Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus.

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones.

Ferroptosis, a cell death process driven by iron-dependent phospholipid peroxidation, has been implicated in various diseases. There are two major surveillance mechanisms to suppress ferroptosis: one mediated by glutathione peroxidase 4 (GPX4) that catalyzes the reduction of phospholipid peroxides and the other mediated by enzymes, such as FSP1, that produce metabolites with free radical-trapping antioxidant activity. In this study, through a whole-genome CRISPR activation screen, followed by mechanistic investigation, we identified phospholipid-modifying enzymes MBOAT1 and MBOAT2 as ferroptosis suppressors. MBOAT1/2 inhibit ferroptosis by remodeling the cellular phospholipid profile, and strikingly, their ferroptosis surveillance function is independent of GPX4 or FSP1. MBOAT1 and MBOAT2 are transcriptionally upregulated by sex hormone receptors, i.e., estrogen receptor (ER) and androgen receptor (AR), respectively. A combination of ER or AR antagonist with ferroptosis induction significantly inhibited the growth of ER+ breast cancer and AR+ prostate cancer, even when tumors were resistant to single-agent hormonal therapies.

A Somatically Acquired Enhancer of the Androgen Receptor Is a Noncoding Driver in Advanced Prostate Cancer.

Increased androgen receptor (AR) activity drives therapeutic resistance in advanced prostate cancer. The most common resistance mechanism is amplification of this locus presumably targeting the AR gene. Here, we identify and characterize a somatically acquired AR enhancer located 650 kb centromeric to the AR. Systematic perturbation of this enhancer using genome editing decreased proliferation by suppressing AR levels. Insertion of an additional copy of this region sufficed to increase proliferation under low androgen conditions and to decrease sensitivity to enzalutamide. Epigenetic data generated in localized prostate tumors and benign specimens support the notion that this region is a developmental enhancer. Collectively, these observations underscore the importance of epigenomic profiling in primary specimens and the value of deploying genome editing to functionally characterize noncoding elements. More broadly, this work identifies a therapeutic vulnerability for targeting the AR and emphasizes the importance of regulatory elements as highly recurrent oncogenic drivers.

Genomic Hallmarks and Structural Variation in Metastatic Prostate Cancer.

While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.

Nuclear Pores Promote Lethal Prostate Cancer by Increasing POM121-Driven E2F1, MYC, and AR Nuclear Import.

Nuclear pore complexes (NPCs) regulate nuclear-cytoplasmic transport, transcription, and genome integrity in eukaryotic cells. However, their functional roles in cancer remain poorly understood. We interrogated the evolutionary transcriptomic landscape of NPC components, nucleoporins (Nups), from primary to advanced metastatic human prostate cancer (PC). Focused loss-of-function genetic screen of top-upregulated Nups in aggressive PC models identified POM121 as a key contributor to PC aggressiveness. Mechanistically, POM121 promoted PC progression by enhancing importin-dependent nuclear transport of key oncogenic (E2F1, MYC) and PC-specific (AR-GATA2) transcription factors, uncovering a pharmacologically targetable axis that, when inhibited, decreased tumor growth, restored standard therapy efficacy, and improved survival in patient-derived pre-clinical models. Our studies molecularly establish a role of NPCs in PC progression and give a rationale for NPC-regulated nuclear import targeting as a therapeutic strategy for lethal PC. These findings may have implications for understanding how NPC deregulation contributes to the pathogenesis of other tumor types.

Structural Alterations Driving Castration-Resistant Prostate Cancer Revealed by Linked-Read Genome Sequencing.

Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%-87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.

Androgen receptor-mediated CD8+ T cell stemness programs drive sex differences in antitumor immunity.

The incidence and mortality rates of many non-reproductive human cancers are generally higher in males than in females. However, the immunological mechanism underlying sexual differences in cancers remains elusive. Here, we demonstrated that sex-related differences in tumor burden depended on adaptive immunity. Male CD8+ T cells exhibited impaired effector and stem cell-like properties compared with female CD8+ T cells. Mechanistically, androgen receptor inhibited the activity and stemness of male tumor-infiltrating CD8+ T cells by regulating epigenetic and transcriptional differentiation programs. Castration combined with anti-PD-L1 treatment synergistically restricted tumor growth in male mice. In humans, fewer male CD8+ T cells maintained a stem cell-like memory state compared with female counterparts. Moreover, AR expression correlated with tumor-infiltrating CD8+ T cell exhaustion in cancer patients. Our findings reveal sex-biased CD8+ T cell stemness programs in cancer progression and in the responses to cancer immunotherapy, providing insights into the development of sex-based immunotherapeutic strategies for cancer treatment.

Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Minor intron splicing is critical for survival of lethal prostate cancer.

