Testosterone suppression in the treatment of recurrent or metastatic prostate cancer - A Canadian consensus statement.

Testosterone suppression, achieved through orchiectomy or medically induced androgen-deprivation therapy (ADT), is a standard treatment for men with recurrent and metastatic prostate cancer. Current assay methods demonstrate the capacity for testosterone suppression to <0.7 nmol/l, and clinical data support improved outcomes from ADT when lower levels are achieved. Practical clinical guidelines are warranted to facilitate adoption of 0.7 nmol/l as the new standard castrate testosterone level.A pan-Canadian group of experts, representing diverse clinical specialties, identified key clinical issues, searched and reviewed relevant literature, and developed consensus statements on testosterone suppression for the treatment of prostate cancer. The expert panel found that current evidence supports the clinical benefit of achieving low testosterone levels during ADT, and encourage adoption of ≤0.7 nmol/l as a new castrate level threshold. The panel recommends regular monitoring of testosterone (e.g., every 3-6 months) and prostate-specific antigen (PSA) levels as clinically appropriate (e.g., every 3-6 months) during ADT, with reassessment of therapeutic strategy if testosterone is not suppressed or if PSA rises regardless of adequate testosterone suppression. The panel also emphasizes the need for greater awareness and education regarding testosterone assay specifications, and strongly promotes the use of mass spectrometry-based assays to ensure accurate measurement of testosterone at castrate levels.

Maximal testosterone suppression in the management of recurrent and metastatic prostate cancer.

INTRODUCTION: Testosterone suppression, or androgen-deprivation therapy (ADT), is an established treatment for recurrent and metastatic prostate cancer (PCa). Based on the accuracy and sensitivity of early assays (c. 1960-1970), the castrate testosterone level was set at ≤1.7 nmol/l. Improved sensitivity of testosterone assays shows that both surgical and medical castration can achieve levels <0.7 nmol/l. However, the clinical implications and importance of maximum testosterone suppression remains a subject of controversy. This evidence-based review assesses prospective and retrospective clinical data, linking maximum suppression of testosterone with improved outcomes from ADT. METHODS: PubMed and conference proceedings were searched for studies assessing the impact of low testosterone on clinical outcomes from ADT. The key search terms included combinations of prostate cancer and testosterone, predictive/prognostic, and androgen deprivation. Results were limited to studies investigating the relationship between testosterone levels and clinical outcomes. RESULTS: Both prospective and retrospective data support a relationship between testosterone levels below the historical standard of 1.7 nmol/l and improved outcomes. Eight studies showed significant improvements in survival-related outcomes, with the majority of data supporting a testosterone level cutoff of ≤0.7 nmol/l. CONCLUSIONS: Tracking both testosterone and prostate-specific antigen (PSA) levels has significant clinical benefits, and the serum testosterone threshold of ≤0.7 nmol/l is a practical goal. The relative levels of testosterone and PSA may indicate continued hormone responsiveness or progression toward castration-resistant prostate cancer (CRPC) and should, therefore, inform treatment strategy. Standardization of assay methods and clinical coordination to facilitate widespread access to state-of the art laboratory equipment is necessary to ensure accurate decision-making.

Navigating later lines of treatment for advanced colorectal cancer - optimizing targeted biological therapies to improve outcomes.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer among males and second among females worldwide. The treatment landscape for advanced CRC (aCRC) is rapidly evolving and there are now a number of randomized trials assessing treatment of aCRC beyond first-line, prompting important questions about how to optimize therapy and maximize benefit. The availability of targeted agents has increased the complexity of post-progression treatment of aCRC. Targeted biological agents with varying modes of action are now approved for use in second-line and beyond, including the VEGF-inhibitors bevacizumab and aflibercept, the VEGFR/multikinase-inhibitor regorafenib, and the EGFR-inhibitors cetuximab and panitumumab. This article provides a systematic overview of the available phase III trial data, discusses biomarkers predictive of response to treatment, addresses safety concerns associated with specific agents, and provides practical, evidence-based recommendations for the later lines of treatment for patients with unresectable aCRC.

Controlling angiogenesis in breast cancer: a systematic review of anti-angiogenic trials.

