestingly, in AR + TNBC, AR interacts at an ARE located in the promoter of ERβ33, and ERβ also plays a role in regulation of PTEN expression to control tumor growth34. The interaction between AR and PTEN may be context specific and important for predicting outcomes for patients with AR+ disease: where AR expression is associated with disease progression in prostate cancer7, PTEN loss is also correlated with poor outcomes36. In breast cancer, however, loss of PTEN is also correlated with negative ER and PR status, and PTEN loss is associated with breast tumor progression37. Therefore, these results suggest that the function of PTEN may be context specific and understanding the nuances in situational signaling of AR may help elucidate the role for PTEN in AR+ disease progression.There are opposing findings when comparing AR and PTEN transcript expression in prostate verses breast cancer. In breast cancer, there is an AR-binding motif located in the 38. The activation of the Src/ERK pathway is dependent on androgen concentration (0.1–10 nM) and is inhibited at high concentrations (100 nM)39. Treatment with dihydrotestosterone (DHT) also induces rapid ERK1/2 phosphorylation; however, MAPK activation can be blocked pharmacologically using a MEK inhibitor, suggesting AR is activating the Raf1-MEK pathway resulting in MAPK activation40. Further, AR can also activate the phosphatidyl-inositol 3-kinase (PI3K)/Akt pathway leading to activation of mammalian target of rapamycin (mTOR)41. In addition, androgens can interact at the plasma membrane which is associated with the modulation of intracellular calcium and cAMP levels42. Many membrane-bound G-protein coupled receptors are also responsive to androgen treatment, leading to an increase in apoptosis43, phosphorylation of ERK44, or reduced cell migration and metastasis45. Together, these findings suggest that AR may also function within the cytoplasm or at the membrane to activate non-genomic functions.Prostate cancer cells exhibit rapid proliferation responses in response to androgen stimulation, suggesting non-genomic AR signaling. Upon activation with androgens or estrogens, cytoplasmic AR can activate MAPK/ERK signaling through an association with Src46. Further, inhibition of AR resulted in decreased levels of phospho-Elk1, phospho-RSK, and c-FOS in xenograft tumors and in patient tumors, corresponding to a decrease in ERK target proteins46. In TNBC, AR inhibition has also been shown to modulate the activity of the Ca2+-activated K+ channel, KCa1.1, which is associated with breast cancer invasion and metastasis48. Multiple groups have also studied the role of cytoplasmic AR phosphorylation50; however, additional work is required to understand how AR modifications influence cellular function and localization. At the membrane, many receptors mediate rapid responses to androgen signaling, representing novel membrane-ARs52. These signals, however, are complex as agonistic verses antagonistic effects are dependent on receptor stoichiometry52. Furthermore, AR is expressed in fibrosarcoma cells; however, a significant portion of AR is transcriptionally incompetent and does not bind to AREs upon activation. Rather, there is crosstalk between EGFR and AR, and treatment with bicalutamide decreases xenograft tumor growth53. Together these data from multiple cancer models suggest that AR has non-genomic functions affecting tumor growth both in prostate and breast cancer which warrant further investigation.Similar to non-genomic AR functions in prostate cancer, the cytoplasmic roles of AR have also been investigated in breast cancer. Chia et al. demonstrated that AR is necessary and sufficient for ERK phosphorylation following DHT stimulation in MDA-MB-453 and HCC-1954 cells54. Aberrant splicing of AR pre-mRNA, however, can result in the production of AR isoforms that are constitutively active. These isoforms can drive an AR transcriptional program even in the absence of androgen signaling, resulting in androgen independent tumor growth56. AR variants (ARVs) are present both in prostate cancer and breast cancer, and these variants commonly are truncated or have mutations in the AR LBD57. In addition, AR transcripts can have aberrant splicing, resulting in skipped exons58. ChIP studies have demonstrated that splice variants, including AR-V7, are able to bind canonical AREs as well as unique regions of additional genes58. AR splice variants have been shown to require expression of full-length AR (AR-FL) suggesting that a balance between ARV and AR-FL expression is required for resistance in prostate cancer models59.A number of AR splice variants have been identified, and they play an important role in the development of CRPC. The gene encoding AR is located on the X chromosome, encoding nine exons that produce the full-length AR transcript60. Clinically, in a cohort of prostate cancer patients treated with enzalutamide or abiraterone acetate, 39 patients (19%) had detectable levels of AR-V7 in circulating tumor cells (CTC)61. Patients with AR-V7 expression had lower PSA response rates and worse survival compared to AR-V7 negative patients61. In addition to reliance on AR-splice variants, resistance to ADT is also mediated through signaling of additional hormone receptors. The GR has been shown to be increasingly present in androgen-deprived prostate cancer patients (78% vs. 38% of untreated patients)62, and expression of GR is increased in xenografts that are resistant to ARN-509 63. In addition, there is overlap between AR and GR binding at classic response elements as well as regulation by both DHT and dexamethasone, a GR agonist63. In prostate cancer cells, stimulation with dexamethasone in the presence of enzalutamide resulted in expression of AR target genes63, providing further evidence that GR signaling could compensate for AR in the presence of AR-antagonists.The most common splice variant, AR-V7, lacks an LBD60. Little is known about how AR-V7 may be contributing to antiandrogen resistance in AR+ TNBC or if it is functioning similarly to its observed role in CRPC64. In HER2-enriched patients, however, high AR-V7 expression is associated with significantly higher metastasis-free survival and disease-specific survival60. Therefore, the ability of a tumor to produce its own androgens, as well as its reliance on splice variants may also play an important role in understanding how AR is functioning to drive tumor growth in the context of ADT or antiandrogen therapies.There are significantly fewer AR mutations observed in TNBC compared to CRPC; however, AR splice variants are still common. In breast cancer, AR-V7 is most highly expressed splice variant in basal tumors compared to other tumor types, with the lowest expression in luminal tumors65. Notably, MDA-MB-453 cells contain the AR-Q865H variant which harbors a mutation in the AR LBD66, demonstrating the importance of considering the influence of ARV expression in laboratory studies. Furthermore, understanding the similarities and differences of how ARVs may be influencing AR expression and contributing to breast tumorigenesis will be important.Importantly, differences also exist in preclinical cell lines used to study AR+ breast cancers. While common cell lines, including MDA-MB-453, MDA-MB-231, ZR-75-1, MFM-223, MCF-7, and T47D, have varying levels of AR-FL expression, ARV expression also varies widely among cell lines—both in total ARV expression and expression of specific ARVs67, and AR status is related to ER and PR status but independent of the status of HER268. Interestingly, patients with AR:ER ratios ≥2 have worse disease-free survival compared to patients with lower AR:ER ratios in the presence of antiestrogen therapies or chemotherapy treatment69. Defining expression of AR and ER, however, is challenging, and results vary widely depending on the assays  and cut-offs used to define positivity. Clinically, ER expression is measured by IHC, and ER+ tumors are defined as those with >1% of tumor cells with positive nuclei70. AR positivity, however, has been defined with varying cut-off levels from 1 to 75%71. The prognostic role of AR in breast cancer remains unclear. A recent study has demonstrated that >78% AR positivity is required to accurately assess the prognostic role of AR in ER+ cancers, with ER+ patients that have ≥78% AR positivity having the best survival outcomes72. In other studies, however, breast cancer patients with AR+ tumors have better overall survival at both 3 and 5 years compared to patients with AR− tumors, regardless of ER expression67. These data suggest that the role of AR in driving breast cancer growth may differ in the presence or absence of ER and that antagonizing AR may have different effects depending on the level of AR expression.Breast tumors that are ER+ are more likely to be AR+ compared to tumors that are ER−33. In ER+ breast cancer, AR binds at an ARE located in the promoter of the ERβ gene, resulting in increased ERβ expression33. Interestingly, the presence of ERβ has been shown to inhibit transcriptional activity of ERα73, therefore, suggesting that AR-regulated increased activity of ERβ may indirectly influence ERα activity. Similarly, in prostate tissue, ERβ is thought to play an antagonistic role to AR, resulting in the suppression of cellular proliferation and the promotion of apoptosis74. ERβ is also important for the control of cell cycle progression and arrest76, indicating that increasing ERβ expression may be a therapeutic strategy in prostate cancer77.AR expression in ER+ breast cancers antagonizes the signaling of mitogenic ERα, and AR expression leads to the upregulation of ERβ78. Patients resistant to tamoxifen with AR:ERα ratios ≥2 have worse disease-free survival, and disease-specific survival79. Interestingly, in tamoxifen-resistant MCF-7 cells, loss of AR signaling by AR knockdown, but not treatment with enzalutamide, restored sensitivity to tamoxifen80. These results suggest that AR expression may be a mechanism of hormone therapy resistance, and therefore a therapeutic target in resistant hormone receptor positive breast cancers.In contrast to early studies suggesting high AR expression is associated with improved outcomes, recent