The evolutionarily conserved minor spliceosome (MiS) is required for protein expression of ∼714 minor intron-containing genes (MIGs) crucial for cell-cycle regulation, DNA repair, and MAP-kinase signaling. We explored the role of MIGs and MiS in cancer, taking prostate cancer (PCa) as an exemplar. Both androgen receptor signaling and elevated levels of U6atac, a MiS small nuclear RNA, regulate MiS activity, which is highest in advanced metastatic PCa. siU6atac-mediated MiS inhibition in PCa in vitro model systems resulted in aberrant minor intron splicing leading to cell-cycle G1 arrest. Small interfering RNA knocking down U6atac was ∼50% more efficient in lowering tumor burden in models of advanced therapy-resistant PCa compared with standard antiandrogen therapy. In lethal PCa, siU6atac disrupted the splicing of a crucial lineage dependency factor, the RE1-silencing factor (REST). Taken together, we have nominated MiS as a vulnerability for lethal PCa and potentially other cancers.

Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes.

Steroid receptors activate gene transcription by recruiting coactivators to initiate transcription of their target genes. For most nuclear receptors, the ligand-dependent activation function domain-2 (AF-2) is a primary contributor to the nuclear receptor (NR) transcriptional activity. In contrast to other steroid receptors, such as ERα, the activation function of androgen receptor (AR) is largely dependent on its ligand-independent AF-1 located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different AF domain from other receptors despite that NRs share similar domain organizations. Here, we present cryoelectron microscopy (cryo-EM) structures of DNA-bound full-length AR and its complex structure with key coactivators, SRC-3 and p300. AR dimerization follows a unique head-to-head and tail-to-tail manner. Unlike ERα, AR directly contacts a single SRC-3 and p300. The AR NTD is the primary site for coactivator recruitment. The structures provide a basis for understanding assembly of the AR:coactivator complex and its domain contributions for coactivator assembly and transcriptional regulation.

YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth.

Hippo signaling restricts tumor growth by inhibiting the oncogenic potential of YAP/TAZ-TEAD transcriptional complex. Here, we uncover a context-dependent tumor suppressor function of YAP in androgen receptor (AR) positive prostate cancer (PCa) and show that YAP impedes AR+ PCa growth by antagonizing TEAD-mediated AR signaling. TEAD forms a complex with AR to enhance its promoter/enhancer occupancy and transcriptional activity. YAP and AR compete for TEAD binding and consequently, elevated YAP in the nucleus disrupts AR-TEAD interaction and prevents TEAD from promoting AR signaling. Pharmacological inhibition of MST1/2 or LATS1/2, or transgenic activation of YAP suppressed the growth of PCa expressing therapy resistant AR splicing variants. Our study uncovers an unanticipated crosstalk between Hippo and AR signaling pathways, reveals an antagonistic relationship between YAP and TEAD in AR+ PCa, and suggests that targeting the Hippo signaling pathway may provide a therapeutical opportunity to treat PCa driven by therapy resistant AR variants.

Testosterone acts through membrane protein GPRC6A to cause cardiac edema in zebrafish embryos.

Androgens are classically thought to act through intracellular androgen receptors (AR/NR3C4), but they can also trigger non-genomic effects via membrane proteins. Although several membrane androgen receptors have been characterized in vitro, their functions in vivo remain unclear. Using a chemical-genetic screen in zebrafish, we found that GPRC6A, a G-protein coupled receptor, mediates non-genomic androgen actions during embryonic development. Exposure to androgens (androstanedione, DHT, and testosterone) caused cardiac edema or tail curvature in wild-type embryos, as well as in ar mutants, suggesting AR-independent pathways. We then mutated putative membrane androgen receptors (gprc6a, hcar1-4, and zip9) and found that only gprc6a mutants exhibited a significant reduction in cardiac edema following testosterone exposure. Additionally, co-treatment of wild-type embryos with testosterone and GPRC6A antagonists significantly suppressed the cardiac edema phenotype. Using RNA-seq and RNA rescue approaches, we found that testosterone-GPRC6A causes cardiac phenotypes by reducing Pak1 signaling. Our results indicate that testosterone induces cardiac edema in zebrafish embryos through GPRC6A, independent of nuclear androgen receptors, highlighting a novel non-genomic androgen signaling pathway in embryonic development.

Maintenance of CTCF- and Transcription Factor-Mediated Interactions from the Gametes to the Early Mouse Embryo.

