HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

ßArrestin1 regulates glucocorticoid receptor mitogenic signaling in castration-resistant prostate cancer.

BACKGROUND: Prostate cancer (PC) is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in males. The disease is initially treated with methods that inhibit androgen receptor (AR) signal transduction. Laboratory-based and clinical studies have identified alternative pathways that cause the failure of AR signal inhibition and consequent development of castration-resistant prostate cancer (CRPC). Glucocorticoid receptor (GR) signaling is activated in certain PC patients and promotes the emergence of CRPC, although by as yet incompletely understood mechanisms. We have previously demonstrated that ubiquitous ßarrestin1 (ßArr1) expression levels are linked to PC progression. Here, we consider the possibility that ßArr1 interacts with and activates GR in model CRPC cells. METHODS: Bioinformatic analysis of tumor xenograft and human PC datasets was used to correlate the expression of ßArr1 and GR. Western blot, immunohistochemistry and immunofluorescence microscopy, and subcellular fractionation were used to determine protein expression level and localization. Immunoprecipitation was applied to detect protein-protein interactions. RNA expression levels were determined using quantitative reverse transcription-polymerase chain reaction. Prostate sphere analysis was used to assess the rate of growth and invasion. The xenograft tumor implantation method was used to determine the tumor growth rate, local invasion, and metastasis. RESULTS: Elevated expression of ßArr1 positively correlated with increased GR expression and function in CRPC xenograft and in human PC patients. ßArr1 is expressed in the cell cytosol and nucleus, and it formed a complex with GR in the nucleus and not cytosol. Depletion of ßArr1 in AR-null CRPC cells inhibited GR function and CRPC growth and invasion in both in vitro and in vivo settings. CONCLUSIONS: ßArr1 binds GR that initiates mitogenic signaling cascades involved in the progression of PC to CRPC. The targeting of the ßArr1-GR axis may provide a new opportunity to better manage the CRPC disease.

Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells.

Uropathogenic Escherichia coli (UPEC) is the causative bacterium in most urinary tract infections (UTIs). UPEC cells adhere to and invade bladder epithelial cells (BECs) and cause uropathogenicity. Invading UPEC cells may encounter one of several fates, including degradation in the lysosome, expulsion to the extracellular milieu for clearance, or survival as an intracellular bacterial community and quiescent intracellular reservoir that can cause later infections. Here we considered the possibility that UPEC cells secrete factors that activate specific host cell signaling networks to facilitate the UPEC invasion of BECs. Using GFP-based reporters and Western blot analysis, we found that the representative human cystitis isolate E. coli UTI89 and its derivative UTI89ΔFimH, which does not bind to BECs, equally activate phosphatidylinositol 4,5-bisphosphate 3-OH kinase (PI3K), Akt kinase, and mTOR complex (mTORC) 1 and 2 in BECs. We also found that conditioned medium taken from UTI89 and UTI89ΔFimH cultures similarly activates epidermal growth factor receptor (EGFR), PI3K, Akt, and mTORC and that inhibition of EGFR and mTORC2, but not mTORC1, abrogates UTI89 invasion in vitro and in animal models of UTI. Our results reveal a key molecular mechanism of UPEC invasion and the host cells it targets, insights that may have therapeutic utility for managing the ever-increasing number of persistent and chronic UTIs.

The stress response neuropeptide CRF increases amyloid-ß production by regulating γ-secretase activity.

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-ß (Aß) production. In cells, CRF treatment increases Aß production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by ß-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aß. These data collectively link CRF to increased Aß through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aß and in some cases preferentially increase Aß42 via complex effects on γ-secretase.

Feedback regulation of G protein-coupled receptor signaling by GRKs and arrestins.

GPCRs are ubiquitous in mammalian cells and present intricate mechanisms for cellular signaling and communication. Mechanistically, GPCR signaling was identified to occur vectorially through heterotrimeric G proteins that are negatively regulated by GRK and arrestin effectors. Emerging evidence highlights additional roles for GRK and Arrestin partners, and establishes the existence of interconnected feedback pathways that collectively define GPCR signaling. GPCRs influence cellular dynamics and can mediate pathologic development, such as cancer and cardiovascular remolding. Hence, a better understanding of their overall signal regulation is of great translational interest and research continues to exploit the pharmacologic potential for modulating their activity.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

BACKGROUND: Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. METHODS: Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. RESULTS: Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. CONCLUSION: Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

Biased α-adrenergic receptor and ßarrestin signaling in a cell culture model of benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is a common disease in older men that involves the enlargement of the prostate gland. This occurs in response to signal transduction initiated by α-adrenergic receptors (α-ARs). When bound to ligands, α-ARs stimulate the mitogenic extracellular signal-regulated kinases 1 and 2 (ERK) pathway, ultimately promoting stromal and epithelial cell hyperplasia in the prostate. Current knowledge of how α-ARs promote prostate cell growth remains incomplete, and despite decades of research, there is no cure for BPH. In this study, we aimed to exploit an in vitro model system of BPH in order to better understand the mechanisms of α-AR signaling in prostatic hyperplasia.