data suggest high AR expression may be associated with therapy resistance, including endocrine therapy resistance. Indeed, De Amicis et al. first reported the positive correlation between high AR expression and tamoxifen resistance, suggesting that tumors with a high AR:ER ratio are more likely to be resistant to antiestrogen therapies, which are common first line of therapy for ER+ tumors81. Enzalutamide has been shown to be effective in AR+ breast tumors, including ER+ (MCF-7) cells and ER− (MDA-MB-453) cells82. ChIP analyses demonstrate that there is extensive overlap between AR and ER binding sites after E2 stimulation in MCF-7 cells81. Interestingly, however, AR binding was different based on stimulation with DHT or E2 in MCF-7 cells suggesting that AR may regulate a unique transcriptional program in the absence of estrogen signaling, providing additional evidence for synergism between antiestrogen and antiandrogen therapies81. These results indicate that targeting AR in combination with anti-ER therapies may be an effective therapeutic strategy for patients with AR+, ER+ breast cancers.Anti-AR therapy is of increasing clinical interest. AR inhibition may be an effective strategy for growth inhibition of AR+, ER+ breast tumors. AR inhibition with enzalutamide has been shown to be synergistic with tamoxifen- or fulvestrant-mediated ER inhibition, in addition to controlling growth of tamoxifen-resistant MCF-7 cells in vitro and in vivo in an AR+, ER+ patient-derived xenograft model83. ER and AR recognize similar sequences of DNA: where ER binds to 5'-AGGTCA-3', AR recognizes the 5'-AGAACA-3' sequence84. Further, in breast cancer, both AR and ER require similar cofactors for the activation of common signaling pathways83. Binding of AR or ER can activate MAPK signaling, among other pathways83, and due to their similar structure and signaling function, both hormone receptors are in competition within the cell for the binding of scaffold proteins and cofactors83. While AR and ER share many similarities, there may be important differences determining their role in driving tumor growth.Functionally, AR and ER share many similarities in their signaling pathways, including the mechanism of receptor activation, as both receptors are activated through ligand binding85. Breast cancers that do not express ER, PR, and HER2 have been traditionally described as TNBC. Recently, however, a subtype of TNBC has been established which is characterized by luminal AR expression87. In studies with AR+ human breast cancer cell lines, androgens had both proliferative and antiproliferative effects depending on the cell line of interest88. More recently, however, multiple groups have demonstrated that targeting AR in AR+ TNBC is an effective treatment strategy both in vitro and in vivo90. Interestingly, in AR+ TNBC, ~30% of patients have expression of ERβ91, and ERβ expression has been shown to increase the efficacy of antiandrogens in AR+ TNBC cells92. Together these data demonstrate the importance of AR in driving growth of AR+ TNBCs.The function of AR in breast cancer appears to be dependent upon its co-expression with ER, as there is evidence for varying effects of AR on the growth of breast cancer cells in the presence or absence of ER. Indeed, while AR is co-expressed with ER in 70–90% of breast tumors, AR is only expressed in 15–30% of ER-negative breast tumors93. In an analysis of AR+, ER− MDA-MB-453 cells, the AR cistrome was found to be more similar to that of ER in MCF-7 (AR−/ER+) cells compared to the AR cistrome in LNCaP prostate cancer cells22. Therefore, AR may function in place of ER in AR+, ER− breast cancer22, having a distinct role in AR+ TNBC compared to prostate cancer. AR may also be important for promoting the cancer stem cell-like (CSC-like) population in TNBC, in addition to reducing the levels of detachment-induced apoptosis in cells grown in forced suspension compared to attachment conditions94. These results suggest that AR blockade may be effective in combination with paclitaxel to target CSC-like cells and reduce tumor recurrence compared to paclitaxel treatment alone94. In addition, AR is commonly enriched in breast cancers overexpressing HER2, indicating a role for AR in activation of HER2 and Wnt signaling95. Therefore, AR expression may be an important target for directing treatments for patients with ER- breast cancer.While AR expression has been increasingly recognized in AR+, ER− breast cancers, the specific role of AR signaling is not well understood. Some studies suggest an important role for AR in signaling in the absence of ER96. Additionally, in response to ionizing radiation, CRPC cells have enhanced DNA repair and decreased DNA damage97. AR activation results in the expression of DNA damage repair genes including PRKDC, encoding DNA-dependent protein kinase catalytic subunit (DNA-PKcs), an essential protein necessary for nonhomologous end joining (NHEJ) repair of double-stranded DNA (dsDNA) breaks96. In addition, treatment with radiation and androgens results in the upregulation of XRCC2 and XRCC3, two genes important for homologous recombination (HR)96. Conversely, antiandrogen treatment results in decreased DNA repair in cells and increased levels of dsDNA breaks97. The same group also showed that treatment with AR inhibitors results in increased radiosensitivity and decreased NHEJ-mediated recombination suggesting that AR is involved in NHEJ-mediated repair of dsDNA breaks97. DNA-PKcs has been shown to function in complex with Ku70 and Ku80 to respond to DNA damage. Interestingly, DNA-PKcs physically interacts with AR; however, this interaction does not require the presence of DNA98. This suggests that AR regulation of the DNA damage response may not be completely dependent on AR-mediated transcriptional regulation of DNA damage response genes. Following androgen stimulation in prostate cancer cells, AR is recruited to enhancer elements, along with DNA-PKcs, coregulator p300, and RNA Pol II suggesting that the interaction of AR and DNA-PKcs may be important for the regulation of specific transcriptional programming98. Therefore, an interaction between AR and DNA-PKcs may also be important for AR’s role in the repair of DNA damage. In patient tissue, castration resulted in the downregulation of Ku70 protein levels, impairing NHEJ99. AR regulates Ku70 levels in prostate tissue, and due to the critical role of Ku70 in effective NHEJ, downregulation of this protein abrogates NHEJ-mediated repair99. Collectively these data suggest that AR signaling plays an important role in the repair of dsDNA breaks, at least in part through interactions with Ku70/Ku80 and DNA-PKcs, members of the NHEJ repair pathway.While the mechanism of AR in response to DNA damage is just beginning to be uncovered in breast cancer, the mechanistic role of AR in DNA damage repair has been more extensively characterized in prostate cancer. Goodwin et al. found that AR is activated in response to reactive oxygen species (ROS) and DNA damage101. In addition, AR mRNA levels correlate with survival following radiation treatment, and AR is important for regulating the DNA damage response in AR+ breast cancer cell lines102. Pharmacologic AR inhibition results in delayed repair of dsDNA breaks following ionizing radiation, suggesting that AR is influencing dsDNA damage repair. Additionally, AR inhibition with enzalutamide decreases levels of phosphorylated DNA-PKcs following radiation, indicating that NHEJ may be important for the repair of radiation-induced dsDNA breaks in breast cancer100. Although some similarities exist between the role of AR in DNA damage repair in prostate and breast cancers, a full characterization of the similarities and differences is still ongoing.Recent data in breast cancer suggest that loss of AR signaling through knockdown or pharmacologic inhibition with enzalutamide or seviteronel results in increased sensitivity to ionizing radiation103. In prostate cancer, AR is regulated in a cell-cycle-dependent manner104. Nuclear transactivation of AR is highest in G1 and decreases in S phase, while the same changes occur in AR phosphorylation and cellular localization104. Further, CDK1 has been shown to phosphorylate AR on S308 in response to ligand binding104. The phosphorylation results in changes in AR chromatin localization104. AR signaling is responsible for the activation of genes controlling the G1–S transition105. Specifically, AR is responsible for G1 CDK activation and the phosphorylation of retinoblastoma (pRb), which is necessary for the activation of CDKs that will drive the G1–S phase progression105. In the absence of androgen signaling, prostate cancer cells will arrest in early G1 phase as they do not have expression of the necessary CDK and cyclin proteins107. AR and pRb have also been shown to interact, and an overexpression of pRb increases the transcriptional activity of AR109.Cell cycle progression is driven by the rising and falling in levels of cyclins and cyclin dependent kinase (CDKs), in addition to the activation of these proteinsCCND1 expression110. Importantly, CCND1 encodes cyclin D1 which has an interaction with pRb that is necessary for cell cycle progression. AR binds to AREs that are located ~570–556 base pairs upstream of the transcription start site of the proximal promoter of CCND1, suggesting that AR plays a regulatory role to influence CCND1 expression110. In prostate cancer cells, following treatment with androgens, there is induction of expression of CDK inhibitors p21 and p27111. Expression of p21 is controlled at the transcriptional level through the presence of an ARE in the promoter region, ~200 base pairs upstream of the proximal promoter112. AR signaling has been shown to be important for control of cell cycle-related gene expression, resulting in growth implications in tumor cells.AR signaling is also important for the regulation of other cell cycle related genes, including the regulation of 113. AR expression is also regulated by AREs as well as MYC binding at the consensus site114. Thus, in addition to its role cell growth and the DNA damage response, AR expression and activation is itself regulated in a cell cycle-dependent