Novel potent liposome agonists of triggering receptor expressed on myeloid cells 2 phenocopy antibody treatment in cells.

The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists.

LncRNA PCAT1 activates AKT and NF-κB signaling in castration-resistant prostate cancer by regulating the PHLPP/FKBP51/IKKα complex.

In PTEN-deficient prostate cancers, AKT signaling may be activated upon suppression of androgen receptor signaling. Activation of AKT as well as NF-κB signaling involves a key regulatory protein complex containing PHLPP, FKBP51 and IKKα. Here, we report a critical role of lncRNA PCAT1 in regulating the PHLPP/FKBP51/IKKα complex and progression of castration-resistant prostate cancer (CRPC). Using database queries, bioinformatic analyses, as well as RIP and RNA pull-down assays, we discovered and validated that the lncRNA-PCAT1 perturbs the PHLPP/FKBP51/IKKα complex and activates AKT and NF-κB signaling. Expression of lncRNA-PCAT1 is positively linked to CRPC progression. PCAT1 binds directly to FKBP51, displacing PHLPP from the PHLPP/FKBP51/IKKα complex, leading to activation of AKT and NF-κB signaling. Targeting PCAT1 restores PHLPP binding to FKBP1 leading to suppression of AKT signaling. Preclinical study in a mouse model of CRPC suggests therapeutic potential by targeting lncRNA PCAT1 to suppress CRPC progression. Together, the newly identified PCAT1/FKBP51/IKKα complex provides mechanistic insight in the interplay between AKT, NF-κB and AR signaling in CRPC, and the preclinical studies suggest that a novel role for PCAT1 as a therapeutic target.

Comprehensive metabolomics analysis of prostate cancer tissue in relation to tumor aggressiveness and TMPRSS2-ERG fusion status.

BACKGROUND: Prostate cancer (PC) can display very heterogeneous phenotypes ranging from indolent asymptomatic to aggressive lethal forms. Understanding how these PC subtypes vary in their striving for energy and anabolic molecules is of fundamental importance for developing more effective therapies and diagnostics. Here, we carried out an extensive analysis of prostate tissue samples to reveal metabolic alterations during PC development and disease progression and furthermore between TMPRSS2-ERG rearrangement-positive and -negative PC subclasses. METHODS: Comprehensive metabolomics analysis of prostate tissue samples was performed by non-destructive high-resolution magic angle spinning nuclear magnetic resonance (1H HR MAS NMR). Subsequently, samples underwent moderate extraction, leaving tissue morphology intact for histopathological characterization. Metabolites in tissue extracts were identified by 1H/31P NMR and liquid chromatography-mass spectrometry (LC-MS). These metabolomics profiles were analyzed by chemometric tools and the outcome was further validated using proteomic data from a separate sample cohort. RESULTS: The obtained metabolite patterns significantly differed between PC and benign tissue and between samples with high and low Gleason score (GS). Five key metabolites (phosphocholine, glutamate, hypoxanthine, arginine and α-glucose) were identified, who were sufficient to differentiate between cancer and benign tissue and between high to low GS. In ERG-positive PC, the analysis revealed several acylcarnitines among the increased metabolites together with decreased levels of proteins involved in ß-oxidation; indicating decreased acyl-CoAs oxidation in ERG-positive tumors. The ERG-positive group also showed increased levels of metabolites and proteins involved in purine catabolism; a potential sign of increased DNA damage and oxidative stress. CONCLUSIONS: Our comprehensive metabolomic analysis strongly indicates that ERG-positive PC and ERG-negative PC should be considered as different subtypes of PC; a fact requiring different, sub-type specific treatment strategies for affected patients.

SPOP promotes transcriptional expression of DNA repair and replication factors to prevent replication stress and genomic instability.

Mutations in SPOP, the gene most frequently point-mutated in primary prostate cancer, are associated with a high degree of genomic instability and deficiency in homologous recombination repair of DNA but the underlying mechanisms behind this defect are currently unknown. Here we demonstrate that SPOP knockdown leads to spontaneous replication stress and impaired recovery from replication fork stalling. We show that this is associated with reduced expression of several key DNA repair and replication factors including BRCA2, ATR, CHK1 and RAD51. Consequently, SPOP knockdown impairs RAD51 foci formation and activation of CHK1 in response to replication stress and compromises recovery from replication fork stalling. An SPOP interactome analysis shows that wild type (WT) SPOP but not mutant SPOP associates with multiple proteins involved in transcription, mRNA splicing and export. Consistent with the association of SPOP with transcription, splicing and RNA export complexes, the decreased expression of BRCA2, ATR, CHK1 and RAD51 occurs at the level of transcription.

Immunoreactivity for prostate specific antigen and Ki67 differentiates subgroups of prostate cancer related to outcome.