ßArrestin1 regulates the guanine nucleotide exchange factor RasGRF2 expression and the small GTPase Rac-mediated formation of membrane protrusion and cell motility.

ßArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that ßarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of ßarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of ßarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of ßarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that ßarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.

Acute activation of ß2-adrenergic receptor regulates focal adhesions through ßArrestin2- and p115RhoGEF protein-mediated activation of RhoA.

ß(2)-Adrenergic receptors (ß(2)ARs) regulate cellular functions through G protein-transduced and ßArrestin-transduced signals. ß(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that ß(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. ß(2)ARs induce activation of RhoA, which is dependent on ßArrestin2 but not G(s). ßArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that ßArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon ß(2)AR activation, both ßArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of ß(2)ARs in cell migration. These results suggest that ßArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.

S-nitrosylation-regulated GPCR signaling.

G protein-coupled receptors (GPCRs) are the most numerous and diverse type of cell surface receptors, accounting for about 1% of the entire human genome and relaying signals from a variety of extracellular stimuli that range from lipid and peptide growth factors to ions and sensory inputs. Activated GPCRs regulate a multitude of target cell functions, including intermediary metabolism, growth and differentiation, and migration and invasion. The GPCRs contain a characteristic 7-transmembrane domain topology and their activation promotes complex formation with a variety of intracellular partner proteins, which form basis for initiation of distinct signaling networks as well as dictate fate of the receptor itself. Both termination of active GPCR signaling and removal from the plasma membrane are controlled by protein post-translational modifications of the receptor itself and its interacting partners. Phosphorylation, acylation and ubiquitination are the most studied post-translational modifications involved in GPCR signal transduction, subcellular trafficking and overall expression. Emerging evidence demonstrates that protein S-nitrosylation, the covalent attachment of a nitric oxide moiety to specified cysteine thiol groups, of GPCRs and/or their associated effectors also participates in the fine-tuning of receptor signaling and expression. This newly appreciated mode of GPCR system modification adds another set of controls to more precisely regulate the many cellular functions elicited by this large group of receptors. This article is part of a Special Issue entitled: Regulation of cellular processes by S-nitrosylation.

PGE2 promotes angiogenesis through EP4 and PKA Cγ pathway.

Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic response is involved in human diseases, including cancer. Proinflammatory prostaglandin E2 (PGE2) is secreted by many cell types and plays important roles in the process of angiogenesis via activation of cognate EP1-4 receptors. Here, we provide evidence that PGE2 promotes the in vitro tube formation of human microvascular endothelial cells, ex vivo vessel outgrowth of aortic rings, and actual in vivo angiogenesis. Use of EP subtype-selective agonists and antagonists suggested EP4 mediates the prostaglandin-induced tube formation, and this conclusion was substantiated with small interfering RNA to specifically knockdown the EP4 expression. EP4 couples to Gαs, leading to activation of protein kinase A (PKA). Inhibition of PKA activity or knockdown of PKA catalytic subunit γ with RNAi attenuates the PGE2-induced tube formation. Further, knocking down the expression of Rap1A, HSPB6, or endothelial NO synthase, which serve as PKA-activatable substrates, inhibits the tube formation, whereas knockdown of RhoA or glycogen synthase kinase 3ß that are inactivated after phosphorylation by PKA increases the tube formation. These results support the existence of EP4-to-PKA angiogenic signal and provide rationale for use of selective EP4 signal inhibitors as a probable strategy to control pathologic angiogenesis.

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. AR KO reduced levels of H3.3 at enhancers, indicating feedback mechanism. In addition, HIRA KO deregulates glucocorticoid-driven transcription, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity. Key points: *H3.3 at enhancers promotes acetylation of H3K27Ac and retention of AR/BRD4 complex for transcription regulation*Knockout of H3.3 chaperone HIRA suppresses PC cells growth and deregulates androgen-induced transcription*H3.3/HIRA pathway regulates both AR and GR, suggesting a common HIRA/H3.3 mechanism of nuclear receptors function.

DAXX drives de novo lipogenesis and contributes to tumorigenesis.