manner which then influences expression of CDK and transcription factors to regulate progression through the cell cycle.Additionally, in prostate cancer cells, the synthetic androgen mibolerone inhibited proliferation and reduced levels of c-MYC transcripts, suggesting that AR is important for regulating c-MYC levels115. Conversely, treatment with bicalutamide resulted in a decrease in cyclin D1 expression, while increasing p73 and p21 levels, implicating a role for AR in the control of cell cycle progression in AR+ TNBC models115. The expression of AR and pRb in breast cancer is also significantly correlated, and AR has been shown to interact with other transcription factors, including MYC, which are important for cell cycle control116. In breast tumors, high AR expression is negatively correlated with MYC overexpression116. MYC expression has been linked to cell proliferation, and inactivation of MYC impairs cell cycle progression as MYC targets cell cycle regulators like cyclins, CDKs, and E2F transcription factors117. Additionally, in breast cancer models, the presence of an ARE −383 to −377 base pairs upstream of the ERβ promoter region results in enhanced control of ERβ expression as a result of AR signaling33. ERβ has been shown to negatively regulate transcription of c-MYC, cyclin D1, and cyclin A, while also increasing transcription of CDK inhibitors like p21 and p2733. In ER+ breast cancer models, DHT-mediated activation of AR has been shown to inhibit ERα signaling and cell cycle progression through a reduction in cyclin D1 transcription118. Further, AR and ER both require the steroid receptor coactivator AIB1118 which is commonly expressed in breast cancers120, and high AIB1 expression is correlated with poor mortality121. Therefore, through direct or indirect mechanisms, AR signaling likely also plays an important role in controlling cell cycle progression in breast cancer.In addition to interactions with cyclins and CDKs, AR also interacts with many other important proteins, including well characterized oncogenes and tumor suppressers. In AR+ TNBC, DHT has been shown to increase levels of cyclin D1, while decreasing p73 and p21 expression122. Gene array and IHC data of both primary and metastatic tumors demonstrate that AR mRNA and protein expression are significantly higher in metastases compared to primary prostate lesions123. In vitro, increased AR expression in prostate tumors also led to the formation of metastases and induction of the epithelial to mesenchymal transition (EMT)123, the process by which cells lose their polarity and gain the ability to migrate and become invasive. In addition, during prostate cancer development, the presence of fibroblasts provides important structural and functional changes that regulate the extracellular matrix124. Expression of nuclear receptors has been shown to be important in squamous cell carcinoma cancer-associated fibroblasts (CAFs) compared to normal-associated fibroblasts, with nuclear receptors influencing many cellular functions including invasiveness125. Additionally, AR expression in prostate CAFs has been shown to promote growth and invasion126. AR activation in the stroma has been shown to be essential for prostate cancer progression and metastasis127. Interestingly, the AR cistrome in prostate CAFs is distinct from the AR cistrome in epithelial cells suggesting a novel role for AR in the microenvironment128. Notably, AR relies on AP-1 in the stroma, rather than FOXA1 as observed in epithelial cells128.AR has been shown to contribute to the formation of metastases. The AR pathway and AR splice variants have been implicated in metastatic phenotypes in prostate cancer129. Therefore, disruption of AR signaling can result in overexpression of ZBTB46 resulting in an increase in transcriptional regulation of SNAI1, a driver of EMT, resulting in metastasis formation129. Further, AR inhibition with enzalutamide has been shown to increase metastases by decreasing EPHB6 suppression leading to JNK signaling resulting in cell invasion130. These findings suggest that AR plays an important role in controlling metastatic progression of prostate tumors, demonstrating the importance of future work in this area.Furthermore, the regulatory role of AR in gene expression has been shown to be important for the regulation of prostate cancer metastases. In this context, AR negatively regulates expression of ZBTB46, a tumor promoter through miR-1131. When MCF-7 cells were treated with DHT, there was also an increase in invasion and migration, as well as a decrease in epithelial markers and an increase in mesenchymal markers132. DHT treatment also induced other markers of EMT suggesting that AR activation may promote EMT in MCF-7 cells132.In patients with breast cancer, metastases are likely to have multiple drivers of disease progression. In preclinical models, AR has also been shown to contribute to invasiveness and migration of TNBC cells through activation of the Src complex134. One study suggests that there is 78.6% agreement in AR status in primary tumor and lymph node metastases135. In the discordant cases, 60/72 had AR- primary tumors, and AR+ lymph node metastases135. Further, IHC analyses in tumors and metastases showed greater than 60% agreement between the expression of AR in primary tumor and metastases136.As with prostate cancer, previous data from breast cancer patients demonstrates that AR expression is conserved from the primary tumor into metastasesPharmacological agents have been developed to inhibit AR binding to androgens and AR activation due to its role in driving cancer development and progression Fig. . Many of137. This treatment attempts to lower levels of serum testosterone in men with prostate cancer to prevent tumor growth138. This is done chemically with the use of luteinizing hormone-releasing hormone (LHRH) or gonadotrophin-releasing hormone (GnRH) antagonists, like Degarelix, Goserelin, and Leuprolide, which are used to suppress the production of androgens137. Many clinical trials also are assessing the efficacy of ADT in combination with other treatment strategies in an attempt to improve ADT efficacy, especially in cases where AR mutations cause castration to be ineffective at controlling disease progression.The use of ADT is universally accepted as a first line therapy for metastatic prostate cancer140. Competitive inhibitors of 5α-reductase, like finasteride or dutasteride, can be used to lower levels of serum and prostate DHT144. The effects of these 5α-reductase inhibitors, however, are complex as they may not exclusively target the enzymatic activity of 5α-reductase and likely have additional off-target AR inhibitory effects as well145.5α-DHT is produced from testosterone in specific tissues, including the prostate, through the enzymatic activity of 5α-reductase. Compared to testosterone, DHT has a slower dissociation rate from AR, suggesting that AR-DHT is a more stable complex, making DHT the preferred AR ligand146. CYP17-lyase inhibitors lower androgen availability to reduce the activation of androgen signaling. Trials in men with chemotherapy-naive CRPC concluded that treatment with abiraterone acetate and prednisone prolongs overall survival compared to treatment with prednisone alone (NCT00887198)147. In a phase II trial for women with triple negative, AR+ locally advanced or metastatic breast cancer (NCT01842321), treatment with abiraterone acetate and prednisone also provided benefit for some patients148. Of 138 patients assessed for the trial, 53 (37.6%) had AR+ TNBC: 34 of these patients were included. This trial assessed the clinical benefit rate (CBR) for 30 of the patients at 6 months with a CBR of 20.0% (95% CI: 7.7–38.6%) including one patient who had a complete response (CR) and 5 patients with stable disease (SD) at ≥6 months148. Secondary outcomes included objective response rate , and progression-free survival 148. These studies suggest that treatment with abiraterone acetate may be a beneficial treatment strategy for both men with CRPC and women with molecular apocrine breast cancer.Abiraterone acetate is a selective inhibitor of cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17), which, through its function, decreases the adrenal and tumoral synthesis of androgens149. Galeterone has been shown to be effective in reduction of PSA levels and was well-tolerated by patients in early clinical trials150. Orteronel treatment is effective at suppressing testosterone levels and shrinking the androgen-dependent organs including the prostate gland151. Phase III clinical trials found that orteronel and prednisone treatment verses placebo and prednisone gave patients longer progression-free survival (PFS); however, men with orteronel and prednisone treatment did not have extended overall survival152. In breast cancer, there are currently phase I and II clinical trials assessing the use of orteronel in patients with metastatic breast cancers that express AR . NCT01808040 assesses the safety of orteronel use for the treatment of postmenopausal women with hormone receptor positive metastatic breast cancer in addition to measuring the estradiol levels in these patients following treatment153. NCT01990209 is a phase II trial for male or female patients with metastatic AR+ BC (TNBC or ER+ and/or PR+ BC) with primary outcome measures of response and disease control rates. This trial will also assess safety, PFS, OS, and serum hormone levels in addition to screening tumors for PTEN expression and PIK3CA mutations. Due to failure in phase III clinical trials in men with prostate cancer, orteronel was taken out of development in 2014154.Other CYP17-lyase inhibitors include galeterone (TOK-001) and orteronel (TAK-700)155. Flutamide and bicalutamide are two such agents that have been used to block androgen binding and abrogate nuclear AR signaling. Although AR targeting has been a strategy for over 30 years, original phase II clinical trials with flutamide suggested it did not have antitumor activity which delayed the initiation of further trials with the drug156. Recent studies, however, have shown that flutamide treatment is effective and well-tolerated for treating PSA recurrence following