PURPOSE: Angiogenesis is critical for tumor growth and a promising therapeutic target. This review will summarize and analyze data from clinical trials of anti-angiogenic agents in the treatment of breast cancer (BC). DESIGN: A systematic search of PubMed and conference databases was performed to identify reports of randomized clinical trials investigating specific anti-angiogenic agents in the treatment of BC. RESULTS AND DISCUSSION: Phase III trials in advanced BC have demonstrated a reduction in the risk of disease progression (22-52%), improved response rates and net improvements in progression-free survival of 1.2 to 5.5 months, but no significant improvements in overall survival with the addition of bevacizumab to chemotherapy. Results of phase III trials in early breast cancer have been inconsistent. Bevacizumab-containing regimens have also been associated with higher overall adverse event rates compared to chemotherapy alone. Phase III trials of the tyrosine kinase inhibitor sunitinib were negative, while randomized phase II trials of sorafenib and pazopanib have improved some outcomes when combined with chemotherapy or targeted therapy compared to controls. In addition to expected vascular class safety signals, tyrosine kinase inhibitors show "off-target" side effects. Ongoing clinical trials evaluating combinatorial strategies based on biological synergies and translational studies identifying biological predictors of response will be crucial to establish meaningful clinical benefits in selected BC populations. CONCLUSION: Most trials of anti-angiogenic agents in BC have reported improved response rate and progression-free survival but no increase in overall survival compared to chemotherapy alone. Optimizing the therapeutic indices of these agents is a focus of ongoing research and will be critical to their future development.

The roles of testicular nuclear receptor 4 (TR4) in male fertility-priapism and sexual behavior defects in TR4 knockout mice.

BACKGROUND: Successful reproductive efforts require the establishment of a situation favorable for reproduction that requires integration of both behavior and internal physiological events. TR4 nuclear receptor is known to be involved in male fertility via controlling spermatogenesis, yet its roles in regulating other biological events related to reproduction have not been completely revealed. METHODS: Male TR4 knockout (TR4 -/-) and wild type mice were used for the sexual behavior and penile dysfunction studies. Mice were sacrificed for histological examination and corresponding genes profiles were analyzed by quantitative RT-PCR. Reporter gene assays were performed. RESULTS: We describe an unexpected finding of priapism in TR4 -/- mice. As a transcriptional factor, we demonstrated that TR4 transcriptionally modulates a key enzyme regulating penis erection and neuronal nitric oxide synthese NOS (nNOS). Thereby, elimination of TR4 results in nNOS reduction in both mRNA and protein levels, consequently may lead to erectile dysfunction. In addition, male TR4 -/- mice display defects in sexual and social behavior, with increased fear or anxiety, as well as reduced mounting, intromission, and ejaculation. Reduction of ER alpha, ER beta, and oxytocin in the hypothalamus may contribute to defects in sexual behavior and stress response. CONCLUSIONS: Together, these results provide in vivo evidence of important TR4 roles in penile physiology, as well as in male sexual behavior. In conjunction with previous finding, TR4 represents a key factor that controls male fertility via regulating behavior and internal physiological events.

TCDD exposure disrupts mammary epithelial cell differentiation and function.

Mammary gland growth and differentiation during pregnancy is a developmental process that is sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a widespread environmental contaminant and a potent ligand for the aryl hydrocarbon receptor (AhR). We demonstrate reduced beta-casein protein induction in mouse mammary glands and in cultured SCp2 mammary epithelial cells following exposure to TCDD. SCp2 cells exposed to TCDD also show reduced cell clustering and less alveolar-like structure formation. SCp2 cells express transcriptionally active AhR, and exposure to TCDD induces expression of the AhR target gene CYP1B1. Exposure to TCDD during pregnancy reduced expression of the cell adhesion molecule E-cadherin in the mammary gland and decreased phosphorylation of STAT5, a known regulator of beta-casein gene expression. These data provide morphological and molecular evidence that TCDD-mediated AhR activation disrupts structural and functional differentiation of the mammary gland, and present an in vitro model for studying the effects of TCDD on mammary epithelial cell function.

Activation of the aryl hydrocarbon receptor during different critical windows in pregnancy alters mammary epithelial cell proliferation and differentiation.