The epigenetic information present in mammalian gametes and whether it is transmitted to the progeny are relatively unknown. We find that many promoters in mouse sperm are occupied by RNA polymerase II (Pol II) and Mediator. The same promoters are accessible in GV and MII oocytes and preimplantation embryos. Sperm distal ATAC-seq sites containing motifs for various transcription factors are conserved in monkeys and humans. ChIP-seq analyses confirm that Foxa1, ERα, and AR occupy distal enhancers in sperm. Accessible sperm enhancers containing H3.3 and H2A.Z are also accessible in oocytes and preimplantation embryos. Furthermore, their interactions with promoters in the gametes persist during early development. Sperm- or oocyte-specific interactions mediated by CTCF and cohesin are only present in the paternal or maternal chromosomes, respectively, in the zygote and 2-cell stages. These interactions converge in both chromosomes by the 8-cell stage. Thus, mammalian gametes contain complex patterns of 3D interactions that can be transmitted to the zygote after fertilization.

The androgen receptor in mesenchymal progenitors regulates skeletal muscle mass via Igf1 expression in male mice.

Androgens exert their effects primarily by binding to the androgen receptor (AR), a ligand-dependent nuclear receptor. While androgens have anabolic effects on skeletal muscle, previous studies reported that AR functions in myofibers to regulate skeletal muscle quality, rather than skeletal muscle mass. Therefore, the anabolic effects of androgens are exerted via nonmyofiber cells. In this context, the cellular and molecular mechanisms of AR in mesenchymal progenitors, which play a crucial role in maintaining skeletal muscle homeostasis, remain largely unknown. In this study, we demonstrated expression of AR in mesenchymal progenitors and found that targeted AR ablation in mesenchymal progenitors reduced limb muscle mass in mature adult, but not young or aged, male mice, although fatty infiltration of muscle was not affected. The absence of AR in mesenchymal progenitors led to remarkable perineal muscle hypotrophy, regardless of age, due to abnormal regulation of transcripts associated with cell death and extracellular matrix organization. Additionally, we revealed that AR in mesenchymal progenitors regulates the expression of insulin-like growth factor 1 (Igf1) and that IGF1 administration prevents perineal muscle atrophy in a paracrine manner. These findings indicate that the anabolic effects of androgens regulate skeletal muscle mass via, at least in part, AR signaling in mesenchymal progenitors.

Streamlined identification of clinically and functionally relevant genetic regulators of lower-tract urogenital development.

Congenital anomalies of the lower genitourinary (LGU) tract are frequently comorbid due to genetically linked developmental pathways, and are among the most common yet most socially stigmatized congenital phenotypes. Genes involved in sexual differentiation are prime candidates for developmental anomalies of multiple LGU organs, but insufficient prospective screening tools have prevented the rapid identification of causative genes. Androgen signaling is among the most influential modulators of LGU development. The present study uses SpDamID technology in vivo to generate a comprehensive map of the pathways actively regulated by the androgen receptor (AR) in the genitalia in the presence of the p300 coactivator, identifying wingless/integrated (WNT) signaling as a highly enriched AR-regulated pathway in the genitalia. Transcription factor (TF) hits were then assayed for sexually dimorphic expression at two critical time points and also cross-referenced to a database of clinically relevant copy number variations to identify 252 TFs exhibiting copy variation in patients with LGU phenotypes. A subset of 54 TFs was identified for which LGU phenotypes are statistically overrepresented as a proportion of total observed phenotypes. The 252 TF hitlist was then subjected to a functional screen to identify hits whose silencing affects genital mesenchymal growth rates. Overlap of these datasets results in a refined list of 133 TFs of both functional and clinical relevance to LGU development, 31 of which are top priority candidates, including the well-documented renal progenitor regulator, Sall1. Loss of Sall1 was examined in vivo and confirmed to be a powerful regulator of LGU development.

The decision of male medaka to mate or fight depends on two complementary androgen signaling pathways.

Adult male animals typically court and attempt to mate with females, while attacking other males. Emerging evidence from mice indicates that neurons expressing the estrogen receptor ESR1 in behaviorally relevant brain regions play a central role in mediating these mutually exclusive behavioral responses to conspecifics. However, the findings in mice are unlikely to apply to vertebrates in general because, in many species other than rodents and some birds, androgens-rather than estrogens-have been implicated in male behaviors. Here, we report that male medaka (Oryzias latipes) lacking one of the two androgen receptor subtypes (Ara) are less aggressive toward other males and instead actively court them, while those lacking the other subtype (Arb) are less motivated to mate with females and conversely attack them. These findings indicate that, in male medaka, the Ara- and Arb-mediated androgen signaling pathways facilitate appropriate behavioral responses, while simultaneously suppressing inappropriate responses, to males and females, respectively. Notably, males lacking either receptor retain the ability to discriminate the sex of conspecifics, suggesting a defect in the subsequent decision-making process to mate or fight. We further show that Ara and Arb are expressed in intermingled but largely distinct populations of neurons, and stimulate the expression of different behaviorally relevant genes including galanin and vasotocin, respectively. Collectively, our results demonstrate that male teleosts make adaptive decisions to mate or fight as a result of the activation of one of two complementary androgen signaling pathways, depending on the sex of the conspecific that they encounter.