prostatectomy, radiation therapy, or cryotherapy for patients with prostate cancer157. In addition, in breast cancer, bicalutamide has been shown to have a CBR of 19% in patients with AR+, ER−, PR− metastatic breast cancer where 12% of tumors were AR+158. These results suggest that antiandrogen therapies are effective for the treatment of patients with traditionally hormone receptor-negative breast cancers. Unfortunately, in prostate cancer, it has been shown that exposure to antiandrogens can augment frequency of AR mutations and variants159, and metabolites of antiandrogens can result in stimulation of prostate cancer cell growth as flutamide metabolites function as an AR agonist160. There are additional ongoing clinical trials that are assessing the use of flutamide as a second line treatment of patients with CRPC who have relapsed after ADT and bicalutamide treatment (NCT02918968) or using flutamide treatment to prevent prostate cancer in patients with neoplasia of the prostate (NCT00006214). In addition, NCT02910050 is investigating the use of bicalutamide with AIs in AR+, ER+ breast cancers161.Antiandrogens are a class of agents which act as nonsteroidal competitive inhibitors of the AR146. In addition to growth inhibition, enzalutamide also can inhibit tumor cell migration and invasion162. In mCRPC patients who had previously received chemotherapy treatment, treatment with enzalutamide also contributed to prolonged survival (NCT00974311)163. In breast cancer, a Phase II trial (NCT01889238) for women with advanced, AR+ TNBC tested the use of enzalutamide for improving outcomes and CBR for patients at 16 weeks (CBR16) as well as assessing clinical benefit at 24 weeks (CBR24), PFS, response rates, and safety of enzalutamide treatment164. This study also found that 47% of the 118 enrolled patients had an AR related gene signature, and clinical outcomes were better for patients with AR+ disease164. No new side effects were reported from enzalutamide treatment in this trial, indicating its potential use as a therapeutic for women with TNBC164.Four FDA-approved second generation antiandrogens, abiraterone acetate, apalutamide, darolutamide, and enzalutamide, improve upon the first-generation antiandrogens. Enzalutamide is able to inhibit the growth of both ER+ and ER− breast tumors by inhibiting AR nuclear translocation149. Apalutamide has a seven- to ten-fold increased binding affinity to AR compared to bicalutamide165. In preclinical studies, apalutamide had antitumor activity in a castration-sensitive model of prostate cancer166. There were also lower levels of apalutamide in mouse steady-state plasma and brain levels compared to enzalutamide treatment, which could indicate lower frequency of seizures with apalutamide166. In preclinical studies, apalutamide also had antitumor and growth inhibitory effects in AR+ TNBC cells166. Results from the SPARTAN trial, a Phase III clinical trial (NCT01946204) for men with nonmetastatic castration-resistant prostate cancer (nmCRPC), demonstrated improved metastasis-free survival in patients treated with apalutamide compared to placebo167. Following this trial, apalutamide was approved by the FDA for treatment of nmCRPC165. To date, there have been no trials with apalutamide in patients with AR+ breast cancer.Apalutamide (ARN-509) is a second generation AR antagonist, similar to enzalutamide, that binds to the LBD of AR to inhibit nuclear translocation and ARE binding149. In addition, darolutamide can be effective against mutant ARVs which can develop with resistance to enzalutamide and apalutamide therapy168. In prostate models, darolutamide has low brain-penetrance and treatment does not produce an increase in mouse serum testosterone levels168. Recently, results from the ARAMIS trial (NCT02200614), a phase III trial for nmCRPC patients, demonstrate that darolutamide provides better metastasis-free survival compared to placebo169. The START trial is a phase II trial for women with AR+ TNBC comparing darolutamide treatment with capecitabine, an antimetabolite chemotherapeutic (NCT03383679). This trial investigates CBR16 as a primary objective, and CBR24, response rates, overall survival, PFS, and safety as secondary objectives for women with locally recurrent or metastatic AR+ TNBC.Darolutamide (ODM-201) is an AR inhibitor that binds wild-type AR with a higher affinity than enzalutamide to block AR nuclear translocation170, rendering it a potentially effective alternative to agents which either inhibit androgen production or AR activation. Clinical trials for patients with ER+ or TNBC indicated that seviteronel was well-tolerated in women, with the majority of adverse events (AEs) being Grade 1/2, in addition to four Grade 3/4 AEs that may be related to seviteronel treatment171. Phase I trials in CRPC patients suggest that seviteronel may be an effective treatment alternative for men who are not responsive on other therapies with most reported AEs being Grade 1/2172. Preclinical work in AR+ TNBC demonstrates that seviteronel inhibits cell proliferation and growth on soft agar173. ChIP-seq and RNA-seq analyses demonstrate that AR-regulated genes are increased with DHT stimulation and decreased in mice treated with seviteronel173. Trials with seviteronel continue to be ongoing for patients with CRPC, AR+ TNBC, or men with ER+ breast cancer, who had previously been treated with enzalutamide . The CLARITY-01 trial (NCT02580448) is assessing the CBR at 16 or 24 weeks for women with ER+ or TNBC or men with locally advanced or metastatic breast cancer who are receiving seviteronel treatment174. Of the patients enrolled for stage 1, CBR16 for TNBC patients was 2 of 6, and CBR24 for ER+ patients was 2 of 11174. Of patients with CTCs, 7 of 10 had a CTC decline at C2D1174. Patients receiving seviteronel also had a decline from baseline in concentrations of estradiol and testosterone175. The most common AEs were tremor, pain, fatigue and dyspnea, nausea, AST increase, ALT increase and abdominal pain, suggesting that seviteronel was well-tolerated175. These results indicate that seviteronel may be a potential therapeutic option for the treatment of AR+ disease.A number of novel compounds have also been developed to block or abrogate androgen signaling. Seviteronel (VT-464) is a nonsteroidal selective CYP17-lyase inhibitor and AR antagonist that both blocks testosterone and estrogen production and inhibits AR activation176. When injected, 4-OHT is converted to 4-hydroxyandrostenedione (4-OHA), a previously used form of AI that was injected for the treatment of breast cancer176. Both 4-OHT and 4-OHA are unable to be made into estrogens in vivo176. Preclinically, CR1447 has been shown to inhibit growth of AR+ BC cell lines, but not AR knockout cell lines, or those with siRNA-mediated AR knockdown176. Results from a Phase I clinical trial (NCT02067741) indicate that, when topically administered, CR1447 was well-tolerated with grade 1/2 AEs and no dose-limiting toxicities (DLTs) in 12 patients with ER+/HER2− breast cancer176. Two patients (17%) had stable disease after 12 weeks of treatment176. Therefore, CR1447 may also be viable treatment option.CR1447 (4-hydroxytestosterone [4-OHT]) is a novel AR inhibitor that acts both as a steroidal AI as well as an AR antagonist by binding to AR177. Enobosarm has tissue specific activity, with anabolic activity in muscles and bone without affecting growth of hair in women and prostate in men178. It has been well-tolerated by both men and women; additionally, in patients with advanced cancer, treatment with enobosarm leads to an increase in lean body mass179. Enobosarm has also been well-tolerated as an androgen agonist in women with AR+ metastatic breast cancer180. Androgen-based AR agonists have previously been shown to be effective for the treatment of breast cancer181, and enobosarm similarly stimulates AR, but unlike androgens, does not have masculinizing side effects182. A phase II trial (NCT01616758) assesses CBR, and PSA is evaluated as a biomarker of AR activity. In addition, NCT02971761 is investigating the use of pembrolizumab with enobosarm for AR+ TNBC patients183. Enobosarm may soon join the treatment armamentarium.Enobosarm is a selective androgen receptor modulator (SARM) that was originally tested in Phase I, II, and III clinical trials for its use in improving lean body mass and treating cachexia185. In addition, ASOs used against AR mRNA were able to shut down the downstream activation of AR-mediated genes in hormone-independent conditions186. ASO administration in mouse models did not have any observed side effects and, compared to castration, did not result in shrinking of mouse prostates185. Use of ASOs may also be a method for targeting AR splice variants as two ASOs have been used to effectively silence AR-V7, but not AR-FL, signaling in CRPC cell lines187. Therefore, these findings suggest that the use of ASOs may be a useful strategy for overcoming the resistance that often develops to antiandrogens in prostate cancer. In addition, ASOs may also be an effective treatment strategy for targeting mutant ARVs.Antisense oligonucleotides (ASOs) have also been used to inhibit AR-driven gene expression, especially in contexts where AR is activated independent of hormone binding. ASOs bind to mRNA, causing the mRNA to be degraded, therefore reducing levels available for protein synthesis. Prostate cancer models have shown that ASOs are able to reduce AR expression, resulting in decreased cell growth188. Upon ligand binding to the protein of interest, the protein is ubiquitinated by the E3 ubiquitin ligase resulting in degradation by the 26S proteasome. Multiple AR degraders have been developed using PROTAC for use in prostate cancer190, and they have been shown to be more effective than enzalutamide in vitro and in vivo in models of enzalutamide-sensitive and resistant prostate cancer192. Enhanced efficacy of AR degraders in prostate cancer models may demonstrate the importance of removing AR protein as opposed to pharmacologically inhibiting AR activity for the treatment of resistant prostate cancers. In the future, pharmacologic AR degraders may be introduced clinically for the treatment of aggressive