Exposure to the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy causes severe defects in mammary gland development and function; however, the underlying mechanism remains unclear. Alterations in epithelial cell proliferation, differentiation, and apoptosis during pregnancy-related mammary development can lead to failed lactogenesis. To determine which of these processes are affected and at what time periods, we examined proliferation, differentiation and apoptosis in mammary glands following exposure to TCDD during early, mid or throughout pregnancy. Although AhR activation throughout pregnancy did not cause early involution, there was a 50% decrease in cell proliferation, which was observed as early as the sixth day of pregnancy (DP). TCDD treatment on the day of impregnation only reduced development and proliferation in early and mid-pregnancy, followed by partial recovery by DP17. However, when AhR activation was delayed to DP7, developmental impairment was not observed in mid-pregnancy, but became evident by DP17, whereas proliferation was reduced at all times. Thus, early exposure to TCDD was neither necessary nor sufficient to cause persistent defects in lactogenesis. These varying outcomes in mammary development due to exposure at different times in pregnancy suggest there are critical windows during which AhR activation impairs mammary epithelial cell proliferation and differentiation.

Expression and activity of aryl hydrocarbon receptors in development and cancer.

Although the aryl hydrocarbon receptor (AhR) has been known as the mediator of the toxicity of particular xenobiotics such as the dioxins, the normal role of this transcription factor in a number of biological processes is just beginning to be recognized. Knowledge of AhR-targeted genes and signaling pathways indicates involvement of AhR in fundamental cell-regulatory pathways. Noted defects in the morphology and functions of certain tissues in the absence of AhR point to critical roles for this protein in developmental processes. Together, the data suggest that the AhR has an important function in controlling the balance among processes involved in cell proliferation, death, and differentiation rather than being essential for them. On the other hand, deregulation of these processes is known to contribute to events such as tumor initiation, promotion, and progression that ultimately lead to malignant tumor formation. Epidemiological and experimental animal data, along with a more detailed understanding of how AhR is involved in regulating particular signaling pathways, provide substantial support for an association between abnormal AhR function and cancer. Here we describe the current understanding of how the AhR may function to regulate both normal and cancerous tissue growth and development.

The roles of testicular orphan nuclear receptor 4 (TR4) in cerebellar development.

Since Testicular Receptor 4 (TR4) was cloned, efforts have been made to elucidate its physiological function. To examine the putative functions of TR4, the conventional TR4 knockout (TR4(-/-)) mouse model was generated. Throughout postnatal and adult stages, TR4(-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal and adult TR4(-/-) cerebellum revealed gross abnormalities in foliation. Further analyses demonstrated changes in the lamination of the TR4(-/-) cerebellar cortex, including reduction in the thickness of both the molecular layer (ML) and the internal granule layer (IGL). Analyses of the developing TR4(-/-) cerebellum indicate that the lamination irregularities observed may result from disrupted granule cell proliferation within the external granule cell layer (EGL), delayed inward migration of post-mitotic granule cells, and increased apoptosis during cerebellar development. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4(-/-) cerebellum, as indicated by aberrant dendritic arborization. In postnatal, neuronal-specific TR4 knockout mice, architectural changes in the cerebellum were similar to those seen in TR4(-/-) animals, suggesting that TR4 function in neuronal lineages might be important for cerebellar morphogenesis, and that the effect on Purkinje cell development is likely mediated by changes elsewhere, such as in granule cells, or is highly dependent on developmental stage. Together, our findings from various TR4 knockout mouse models suggest that TR4 is required for normal cerebellar development and that failure to establish proper cytoarchitecture results in dysfunction of the cerebellum and leads to abnormal behavior.

2,3,7,8-Tetracholorodibenzo-p-dioxin exposure disrupts granule neuron precursor maturation in the developing mouse cerebellum.