A molecular switch from tumor suppressor to oncogene in ER+ve breast cancer: Role of androgen receptor, JAK-STAT, and lineage plasticity.

Cancers develop resistance to inhibitors of oncogenes mainly due to target-centric mechanisms such as mutations and splicing. While inhibitors or antagonists force targets to unnatural conformation contributing to protein instability and resistance, activating tumor suppressors may maintain the protein in an agonistic conformation to elicit sustainable growth inhibition. Due to the lack of tumor suppressor agonists, this hypothesis and the mechanisms underlying resistance are not understood. In estrogen receptor (ER)-positive breast cancer (BC), androgen receptor (AR) is a druggable tumor suppressor offering a promising avenue for this investigation. Spatial genomics suggests that the molecular portrait of AR-expressing BC cells in tumor microenvironment corresponds to better overall patient survival, clinically confirming AR's role as a tumor suppressor. Ligand activation of AR in ER-positive BC xenografts reprograms cistromes, inhibits oncogenic pathways, and promotes cellular elasticity toward a more differentiated state. Sustained AR activation results in cistrome rearrangement toward transcription factor PROP paired-like homeobox 1, transformation of AR into oncogene, and activation of the Janus kinase/signal transducer (JAK/STAT) pathway, all culminating in lineage plasticity to an aggressive resistant subtype. While the molecular profile of AR agonist-sensitive tumors corresponds to better patient survival, the profile represented in the resistant phenotype corresponds to shorter survival. Inhibition of activated oncogenes in resistant tumors reduces growth and resensitizes them to AR agonists. These findings indicate that persistent activation of a context-dependent tumor suppressor may lead to resistance through lineage plasticity-driven tumor metamorphosis. Our work provides a framework to explore the above phenomenon across multiple cancer types and underscores the importance of factoring sensitization of tumor suppressor targets while developing agonist-like drugs.

Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments.

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.

ZFX Mediates Non-canonical Oncogenic Functions of the Androgen Receptor Splice Variant 7 in Castrate-Resistant Prostate Cancer.

Androgen receptor splice variant 7 (AR-V7) is crucial for prostate cancer progression and therapeutic resistance. We show that, independent of ligand, AR-V7 binds both androgen-responsive elements (AREs) and non-canonical sites distinct from full-length AR (AR-FL) targets. Consequently, AR-V7 not only recapitulates AR-FL's partial functions but also regulates an additional gene expression program uniquely via binding to gene promoters rather than ARE enhancers. AR-V7 binding and AR-V7-mediated activation at these unique targets do not require FOXA1 but rely on ZFX and BRD4. Knockdown of ZFX or select unique targets of AR-V7/ZFX, or BRD4 inhibition, suppresses growth of castration-resistant prostate cancer cells. We also define an AR-V7 direct target gene signature that correlates with AR-V7 expression in primary tumors, differentiates metastatic prostate cancer from normal, and predicts poor prognosis. Thus, AR-V7 has both ARE/FOXA1 canonical and ZFX-directed non-canonical regulatory functions in the evolution of anti-androgen therapeutic resistance, providing information to guide effective therapeutic strategies.

Sarcopenia phenotype and impaired muscle function in male mice with fast-twitch muscle-specific knockout of the androgen receptor.

Sarcopenia is distinct from normal muscle atrophy in that it is closely related to a shift in the muscle fiber type. Deficiency of the anabolic action of androgen on skeletal muscles is associated with sarcopenia; however, the function of the androgen receptor (AR) pathway in sarcopenia remains poorly understood. We generated a mouse model (fast-twitch muscle-specific AR knockout [fmARKO] mice) in which the AR was selectively deleted in the fast-twitch muscle fibers. In young male mice, the deletion caused no change in muscle mass, but it reduced muscle strength and fatigue resistance and induced a shift in the soleus muscles from fast-twitch fibers to slow-twitch fibers (14% increase, P = 0.02). After middle age, with the control mice, the male fmARKO mice showed much less muscle function, accompanied by lower hindlimb muscle mass; this phenotype was similar to the progression of sarcopenia. The bone mineral density of the femur was significantly reduced in the fmARKO mice, indicating possible osteosarcopenia. Microarray and gene ontology analyses revealed that in male fmARKO mice, there was downregulation of polyamine biosynthesis-related geneswhich was confirmed by liquid chromatography-tandem mass spectrometry assay and the primary cultured myofibers. None of the AR deletion-related phenotypes were observed in female fmARKO mice. Our findings showed that the AR pathway had essential muscle type- and sex-specific roles in the differentiation toward fast-twitch fibers and in the maintenance of muscle composition and function. The AR in fast-twitch muscles was the dominant regulator of muscle fiber-type composition and muscle function, including the muscle-bone relationship.