AR-driven cancers.Targeted degradation of proteins with the use of Proteolysis Targeting Chimeras (PROTACs) is a novel method for the inhibition of AR signaling in prostate cancer cell models. PROTAC-mediated degradation takes advantage of E3 ubiquitin ligase activity by linking a ligand for the target protein to a ligand for the E3 ubiquitin ligase193. Ketoconazole has also been shown to inhibit enzymes important for testosterone synthesis194 and is under investigation in combination with docetaxel (NCT00212095)195. In addition, TRC253, a novel competitive inhibitor of AR has been shown to be an antagonist to wild-type AR as well as all tested AR mutants196, including AR F877L, a mutation occurring in the LBD of AR197.There are also additional compounds that have limited use in treating AR-driven disease. Ketoconazole is an antifungal agent that is also able to competitively bind to the AR198. Treatment with enzalutamide was also shown to radiosensitize prostate cancer cells more effectively than ADT199. Combination treatment with enzalutamide and radiation therapy resulted in a significant increase in apoptosis and senescence compared to treatment with enzalutamide or radiation alone199. In prostate cancer, radiosensitization was also observed with ARN-50997. In addition, treatment with antiandrogen therapies resulted in the downregulation of DNA repair genes, thereby promoting radiosensitivity through a decrease in NHEJ activity97.Radiotherapy has been shown to induce AR expression in prostate cancer cells, and ADT sensitizes cancer cells to radiotherapy200. Inhibition of AR with enzalutamide results in increased radiosensitization of AR+ breast cancer cells through the inhibition of AR-activated DNA-PKcs-mediated repair100. Similar results were observed with seviteronel, the dual CYP17 inhibitor and AR antagonist101, however the differences in the mechanisms of radiosensitization with these agents need to be further assessed.The AR has been shown to be a potential mediator of radioresistance and a target for the radiosensitization of AR+ TNBC201. Cancers with mutations to BRCA1 or BRCA2 have HR deficiencies, rendering them increasingly susceptible to treatment with PARP inhibitors. Inhibition of PARP in tumors with BRCA mutations results in synthetic lethality and forces cells to rely on NHEJ for repair of DNA breaks. PARP has been shown to be recruited to sites of AR binding and promotes AR function201. When AR is inhibited, HR deficiency and BRCAness is induced202. Therefore, AR activity is important for the maintenance of HR gene expression. Following ADT, PARP levels are elevated leading to prostate cancer cell survival203. Combination therapy of PARP inhibition with ADT may be important for the impairment of HR before the tumors become castration resistant203.Poly ADP-ribose polymerase (PARP) is a nuclear enzyme that modifies substrates through the addition of PAR moieties204. Antiandrogen therapies induce PARP cleavage, resulting in an increase in dsDNA breaks204. This synergy is a therapeutic target for CRPC patients with mutations in DNA damage repair. Therefore, cancer cells with DNA damage repair mutations are more sensitive to PARP inhibitors due to the role of the AR in the transcriptional regulation of DDR genes205.PARP also plays an important role in the AR signaling cascade. Combination treatment with the PARP inhibitor talazoparib with enzalutamide or abiraterone acetate has significant synergyBRCA1 and BRCA2206. To date, the combination therapy of PARPi with anti-AR therapy has not been tested in breast cancer; however, this combination may be an effective treatment strategy for AR+ BC patients, especially those with BRCA mutated tumors. Because many PARPi can induce PARP trapping, resulting in the formation of dsDNA lesions207, and anti-AR therapies have been demonstrated to result in a delay in dsDNA break repair in the presence of DNA damage, combining these therapies may be effective in creating deleterious lesions for tumor cells. Future work may assess PARPi in combination with anti-AR therapies for the treatment of AR+ breast cancers.PARPi has been established to be an effective treatment strategy for patients with breast cancers harboring mutant 105. Due to crosstalk of AR with CDK/pRb in promoting cell cycle progression, combined AR and CDK4/6 inhibition has also been shown to be a therapeutic strategy in prostate cancer208.AR regulates cell cycle progression through the G1–S phase transition, therefore promoting CDK activity and inducing phosphorylation for the inactivation of pRb209.Palbociclib, ribociclib, and abemaciclib are selective inhibitors of CDK4/6 and are widely used for the treatment for ER+ breast cancer. A Phase I/II clinical trial is currently assessing the use of palbociclib with bicalutamide for treatment of AR+ metastatic TNBC (NCT02605486). This trial will establish recommended doses for the combination therapy in addition to measuring PFS, and secondary outcomes including response rates, CBR, and safety210. Combined inhibition of AR with the PI3K or mTOR pathway suppressed cell proliferation and resulted in an increase in apoptosis and cell cycle arrest in CRPC cells210. An ongoing trial is investigating the treatment of taselisib, a PI3K inhibitor, and enzalutamide in patients with AR+ metastatic TNBC (NCT02457910). This trial will assess dose-limiting toxicities to determine the maximum tolerated dose in addition to measuring patient response and CBR211.Phosphatindylinositol 3-kinase (PI3K) is an enzyme involved in cellular functions including cell growth, proliferation, and differentiation; however, PI3K is also highly mutated in cancer. Qi et al. found that inhibition of both AR and PI3K can be synergistic as AR and PI3K signaling work through reciprocal feedback loops213. Data recently presented from this trial reported that enzalutamide treatment is feasible and well-tolerated in this patient population. Finally, phase I/II clinical trials continue to inform drug development and clinical practice, including trials of newer generation antiandrogen agents in women with AR+ breast cancer. Additional studies are needed to better understand use of antiandrogen therapies for the treatment of women with AR+ breast cancers.Many antiandrogen treatment strategies have been effectively translated from preclinical studies into clinical use through the use of clinical trials. For women with metastatic, AR+ TNBC, there is a phase III clinical trial (NCT03055312) underway comparing conventional chemotherapy to bicalutamide treatment. This trial will assess the CBR at 16 weeks as well as progression-free survival at 24 months. The ENDEAR trial (NCT02929576) is a phase III trial comparing PFS for patients treated with paclitaxel chemotherapy +/− enzalutamide or enzalutamide followed by paclitaxel treatment; however, this trial was withdrawn. Finally, there is an ongoing feasibility trial (NCT02750358) of enzalutamide in women with AR+ TNBC that should report preliminary DFS and OS data in the coming yearWhile the role of AR in prostate cancer is more completely understood, the importance of AR signaling in breast cancer is an area of increasing investigation. In order to understand the mechanism of AR signaling and to design proper therapies against AR in breast cancer, additional work needs to be done to elucidate the mechanism by which AR is activating its target genes and contributing to tumor growth and metastasis, as well as systemic and radiation therapy resistance. Advancements in this mechanistic understanding will shed light on potential combination therapies and will allow for more effective treatment for patients with AR+ breast cancers. Further, discerning the intricacies and crosstalk between AR and ER signaling may also provide advancements for treatment of AR+, ER+ breast cancers. These outcomes would be impactful not only for the advanced understanding of the role of AR, but also for new ways in which AR signaling can be inhibited to improve outcomes for women with AR+ breast cancer.


Scientific Reports 10.1038/srep11554, published online 25 June 2015Correction to: This Article contains an error in the affiliations for R. Alhabbab, where ‘Centre of Immunology, Division of Applied Medical Sciences, King Abdulaziz University, KSA’ is incorrectly listed as a current address. The correct affiliations appear below:Centre of Immunology, Division of Applied Medical Sciences, King Abdulaziz University, KSA.Medical Research Council Centre for Transplantation, King’s College London, King’s Health Partners, Guy’s Hospital, London, SE1 9RT, UK.


FGFR3-TACC3 (F3T3) fusions. However, their associated molecular and clinical features are poorly understood. Here we analyze the frequency of F3T3-fusion positivity, its associated genetic and methylation profiles, and its impact on survival in 906 IDH-wildtype GBM patients. We establish an F3T3 prevalence of 4.1% and delineate its associations with cancer signaling pathway alterations. F3T3-positive GBMs had lower tumor mutational and copy-number alteration burdens than F3T3-wildtype GBMs. Although F3T3 fusions were predominantly mutually exclusive with other oncogenic RTK pathway alterations, they did rarely co-occur with EGFR amplification. They were less likely to harbor TP53 alterations. By methylation profiling, they were more likely to be assigned the mesenchymal or RTK II subclass. Despite being older at diagnosis and having similar frequencies of MGMT promoter hypermethylation, patients with F3T3-positive GBMs lived about 8 months longer than those with F3T3-wildtype tumors. While consistent with IDH-wildtype GBM, F3T3-positive GBMs exhibit distinct biological features, underscoring the importance of pursuing molecular studies prior to clinical trial enrollment and targeted treatment.A subset of glioblastomas (GBMs) harbors potentially druggable oncogenic The online version of this article (10.1186/s40478-020-01058-6) contains supplementary material, which is available to authorized users. FGFR3) gene and the coiled-coil domain of the transforming acidic coiled-coil-containing protein 3 (TACC3) gene ) and TACC3 exons 11 (37.8% [14/37]) or 10 (24.3% [9/37]) , and 95p = 0.02) (Table p = 0.96). There was no difference in laterality or anatomic localization (p > 0.05 for both comparisons). All tumors were reviewed by a board-certified neuropathologist and found to meet histologic criteria for GBM, WHO grade IV  ). In contrast, 14.3% (5/35) of EGFR-amplified cases tested and 50.0% (6/12) of PDGFRA-amplified cases tested were assigned the RTK I subclass. When stratified by F3T3-fusion status and considered alongside 446 previously published GBM methylation profiles from the Heidelberg cohort, dimensionality reduction with principal component analysis and t-distributed stochastic neighbor confirmed these findings  to be assigned to the mesenchymal or RTK II subclass and 0.1 ×  as likely to be assigned to any other subclass.A subset of the F3T3-positive (91.9% [34/37]) and -wildtype (11.5% 100/867]) GBMs was analyzed on the Infinium platform [0/867) GBp = 0.006)   had concurrent chromosome 7 gain and 10q loss, 9.4% (3/32) had chromosome 7 gain without 10q loss, 6.3% (2/32) had 10q loss without chromosome 7 gain, and 6.3% (2/32) had concurrent loss of 10q, 13q, and 14q, all consistent with the integrated diagnosis of IDH-wildtype GBM. The overall TCB was assessed using the larger MSK-IMPACT dataset that included all 904 patients. Because the F3T3 fusion protein is thought to cause aneuploidy, we hypothesized that F3T3-positive GBMs would have higher TCBs than F3T3-wildtype GBMs. Unexpectedly, F3T3-positive tumors had significantly lower TCBs. The median TCB for the F3T3-positive tumors was 15.0%  while that for the F3T3-wildtype tumors was 17.4%  (p = 0.9). In all, 64.9% (24/37) of F3T3-positive and 71.5% (620/867) of F3T3-wildtype patients died during follow up (p = 0.36). On univariate Kaplan–Meier analysis, F3T3-positive patients lived slightly longer than F3T3-wildtype patients , corresponding to a hazard ratio of 0.69  , corresponding to a hazard ratio of 0.43 . In a multivariable model adjusted for age, sex, race, and MGMT status, the hazard ratio for F3T3 positivity was 0.68 , similar to the association identified on univariate analysis.Follow-up data were sought for all patients through a comprehensive medical-record and obituary search. The median follow-up time was 55.6 months  among F3T3-positive and 52.0 months  among F3T3-wildtype patients (4.9% 24/3 of F3T3-Treatment data were available for 91.9% (34/37) of the patients with F3T3-positive GBMs, all of whom received upfront radiotherapy and TMZ (concurrently and/or as adjuvant therapy). Only a minority of patients received a therapy targeting FGF/FGFR signaling. Specifically, 5.9% (2/34) received a selective FGFR inhibitor and 5.9% (2/34) received a multikinase inhibitor/non-selective FGFR inhibitor. The small number of patients receiving targeted therapy precluded investigating its association with overall survival.This genomic landscape study of 906 patients with IDH-wildtype GBMs treated at a tertiary cancer referral center demonstrated that 4.1% harbored potentially druggable activating F3T3 fusions with recurrent structural isoforms; characteristic associated mutational, copy-number, and methylation profiles; and clinical outcomes slightly better than patients with F3T3-wildtype tumors despite occurring in older individuals.Our understanding of F3T3-positive GBMs has increased since the initial discovery that demonstrated F3T3 fusions comprised a rare yet recurrent oncogenic structural event resulting in an in-frame fusion protein with constitutive kinase activity and mitogenic effects, which were reversible by FGFR kinase inhibition , 39. TogFGFR3 exons 17 or 18 were most common [TACC3 exon, ranging from exons 3–12. Overall, FGFR3 exon 17 to TACC3 exon 11 and FGFR3 exon 17 to TACC3 exon 10 fusions were most common.The present study confirms molecular features of F3T3-positive GBMs suggested in prior reports, including the diversity of structural isoforms of the F3T3 fusion gene itself. Prior studies established that isoforms involving t common , 39, in EGFR, PDGFRA, or MET amplification [CDK4 and MDM2 amplification [PDGFRA or MET. Similarly, we found that F3T3-positive GBMs were less likely to exhibit concurrent EGFR amplification (5.6% vs. 43.3%); however, our data revealed that these events were not truly mutually exclusive, as two cases with F3T3 fusions exhibited bona fide amplification in EGFR. However, it is possible that the F3T3 fusion and the EGFR amplification events were present in different subclones, as has been shown for amplification of diverse RTKs [CDK4 or MDM2 amplification among F3T3-positive GBMs in our study.However, our data reveal new insights into the molecular alterations associated with F3T3 fusions in IDH-wildtype GBMs, in some instances contrasting with prior smaller studies that employed more targeted sequencing methods. For example, prior reports suggested that the F3T3 fusion was mutually exclusive with fication , 34, andfication , 17. In rse RTKs , 41. FurTP53 and MDM4 were less common among F3T3-positive tumors. Combined with the lower median TMB and TCB observed in F3T3-positive GBMs, these data suggest that F3T3-positivity itself may be enough to drive oncogenesis in the relative absence of other concurrent driver alterations. The relatively genomically quiet nature of the F3T3-positive GBMs was unexpected given that experimental data suggest that the F3T3 fusion protein recruits endogenous TACC3 away from the mitotic spindle, leading to delayed mitotic progression and aneuploidy [TP53 and MDM4 alterations identified in this study) may be engaged in vivo.The present study utilized a hybridization capture-based NGS assay (MSK-IMPACT) for sequencing all exons and selected introns of up to 468 genes with paired tumor-matched normal tissue analysis enabling unambiguous somatic mutation detection , 42, alleuploidy ; our datMGMT promoter hypermethylation does not differ between F3T3-positive and F3T3-wildtype GBMs [EGFR or PDGFRA amplifications were assigned the RTK I subclass in 14.3% and 50.0% of cases, respectively, consistent with prior reports [TACC3 in regulating the epithelial-mesenchymal transition may relate to the methylation characteristics of F3T3-positive GBM [DNA methylation-based CNS tumor classification is becoming increasingly employed as a diagnostic tool in many neuropathology practices worldwide. The present study provides insight into the F3T3-positive GBM methylome using a previously validated machine-learning-based tumor classifier developed at University Hospital Heidelberg . Aside fype GBMs , the pre reports . While itive GBM , it has tive GBM , 19, 25.p = 0.07), a difference that persisted after adjustment for potential confounders including age, sex, race, and MGMT status. Although this difference did not meet formal statistical significance, at the very least, we can conclude that patients with F3T3-positive tumors have clinical features and outcomes similar to, or perhaps slightly better than, those with non-F3T3-driven, IDH-wildtype GBMs.The present study is among the first to provide long-term clinical follow-up data on a large cohort of patients with F3T3-positive tumors, with a median follow-up time of 55.6 months. Patients with F3T3-positive tumors were slightly older at initial diagnosis (63.6 vs. 59.4 years). Although older age at diagnosis is a well-established risk factor for poorer outcome and shorter overall survival in patients with GBM, patients with F3T3-positive GBMs in this study lived slightly longer than those without F3T3 fusions  [Importantly, given the difference in overall survival suggested by this study, our findings raise the possibility that the underlying biology of F3T3-positive GBM may be a confounding factor in interpreting outcome data in the setting of pan-GBM clinical trials. Considering ongoing research into therapeutic inhibition of tyrosine kinase signaling in general and FGFR3 signaling specifically, identification of F3T3 fusions in patients with IDH-wildtype GBMs could soon have therapeutic implications. For example, the recent phase 2 REGOMA trial showed that median overall survival was approximately 2 months longer in patients with first recurrence of GBM after surgery and radiotherapy/TMZ receiving the oral multikinase inhibitor regorafenib rather than lomustine  . NotablyFGFR signaling has been implicated in a variety of human cancers. FGFR pathway inhibition as a therapeutic strategy remains an area of active investigation, including in ongoing and recently completed clinical trials of FGFR inhibitors in brain tumors . Other inhibitors of FGFR3 and F3T3 have also been tested in preclinical models and in trials in other tumor types, especially urothelial carcinoma and cholangiocarcinoma, for which therapeutics such as erdafitinib, rogaratinib, infigratinib, and the monoclonal antibody vofatamab have shown promise . Recent This study has limitations. Although 906 cases were sequenced, only 137 underwent methylation array analysis. Also, an insufficient number of F3T3-positive patients in this cohort received F3T3-specific targeted therapy to evaluate the potential efficacy of such an approach, the determination of which awaits future clinical trials. Last, detailed treatment information was not available for all F3T3-wildtype patients, thus chemoradiotherapy treatment status was not adjusted for in the Cox proportional hazards regression models.TP53. As histologic features lack specificity for identifying F3T3-positive tumors, comprehensive NGS and methylation analysis should be considered for all IDH-wildtype GBMs to identify patients for potential clinical trial enrollment and targeted treatment.In conclusion, approximately 4% of IDH-wildtype GBMs in this study harbored potentially targetable F3T3 fusions. While their clinical and molecular characteristics were largely in keeping with other IDH-wildtype GBMs, they demonstrated characteristic associated mutational, copy-number, and methylation profiles, and patients with F3T3-positive tumors had clinical outcomes slightly better than patients with F3T3-wildtype tumors. F3T3-positivity was predominantly mutually exclusive with other RTK drivers, and F3T3-wildtype tumors were enriched for mutations in Additional file 1. Online supplementary material.