The widespread environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been linked to developmental neurotoxicity associated with abnormal cerebellar maturation in both humans and rodents. TCDD mediates toxicity via binding to the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of xenobiotic metabolizing enzymes and growth regulatory molecules. Our previous studies demonstrated that cerebellar granule neuron precursor cells (GNPs) express transcriptionally active AhR during critical developmental periods. TCDD exposure also impaired GNP proliferation and survival in vitro. Therefore, this study tested the hypothesis that TCDD exposure disrupts cerebellar development by interfering with GNP differentiation. In vivo experiments indicated that TCDD exposure on postnatal day (PND) 6 resulted in increased expression of a mitotic marker and increased thickness of the external granule layer (EGL) on PND10. Expression of the early differentiation marker TAG-1 was also more pronounced in postmitotic, premigratory granule neurons of the EGL, and increased apoptosis of GNPs was observed. On PND21, expression of the late GNP differentiation marker GABA(A alpha 6) receptor (GABAR(A alpha 6)) and total estimated cell numbers were both reduced following exposure on PND6. Studies in unexposed adult AhR(-/-) mice revealed lower GABAR(A alpha 6) levels and DNA content. In vitro studies showed elevated expression of the early differentiation marker p27/Kip1 and the GABAR(A alpha 6) in GNPs following TCDD exposure, and the expression patterns of proteins related to granule cell neurite outgrowth, beta III-tubulin and polysialic acid neural cell adhesion molecule, were consistent with enhanced neuroblast differentiation. Together, our data suggest that TCDD disrupts a normal physiological role of AhR, resulting in compromised GNP maturation and neuroblast survival, which impacts final cell number in the cerebellum.

Loss of TR4 orphan nuclear receptor reduces phosphoenolpyruvate carboxykinase-mediated gluconeogenesis.

OBJECTIVE: Regulation of phosphoenolpyruvate carboxykinase (PEPCK), the key gene in gluconeogenesis, is critical for glucose homeostasis in response to quick nutritional depletion and/or hormonal alteration. RESEARCH DESIGN/METHODS AND RESULTS: Here, we identified the testicular orphan nuclear receptor 4 (TR4) as a key PEPCK regulator modulating PEPCK gene via a transcriptional mechanism. TR4 transactivates the 490-bp PEPCK promoter-containing luciferase reporter gene activity by direct binding to the TR4 responsive element (TR4RE) located at -451 to -439 in the promoter region. Binding to TR4RE was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Eliminating TR4 via knockout and RNA interference (RNAi) in hepatocytes significantly reduced the PEPCK gene expression and glucose production in response to glucose depletion. In contrast, ectopic expression of TR4 increased PEPCK gene expression and hepatic glucose production in human and mouse hepatoma cells. Mice lacking TR4 also display reduction of PEPCK expression with impaired gluconeogenesis. CONCLUSIONS: Together, both in vitro and in vivo data demonstrate the identification of a new pathway, TR4 --> PEPCK --> gluconeogenesis --> blood glucose, which may allow us to modulate metabolic programs via the control of a new key player, TR4, a member of the nuclear receptor superfamily.

Abnormal cerebellar cytoarchitecture and impaired inhibitory signaling in adult mice lacking TR4 orphan nuclear receptor.

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological functions remain largely unknown. In this study, the TR4 knockout (TR4(-/-)) mouse model was used to investigate the role of TR4 in the adult cerebellum. Behaviorally, these null mice exhibit unsteady gait, as well as involuntary postural and kinetic movements, indicating a disturbance of cerebellar function. In the TR4(-/-) brain, cerebellar restricted hypoplasia is severe and cerebellar vermal lobules VI and VII are underdeveloped, while no structural alterations in the cerebral cortex are observed. Histological analysis of the TR4(-/-) cerebellar cortex reveals reductions in granule cell density, as well as a decreased number of parallel fiber boutons that are enlarged in size. Further analyses reveal that the levels of GABA and GAD are decreased in both Purkinje cells and interneurons of the TR4(-/-) cerebellum, suggesting that the inhibitory circuits signaling within and from the cerebellum may be perturbed. In addition, in the TR4(-/-) cerebellum, immunoreactivity of GluR2/3 was reduced in Purkinje cells, but increased in the deep cerebellar nuclei. Together, these results suggest that the behavioral phenotype of TR4(-/-) mice may result from disrupted inhibitory pathways in the cerebellum. No progressive atrophy was observed at various adult stages in the TR4(-/-) brain, therefore the disturbances most likely originate from a failure to establish proper connections between principal neurons in the cerebellum during development.

Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4.

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4-/- cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4-/- cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4-/- cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4-/- cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.

Growth retardation and abnormal maternal behavior in mice lacking testicular orphan nuclear receptor 4.