Cancer cells exhibit elevated lipid synthesis. In breast and other cancer types, genes involved in lipid production are highly upregulated, but the mechanisms that control their expression remain poorly understood. Using integrated transcriptomic, lipidomic, and molecular studies, here we report that DAXX is a regulator of oncogenic lipogenesis. DAXX depletion attenuates, while its overexpression enhances, lipogenic gene expression, lipogenesis, and tumor growth. Mechanistically, DAXX interacts with SREBP1 and SREBP2 and activates SREBP-mediated transcription. DAXX associates with lipogenic gene promoters through SREBPs. Underscoring the critical roles for the DAXX-SREBP interaction for lipogenesis, SREBP2 knockdown attenuates tumor growth in cells with DAXX overexpression, and DAXX mutants unable to bind SREBP1/2 have weakened activity in promoting lipogenesis and tumor growth. Remarkably, a DAXX mutant deficient of SUMO-binding fails to activate SREBP1/2 and lipogenesis due to impaired SREBP binding and chromatin recruitment and is defective of stimulating tumorigenesis. Hence, DAXX's SUMO-binding activity is critical to oncogenic lipogenesis. Notably, a peptide corresponding to DAXX's C-terminal SUMO-interacting motif (SIM2) is cell-membrane permeable, disrupts the DAXX-SREBP1/2 interactions, and inhibits lipogenesis and tumor growth. These results establish DAXX as a regulator of lipogenesis and a potential therapeutic target for cancer therapy.

Prostaglandin E2 regulates renal cell carcinoma invasion through the EP4 receptor-Rap GTPase signal transduction pathway.

Prognosis for patients with early stage kidney cancer has improved, but the treatment options for patients with locally advanced disease and metastasis remain few. Understanding the molecular mechanisms that regulate invasion and metastasis is critical for developing successful therapies to treat these patients. Proinflammatory prostaglandin E(2) plays an important role in cancer initiation and progression via activation of cognate EP receptors that belong to the superfamily of G protein-coupled receptors. Here we report that prostaglandin E(2) promotes renal cancer cell invasion through a signal transduction pathway that encompasses EP4 and small GTPase Rap. Inactivation of Rap signaling with Rap1GAP, like inhibition of EP4 signaling with ligand antagonist or knockdown with shRNA, reduces the kidney cancer cell invasion. Human kidney cells evidence increased EP4 and decreased Rap1GAP expression levels in the malignant compared with benign samples. These results support the idea that targeted inhibition of EP4 signaling and restoration of Rap1GAP expression constitute a new strategy to control kidney cancer progression.

Dynamin2 S-nitrosylation regulates adenovirus type 5 infection of epithelial cells.

Dynamin2 is a large GTPase that regulates vesicle trafficking, and the GTPase activity of dynamin2 is required for the multistep process of adenovirus infection. Activity of dynamin2 may be regulated by post-translational phosphorylation and S-nitrosylation modifications. In this study, we demonstrate a role for dynamin2 S-nitrosylation in adenovirus infection of epithelial cells. We show that adenovirus serotype 5 (Ad5) infection augments production of nitric oxide (NO) in epithelial cells and causes the S-nitrosylation of dynamin2, mainly on cysteine 86 (C86) and 607 (C607) residues. Forced overexpression of dynamin2 bearing C86A and/or C607A mutations decreases Ad5 infection. Diminishing NO synthesis by RNAi-induced knockdown of endogenous endothelial NO synthase (eNOS) expression attenuates virus infection of target cells. Ad5 infection promotes the kinetically dynamic S-nitrosylation of dynamin2 and eNOS: there is a rapid decrease in eNOS S-nitrosylation and a concomitant increase in the dynamin2 S-nitrosylation. These results support the hypothesis that dynamin2 S-nitrosylation following eNOS activation facilitates adenovirus infection of host epithelial cells.

G-protein coupled receptor kinase 5 regulates prostate tumor growth.

PURPOSE: The limited success of cancer therapeutics is largely attributable to the ability of cancer to become resistant to conventional cytotoxic chemotherapy. Thus, further identification of signaling molecules and pathways that influence tumorigenesis is needed to increase the overall therapeutic options. GRKs, originally recognized for their conserved role in GPCR signal control, have now emerged as regulators of additional biological molecules and functions. MATERIALS AND METHODS: We used Western blot analysis to determine GRK expression in prostate cancer and RNA interference to establish the role of GRK5 in prostate cancer growth and progression through the cell cycle. RESULTS: GRK5 was expressed highly in the aggressive prostate cancer PC3 cell line and its silencing by RNA interference attenuated in vitro cell proliferation. PC3 cells that stably expressed lentiviral small hairpin RNA and targeted GRK5 evidence reduced xenograft tumor growth in mice. This was reversed by rescuing expression with wild-type but not with kinase inactive K215R GRK5, implying the need of GRK5 kinase activity for tumor growth. To investigate possible cellular mechanism(s) for GRK5 in cell growth regulation we tested whether kinase activity would impact cell cycle progression. Like forced over expression of kinase-inactive K215R GRK5, GRK5 knockdown led to G2/M arrest in the cell cycle. Also, evidence revealed that the loss of GRK5 activity resulted in decreased cyclin D1 expression, Rb protein phosphorylation and E2F target gene expression involved in cell cycle control. CONCLUSIONS: Results provide direct evidence that GRK5 has an immediate role in the regulation of prostate tumor growth.