To systematically analyze the existing intelligent rehabilitation mobile applications (APPs) related to distal radius fracture (DRF) and evaluate their features and characteristics, so as to help doctors and patients to make evidence-based choice for appropriate intelligent-assisted rehabilitation.  Literatures which in regard to the intelligent rehabilitation tools of DRF were systematic retrieved from the PubMed, the Cochrane library, Wan Fang, and VIP Data. The effective APPs were systematically screened out through the APP markets of iOS and Android mobile platform, and the functional characteristics of different APPs were evaluated and analyzed.  A total of 8 literatures and 31 APPs were included, which were divided into four categories: intelligent intervention, angle measurement, intelligent monitoring, and auxiliary rehabilitation games. These APPs provide support for the patients' home rehabilitation guidance and training and make up for the high cost and space limitations of traditional rehabilitation methods. The intelligent intervention category has the largest download ratio in the APP market. Angle measurement tools help DRF patients to measure the joint angle autonomously to judge the degree of rehabilitation, which is the most concentrated type of literature research. Some of the APPs and tools have obtained good clinical verification. However, due to the restrictions of cost, geographic authority, and applicable population, a large number of APPs still lack effective evidence to support popularization.  Patients with DRF could draw support from different kinds of APPs in order to fulfill personal need and promote self-management. Intelligent rehabilitation APPs play a positive role in the rehabilitation of patients, but the acceptance of the utilization for intelligent rehabilitation APPs is relatively low, which might need follow-up research to address the conundrum. Distal radius fracture (DRF) is one of the common upper limb fractures, which could be accounted for about 15.7% of upper limb fractures  OR application [tiab] OR app∗ [tiab] OR iphone [tiab]) NOT (Amyloid precursor protein [tiab]).The intelligent rehabilitation tools on the APP market are mainly divided into several categories such as health education and functional exercise. “distal radius fracture”, “rehabilitation”, “functional exercise” or “health education” were searched in Chinese and English separately in APP store, Google player, and Huawei platform.Inclusion criteria include the following: (1) the study about the intelligent rehabilitation APPs or tools for the DRF patient, especially intervention research; (2) APPs or tools that assist the intelligent rehabilitation for the DRF patient on any platform; (3) rehabilitation is the main function or included function of the APPs or tools; and (4) the APP was applicable from intelligent mobile terminals.Exclusion criteria include the following: (1) APPs or tools limited to the operation and use of medical staff, (2) APPs with no score or score less than 3 and download volume less than 100, (3) APPs or tools for rehabilitation of other fracture types, and (4) APPs or tools without detailed description and usage records.Data from the included studies were extracted by the two investigators (J.Y. and D.C.) using standardized and piloted design formats. The preset extraction indicators mainly include basic information such as the name of the APP, the platform used, and the function. The literature research also needs to extract the clinical research methods and results, and the platform information needs to include user usage, downloads, and ratings. Discrepancies in the process of study selection and data extraction were resolved through a group discussion with two other authors (Y.C. and G.W.).The extraction of the intelligent rehabilitation APPs involved in the literature, classified them according to the functions and types of different APPs, and analyzed the advantages and disadvantages.The flow of the search strategy is shown in It can be seen from the result that 25 (68%) smart APPs are applicable to both iOS and Android, with 7 (19%) smart APPs are suitable for iOS system, and 5 (13%) smart APPs are suitable for Android system . Most ofThe number of APP downloads from the APP market is concisely described in Intelligent rehabilitation APPs were divided into five categories according to different functions: intelligent intervention, angle measurement, intelligent monitoring, and rehabilitation game  Figure .Intelligent intervention software was accounted for the most of the results retrieved in the APP market. Intelligent intervention is mainly used in the later stage of fracture, which is a critical period for functional exercise after reaching the clinical healing standard. Such APPs would be able to provide patients with functional exercise and develop personalized rehabilitation services by combining physiotherapy (PT) and home exercise programs (Heps).The second proportion of the results retrieved in the APP market is the angle measurement APP which holds the largest proportion among selected literatures regarding intelligent rehabilitation APPs. The degree of rehabilitation of DRF patients is generally diagnosed by the angle of extension, flexion, pronation, and supination of the wrist and forearm. The angle measurement tool allows the patient to measure and record the rehabilitation angle without leaving home and can make independent judgments or share the data with the doctor in real time so that the doctor can understand the degree of the patient's rehabilitation. The ulnar deviation measurement of the two APPs is demonstrated in There are not many rehabilitation games related to fractures in the APP store and literature studies. The way of games is conducive to divert the patient's attention, in the process of playing games to enhance wrist finger movement and joint training and reduce the discomfort in the training process.The intelligent monitoring APP involves more complicated processes and links, providing patients with an emerging medical service model . It can The popularization of mHealth technology has gradually brought a new experience and service mode to the public, which would help to address the disadvantages of the existing medical system , 19. ThiFrom the research results of the APP market, the most involved are intelligent intervention APPs, the average downloads are over 1000. The most popular APP is the intelligent monitoring APP, which scores more than 4 points. In contrast, the most involved in literatures are the angle measurement APPs, followed by rehabilitation game APPs. This may indicate that angular measurement and rehabilitation game APPs have more research value; meanwhile, intelligent intervention and intelligent monitoring are more valuable regarding commercial and economic areas. The APP involved in some literatures does not yet exist in the APP market, which may indicate that some APPs are still in the development stage. However, most of the APPs in the APP market lack effective evidence and real data support, and the actual medical value needs further verification.Studies have shown that the prognosis of patient with DRF would benefit from the combination of supervised PT and Heps . IntelliMost of the patients with DRF would self-diagnose their condition and recovery process by judging proprioceptive recovery such as grip strength, pinch force, and wrist movement. Few patients would determine their rehabilitation process by measuring the angle of wrist movement , 25. NevPatients with DRF usually experience symptoms such as limited wrist movement , pain, wThe advent of the Internet era would allow non-text-based interactive information, such as video and images, to become more acceptable . RehabilmHealth care can provide personalized precision treatment and better people-centered care , 39. ThiThe market prospect of developing intelligent auxiliary rehabilitation tools from the perspective of patients is very broad. This study provides reference for patients with different needs to choose appropriate intelligent auxiliary rehabilitation tools: the services provided by intelligent intervention and monitoring APPs are more humanized with easier instruction to operate, which is suitable for middle-aged and elderly patients who cannot master APP skillfully; the rehabilitation gaming APPs are more interesting, but the operation and use process is relatively complicated, which are more suitable for younger patients; and the angle measurement APPs require relevant rehabilitation theory knowledge and are more suitable for patients to utilize under the guidance of medical staff or with the assistance of family members. Meanwhile, through this research, detailed information in regard to APP instruction and operation modes of different types of rehabilitation software was obtained, which provides a basis for subsequent clinical promotion and utilization.With the continuous deepening of intelligent medical reform, artificial intelligence algorithms have been widely used in auxiliary diagnostic methods of clinical medicine , 42. APPWith the development of the mHealth medical model, intelligent rehabilitation APPs and tools are gradually being used in clinical and patient independent health management. Through systematic evidence-based analysis based on literature and APP platforms, this study integrates different types of intelligent rehabilitation APPs that are suitable for DRF rehabilitation and explores its effects on the rehabilitation of such fractures from different perspectives. Although the evidence is limited, it can still be clearly shown that APP-based rehabilitation intervention, angle measurement, and monitoring management can all improve the effect of rehabilitation training and actively promote patients' self-rehabilitation management. This also enables clinical medical staff and DRF patients to make evidence-based choice according to the different characteristics and needs of APPs, which could meet the individual needs and improve the effectiveness of self-rehabilitation management. However, due to the restrictions of cost, geographic authority, and applicable population, a large number of APPs still lack effective evidence to support popularization. Therefore, in future research, large-scale user-centered clinical trials that will be added to evaluate the effectiveness and practicality of APPs are particularly important.