Testicular orphan nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily for which a ligand has not yet been found. In vitro data obtained from various cell lines suggest that TR4 functions as a master regulator to modulate many signaling pathways, yet the in vivo physiological roles of TR4 remain unclear. Here, we report the generation of mice lacking TR4 by means of targeted gene disruption (TR4(-/-)). The number of TR4(-/-) pups generated by the mating of TR4(+/-) mice is well under that predicted by the normal Mendelian ratio, and TR4(-/-) mice demonstrate high rates of early postnatal mortality, as well as significant growth retardation. Additionally, TR4(-/-) females show defects in reproduction and maternal behavior, with pups of TR4(-/-) dams dying soon after birth with no indication of milk intake. These results provide in vivo evidence that TR4 plays important roles in growth, embryonic and early postnatal pup survival, female reproductive function, and maternal behavior.

Disruption of TR4 orphan nuclear receptor reduces the expression of liver apolipoprotein E/C-I/C-II gene cluster.

Apolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism.

Isolation and characterization of androgen receptor mutant, AR(M749L), with hypersensitivity to 17-beta estradiol treatment.

Estrogens, primarily 17beta-estradiol (E(2)), may play important roles in male physiology via the androgen receptor (AR). It has already been shown that E(2) modulates AR function in LNCaP prostate cancer cells and xenograft CWR22 prostate cancer tissues. Using a molecular model of E(2) bound-AR-ligand binding domain (LBD) and employing site-directed mutagenesis strategies, we screened several AR mutants that were mutated at E(2)-AR contact sites. We found a mutation at amino acid 749, AR(M749L), which confers AR hypersensitivity to E(2). The reporter assays demonstrate that E(2) can function, like androgen, to induce AR(M749L) transactivation. This E(2)-induced AR mutant transactivation is a direct effect of the AR(M749L), because the transactivation was blocked by antiandrogens. The hypersensitivity of AR(M749L) to E(2) is not due to increased affinity of AR(M749L) for E(2), rather it may be due to the existence of the proper conformation necessary to maintain E(2) binding to the AR-LBD long enough to result in E(2)-induced transactivation. AR(M749L) transactivation can be further enhanced in the presence of AR coregulators, such as ARA70 and SRC-1. Therefore, amino acid 749 may represent an important site within the AR-LBD that is involved in interaction with E(2) that, when mutated, allows E(2) induction of AR transactivation.

Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells.

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.

Spermatogenesis and testis development are normal in mice lacking testicular orphan nuclear receptor 2.

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2(+/-) male mice we found that beta-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.

Mutations in the helix 3 region of the androgen receptor abrogate ARA70 promotion of 17beta-estradiol-induced androgen receptor transactivation.

The influence of estrogen on the development of the male reproductive system may be interrupted in a subset of partial androgen insensitivity syndrome (PAIS) patients. PAIS describes a wide range of male undermasculinization resulting from mutations in the androgen receptor (AR) or steroid metabolism enzymes that perturb androgen-AR regulation of male sex organ development. In this study, we are interested in determining if PAIS-derived AR mutants that respond normally to androgen have altered responses to estrogen in the presence of ARA70, a coregulator previously shown to enhance 17beta-estradiol E2-induced AR transactivation. The wild-type AR (wtAR) and two PAIS AR mutants, AR(S703G) and AR(E709K), all bind to androgen and E2 and subsequently translocate to the nucleus. Whereas ARA70 functionally interacts with the wtAR and the PAIS AR mutants in response to androgen, E2 only promotes the functional interaction between ARA70 and the wtAR but not the PAIS AR mutants. ARA70 increases E2 competitive binding to the wtAR in the presence of low level androgen and also retards E2 dissociation from the wtAR. ARA70 is present in both the cytoplasm and the nucleus of various mouse testicular cells during early embryogenesis day 16, at postpartum day 0 during estradiol synthesis and in the Leydig cells at postpartum day 49. ARA70 may be unable to modulate the PAIS AR mutants-E2 binding, diminishing the effect of E2 via AR during male reproductive system development in patients with such mutations. Therefore, the presence of ARA70 in the testosterone and E2-producing Leydig cells may enhance the overall activity of AR during critical stages of male sex organ development.