Identification of betaArrestin2 as a corepressor of androgen receptor signaling in prostate cancer.

Androgen receptor (AR) signaling regulates the development and homeostasis of male reproductive organs, including the prostate. Deregulation of AR and AR coregulators, expression, or activity is involved in the initiation of prostate cancer and contributes to the transition of the disease to hormone-refractory stage. The ubiquitous betaArrestin proteins are now recognized as bona fide adapters and signal transducers with target effectors found in both the cytosol and nucleus. Here, we provide evidence that betaArrestin2 forms a complex with AR and acts as an AR corepressor in androgen-dependent prostate cancer cells. Accordingly, the forced overexpression of betaArrestin2 diminishes, and knockdown of betaArrestin2 expression with RNAi increases the androgen-induced prostate-specific antigen (PSA) gene expression. betaArrestin2 serves as an adapter, bringing into close proximity the Mdm2 E3 ligase and AR, thereby promoting AR ubiquitylation and degradation. Human prostate tissues evidence an inverse relationship between the expression of betaArrestin2 and AR activity: glands that express high levels of betaArrestin2 exhibit low expression of PSA, and those glands that express low levels of betaArrestin2 evidence elevated PSA levels. We conclude that betaArrestin2 acts as a corepressor of AR by serving as a scaffold for Mdm2 leading to the AR ubiquitylation and degradation.

Inhibition of Mps1 kinase enhances taxanes efficacy in castration resistant prostate cancer.

Androgen ablation therapy is the standard of care for newly diagnosed prostate cancer (PC) patients. PC that relapsed after hormonal therapy, referred to as castration-resistant PC (CRPC), often presents with metastasis (mCRPC) and is the major cause of disease lethality. The few available therapies for mCRPC include the Taxanes Docetaxel (DTX) and Cabazitaxel (CBZ). Alas, clinical success of Taxanes in mCRPC is limited by high intrinsic and acquired resistance. Therefore, it remains essential to develop rationally designed treatments for managing therapy-resistant mCRPC disease. The major effect of Taxanes on microtubule hyper-polymerization is a prolonged mitotic block due to activation of the Spindle Assembly Checkpoint (SAC). Taxane-sensitive cells eventually inactivate SAC and exit mitosis by mitotic catastrophe, resulting in genome instability and blockade of proliferation. Resistant cells remain in mitotic block, and, upon drug decay, resume mitosis and proliferation, underlying one resistance mechanism. In our study we explored the possibility of forced mitotic exit to elevate Taxane efficacy. Inactivation of the SAC component, mitotic checkpoint kinase Mps1/TTK with a small molecule inhibitor (Msp1i), potentiated efficacy of Taxanes treatment in both 2D cell culture and 3D prostasphere settings. Mechanistically, Mps1 inhibition forced mitotic catastrophe in cells blocked in mitosis by Taxanes. Androgen receptor (AR), the main driver of PC, is often mutated or truncated in mCRPC. Remarkably, Mps1i significantly potentiated CBZ cytotoxicity regardless of AR status, in both AR-WT and in AR-truncated CRPC cells. Overall, our data demonstrate that forced mitotic exit by Mps1 inhibition potentiates Taxanes efficacy. Given that several Mps1i's are currently in different stages of clinical trials, our results point to Mps1 as a new therapeutic target to potentiate efficacy of Taxanes in mCRPC patients.

Nuclear ßArrestin1 regulates androgen receptor function in castration resistant prostate cancer.

Progression of prostate cancer (PC) to terminal castration-resistant PC (CRPC) involves a diverse set of intermediates, and androgen receptor (AR) is the key mediator of PC initiation and progression to CRPC. Hence, identification of factors involved in the regulation of AR expression and function is a necessary first-step to improve disease outcome. In this study, we identified ubiquitous ßArrestin 1 (ßArr1) as a regulator of AR function in CRPC. Unbiased gene expression analysis of public datasets revealed increased levels of ARRB1 (the gene encoding ßArr1) in CRPC when compared to normal tissue. Further, ßArr1 expression correlated with enhanced AR transcriptional function in these datasets. The ßArr1 partitions to both nucleus and cytosol and mechanistic studies showed that nuclear, and not cytosolic, ßArr1 formed a complex with AR and AR-coregulator ßCatenin and that the heterotrimeric protein complex was recruited to androgen-response elements of AR-regulated genes. Functionally, we demonstrate that depletion of ßArr1 attenuates PC cell and tumor growth and metastasis, and rescued expression of nuclear, but not cytosolic, ßArr1 restores the PC colony growth and invasion of Matrigel in vitro and tumor growth and metastasis in mice. The targeting of ßArr1-regulated AR transcriptional function may be used in the development of new drugs to treat lethal CRPC.