Many epidemiology studies have shown that maternal polycystic ovary syndrome (PCOS) results in a greater risk of autism spectrum disorders (ASD) development, although the detailed mechanism remains unclear. In this study, we aimed to investigate the potential mechanism and provide a possible treatment for PCOS-mediated ASD through three experiments: Experiment 1: real-time PCR and western blots were employed to measure gene expression in human neurons, and the luciferase reporter assay and chromatin immunoprecipitation (ChIP) was used to map the responsive elements on related gene promoters. Experiment 2: pregnant dams were prenatally exposed to dihydrotestosterone (DHT), androgen receptor (AR) knockdown (shAR) in the amygdala, or berberine (BBR), and the subsequent male offspring were used for autism-like behavior (ALB) assay followed by biomedical analysis, including gene expression, oxidative stress, and mitochondrial function. Experiment 3: the male offspring from prenatal DHT exposed dams were postnatally treated by either shAR or BBR, and the offspring were used for ALB assay followed by biomedical analysis. Our findings showed that DHT treatment suppresses the expression of estrogen receptor β (ERβ) and superoxide dismutase 2 (SOD2) through AR-mediated hypermethylation on the ERβ promoter, and BBR treatment suppresses AR expression through hypermethylation on the AR promoter. Prenatal DHT treatment induces ERβ suppression, oxidative stress and mitochondria dysfunction in the amygdala with subsequent ALB behavior in male offspring, and AR knockdown partly diminishes this effect. Furthermore, both prenatal and postnatal treatment of BBR partly restores prenatal DHT exposure-mediated ALB. In conclusion, DHT suppresses ERβ expression through the AR signaling pathway by hypermethylation on the ERβ promoter, and BBR restores this effect through AR suppression. Prenatal DHT exposure induces ALB in offspring through AR-mediated ERβ suppression, and both prenatal and postnatal treatment of BBR ameliorates this effect. We conclude that BBR ameliorates prenatal DHT exposure-induced ALB through AR suppression, this study may help elucidate the potential mechanism and identify a potential treatment through using BBR for PCOS-mediated ASD. Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by impaired social communication and interaction, together with restrictive and repetitive behaviors  is a common endocrine disorder in women characterized by hyperandrogenism and ovarian dysfunction  is a small isoquinoline alkaloid molecule that can be isolated from plants and holds many pharmacological activities, including antimicrobial, antitumoral, and metabolic modulation effects  were amplified from Ensembl gene ID: ARLNC1-205 ENST00000650661.1 (for AR) and ESR2-201 ENST00000267525.10 (for ERβ) by PCR from human genomic DNA and subcloned into the pGL3-basic vector  using restriction sites of Mlu I and Hind III with the following primers: AR forward: 5′-gcgc-acgcgt- cga ggt tag gag ata aag acc -3′ (Mlu I) and AR reverse: 5′- gtac- aagctt- ctt ttc tgt aca tct cta gat -3′ (Hind III); ERβ forward: 5′-gcgc-acgcgt- atttcaagacgagcctggcca -3′ (Mlu I) and ERβ reverse: 5′- gtac- aagctt- ctg ttt aca ggt aag gtg tgt -3′ (Hind III). To map AR or ERβ promoter activity, the related deletion promoter constructs were generated by PCR methods and subcloned into the pGL3-basic vector  was subcloned into the pLVX-Puro vector (from Clontech) with the restriction sites of Xho1 and Xba1 using the below primers: human AR forward primer: 5′- gtac-ctcgag- atg gaa gtg cag tta ggg ctg -3′ (Xho1) and human AR reverse primer: 5′- gtac-tctaga-tca ctg ggt gtg gaa ata gat -3′ (Xba1). The lentivirus for either AR or empty control (CTL) was expressed by Lenti-X™ Lentiviral Expression Systems (from Clontech).in vivo gene knockdown. The knockdown efficiency was confirmed by more than 65% of mRNA reduction compared to the control group in rat amygdala cells using real-time PCR (see The shRNA lentivirus plasmid for human AR (sc-29204-SH), or non-target control (sc-108060) were purchased from Santa Cruz Biotechnology, and the shRNA lentivirus plasmid for rat AR was a kind gift from Dr. Haimou Zhang (from Hubei University). The related lentivirus for shAR or empty control (CTL) were expressed through Lenti-X™ Lentiviral Expression Systems (from Clontech) per manufacturers’ instructions. The purified and condensed lentivirus was used for  PCR see .5 of treated cells were seeded in a 6-well plate with complete medium to grow until they reached 80% confluence. Cells were then cotransfected by 3 μg of reporter constructs, together with 0.2 μg of pRL-CMV-Luc Renilla plasmid (from Promega). After treatment, the cells were harvested and the luciferase activity assays were carried out using the Dual-Luciferase™ Assay System (Promega), and the transfection efficiencies were normalized using a cotransfected Renilla plasmid according to manufacturers’ instructions. The reporter activities for AR and ERβ were calculated  using the primers provided in ad libitum access to food and water.Sprague–Dawley rats were obtained from Guangdong Medical Animal Center, and maintained under standard 12 h light/dark cycles and given 3 cfu of lentivirus was infused for 1 h. After 1 week of surgery recovery, the female rats were mated with proven male rats, and the successful pregnancy was confirmed by examining the vaginal plugs. The dams were then treated with vehicle  or BBR  by intraperitoneal (i.p.) injection every 2 days starting from day 1. The experimental rats were separated into 4 groups (10 per group). Group 1: CTL rats with empty control lentivirus infusion (CTL); Group 2: DHT rats with empty control lentivirus infusion (DHT); Group 3: DHT rats with shAR knockdown lentivirus infusion (DHT/shAR); Group 4: DHT rats with BBR injection (DHT/BBR). The amygdala neurons were isolated on embryonic day 18 (E18) as described below. After birth, the male offspring were separated from the dams on day 21 and fed until 7–8 weeks old for further experiments. The offspring were then used for ALB tests followed with biomedical analysis , or vehicle pellets (CTL) containing the same matrix but with no hormone  or empty (EMP) lentivirus or treated with vehicle  or BBR  by intraperitoneal (i.p.) injection every 2 days as mentioned in Rat Protocol 1. The rats were separated into the below four groups (10 per group). Group 1: CTL rats with empty control lentivirus infusion (CTL); Group 2: DHT rats with empty control lentivirus infusion (DHT); Group 3: DHT rats with postnatal shAR knockdown lentivirus infusion (DHT/p-shAR); Group 4: DHT rats with postnatal BBR injection (DHT/p-BBR). After 2 weeks of lentivirus and BBR treatment, the offspring were then used for behavior tests and biomedical analysis, as discussed in Rat Protocol 1  were incubated for 12 h at 4°C and subsequently with secondary antibody Alexa Fluor 488. The coverslips were then mounted by antifade Mountant with DAPI . The photographs were taken using a Confocal Laser Microscope  and quantitated using ImageJ software.We evaluated the potential effect of DHT and BBR on the gene expression of AR, SOD2, and ERβ. The human ACS-5003 neurons were treated by either control (CTL), DHT (10 nM), or DHT with AR knockdown by lentivirus (DHT/shAR), or CTL with AR overexpression by lentivirus (CTL/↑AR), CTL with 10 μM BBR (CTL/BBR), or DHT with BBR (DHT/BBR) for 24 h, then cells were harvested for gene expression analysis. Our results showed that DHT treatment decreased expression of SOD2 and ERβ to 66% and 51%, respectively compared to the CTL group, while it did not have any effect on AR expression. We then evaluated the effect of shAR on DHT treatment (DHT/shAR). Our results showed that shAR decreased AR expression to 21%, indicating a successful AR gene manipulation by lentivirus knockdown; very interestingly, shAR completely restored DHT-mediated gene suppression of SOD2 and ERβ. We also measured the effect of AR overexpression (CTL/↑AR) and found that CTL/↑AR increased AR expression to 235% compared to CTL group, indicating a successful gene manipulation by lentivirus overexpression; furthermore, it decreased expression of SOD2 and ERβ to 51% and 47%, respectively. We finally measured the effect of BBR (CTL/BBR), and the results showed that CTL/BBR and DHT/BBR decreased AR expression to 53% and 44%, respectively, and CTL/BBR increased expression of SOD2 and ERβ to 135% and 136%, respectively, compared to the CTL group. Our results indicate that DHT suppresses the expression of SOD2 and ERβ, and AR knockdown restores, while AR expression mimics, this effect; BBR restores this effect by AR suppression see . We thenWe investigated the potential molecular mechanism for DHT-induced ERβ suppression. ERβ promoter deletion constructs were generated and transfected into conditional immortalized human neurons for ERβ reporter activity assay in the presence of either control (CTL) or 10 nM DHT for 24 h, and the DHT-induced relative reporter activities (% control) were calculated. We found that DHT-induced ERβ reporter suppression occurred among the −2,000, −1,600, −1,200, −800, −400, −300 deletion constructs according to Ensembl gene ID: ESR2-201 ENST00000267525.10; the suppression was restored in the −200, −100, and −0 deletion constructs, suggesting that DHT-responsive transcriptional element is located in the range of −300 to 0 on the ERβ promoter see . We thenWe investigated the possible mechanism for BBR-induced AR suppression. AR promoter deletion constructs were generated and transfected into conditional immortalized human neurons for AR BBR reporter activity assay in the presence of either control (CTL) or 10 μM BBR for 24 h, and the BBR-induced relative reporter activities (% control) were calculated. We found that BBR-induced AR reporter suppression occurred among the −2,000, −1,600, −1,200, −800, −400, −300 deletion constructs according to Ensembl gene ID: ARLNC1-205 ENST00000650661.1; the suppression was restored in the −200, −100, and −0 deletion reporter constructs, suggesting that BBR-responsive transcriptional element is located in the area of −300 to 0 on the AR promoter see . We then2.− release  or DHT treatment, which involved either DHT with shAR lentivirus infusion in the amygdala (DHT/shAR) or DHT with BBR injection (DHT/BBR) during the 21-day pregnancy. The subsequent 2-month old male offspring were sacrificed and the brain tissues were isolated for further analysis. We first evaluated the mRNA expression of AR, SOD2, and ERβ in the amygdala. The results showed that prenatal DHT exposure (DHT) decreased expression of SOD2 and ERβ to 54% and 66%, respectively, compared to the CTL group; none of the prenatal treatments had any effect on AR expression, while both the DHT/shAR and DHT/BBR treatments completely restored the DHT-mediated suppression of SOD2 and ERβ see . We thenease see  and 3-niease see  to 202% rker see . The resrker see  and intrrker see  to 65% arker see , while Arker see  comparedF = 2.741, P = 0.041). The Post hoc analysis showed that habituation to the same stimulus conspecific (tests 1–4) was significant in CTL group  = 4.105, P < 0.01), DHT/shAR group  = 3.067, P = 0.044), and DHT/BBR group  = 3.279, P = 0.036), but not in DHT group. Dishabituation was significant in CTL group  = 4.191, P < 0.01), DHT/shAR group  = 3.611, P = 0.024), and DHT/BBR group  = 3.816, P = 0.019), but not significant in DHT group  = 2.947, P = 0.046), and the Post hoc analysis showed that habituation to the same stimulus conspecific (tests 1–4) was significant in CTL group  = 4.264, P < 0.01) and DHT/p-BBR group  = 3.410, P = 0.034), but not in DHT and DHT/p-shAR groups. Dishabituation was significant in CTL group  = 4.361, P < 0.01), and borderline significant in DHT/p-BBR group  = 3.017, P = 0.037), but was not significant in DHT and DHT/p-shAR groups  or DHT treatment during their pregnancy, and the subsequent 2-month-old male offspring from the DHT group received either shAR lentivirus infusion in the amygdala (DHT/p-shAR) or DHT with BBR injection (DHT/p-BBR) for 1 week, and then those male offspring were used for ALB analysis followed by a biomedical analysis of the isolated amygdala. We first evaluated the mRNA expression of AR, SOD2, and ERβ in the amygdala. The results showed that prenatal DHT exposure (DHT) decreased expression of SOD2 and ERβ to 51% and 54%, respectively, but had no effect on AR expression. Additionally, postnatal shAR treatment (DHT/p-shAR) decreased AR expression to 34%, indicating a successful gene manipulation by shAR lentivirus knockdown, but did not affect prenatal DHT exposure-mediated suppression of SOD2 and ERβ. On the other hand, postnatal BBR treatment (DHT/p-BBR) decreased AR expression to 61% and partly restored prenatal DHT exposure-mediated suppression of SOD2 and ERβ see . We thenoups see . Also, toups see  increaseoups see  decreaseoups see . DHT/p-sin vitro experiments demonstrate that DHT suppresses ERβ expression through AR-mediated hypermethylation on ERβ promoter, and BBR restores this effect through AR suppression. Furthermore, the in vivo experiments show that prenatal DHT exposure induces ALB in offspring, and both prenatal and postnatal BBR treatment ameliorates this effect.In this study, the neural progenitor cell ACS-5003 was used to mimic the differentiation of stem cells into neurons during embryo development , and was reviewed and approved by the Institutional Animal Care and Use Committee from Guangzhou University of Chinese Medicine.PY wrote the article. PY, LL, and XL designed, analyzed the data and interpreted the experiments. XL, YL, and ZW performed the vector constructions and gene expression analysis. XC, YL, ZW, and MW performed the statistical analysis and part of the rat experiments. YW, MX, and ML performed gene analysis and part of the mapping analysis. DX, JL, and CW performed the remaining experiments. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.