Based on gene-expression profiles, prostate tumors can be subdivided into subtypes with different aggressiveness and response to treatment. We investigated if similar clinically relevant subgroups can be identified simply by the combination of two immunohistochemistry markers: one for tumor cell differentiation (prostate specific antigen, PSA) and one for proliferation (Ki67). This was analyzed in men with prostate cancer diagnosed at transurethral resection of the prostate 1975-1991 (n = 331) where the majority was managed by watchful waiting. Ki67 and PSA immunoreactivity was related to outcome and to tumor characteristics previously associated with prognosis. Increased Ki67 and decreased PSA were associated with poor outcome, and they provided independent prognostic information from Gleason score. A combinatory score for PSA and Ki67 immunoreactivity was produced using the median PSA and Ki67 levels as cut-off (for Ki67 the upper quartile was also evaluated) for differentiation into subgroups. Patients with PSA low/Ki67 high tumors showed higher Gleason score, more advanced tumor stage, and higher risk of prostate cancer death compared to other patients. Their tumor epithelial cells were often ERG positive and expressed higher levels of ErbB2, phosphorylated epidermal growth factor receptor (pEGF-R) and protein kinase B (pAkt), and their tumor stroma showed a reactive response with type 2 macrophage infiltration, high density of blood vessels and hyaluronic acid, and with reduced levels of caveolin-1, androgen receptors, and mast cells. In contrast, men with PSA high/Ki67 low tumors were characterized by low Gleason score, and the most favorable outcome amongst PSA/Ki67-defined subgroups. Men with PSA low/Ki67 low tumors showed clinical and tumor characteristics intermediate of the two groups above. A combinatory PSA/Ki67 immunoreactivity score identifies subgroups of prostate cancers with different epithelial and stroma phenotypes and highly different outcome but the clinical usefulness of this approach needs to be validated in other cohorts.

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer.

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase ß-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Protein interaction screening for the ankyrin repeats and suppressor of cytokine signaling (SOCS) box (ASB) family identify Asb11 as a novel endoplasmic reticulum resident ubiquitin ligase.

The ankyrin and SOCS (suppressor of cytokine signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting 18 members in humans, the identity of the physiological targets of the Asb proteins remains largely unexplored. To increase our understanding of the function of ASB proteins, we conducted a family-wide SILAC (stable isotope labeling by amino acids in cell culture)-based protein/protein interaction analysis. This investigation led to the identification of novel as well as known ASB-associated proteins like Cullin 5 and Elongins B/C. We observed that several proteins can be bound by more than one Asb protein. The additional exploration of this phenomenon demonstrated that ASB-Cullin 5 complexes can oligomerize and provides evidence that Cullin 5 forms heterodimeric complexes with the Cullin 4a-DDB1 complex. We also demonstrated that ASB11 is a novel endoplasmic reticulum-associated ubiquitin ligase with the ability to interact and promote the ubiquitination of Ribophorin 1, an integral protein of the oligosaccharyltransferase (OST) glycosylation complex. Moreover, expression of ASB11 can increase Ribophorin 1 protein turnover in vivo. In summary, we provide a comprehensive protein/protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases.

OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

BACKGROUND: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored. METHODS: RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth. RESULTS: Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo. CONCLUSIONS: OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

SOCS proteins in regulation of receptor tyrosine kinase signaling.

Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment. The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also evident that RTKs can sometimes bypass SOCS regulation and SOCS proteins can even potentiate RTKs-mediated mitogenic signaling. Thus, apart from negative regulation of receptor signaling, SOCS proteins may also influence signaling in other ways.

SOCS2 mediates the cross talk between androgen and growth hormone signaling in prostate cancer.

UNLABELLED: Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa. SUMMARY: Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

TNIK drives castration-resistant prostate cancer via phosphorylating EGFR.

The development of castration-resistant prostate cancer (CRPC) is driven by intricate genetic and epigenetic mechanisms. Traf2- and Nck-interacting kinase (TNIK) has been reported as a serine/threonine kinase associated with tumor cell proliferation or unfavorable cancer behavior. The microarray approach revealed a substantial upregulation of TNIK expression levels, enabling us to investigate the functional behaviors of the TNIK gene in CRPC. Specifically, we discovered that AR suppresses TNIK gene transcription in LNCaP and C4-2 cells by forming a complex with H3K27me3. Following the reduction of AR levels induced by androgen deprivation therapy (ADT), TNIK is recruited to activate EGFR signaling through phosphorylation in C4-2 cells, thereby promoting CRPC progression. Our findings unveil a regulatory role of AR as a repressor for TNIK while also highlighting how TNIK activates the EGFR pathway via phosphorylation to drive CRPC progression. Consequently, targeting TNIK may represent an appealing therapeutic strategy for CRPC.

Suppressor of cytokine signaling 6 (SOCS6) negatively regulates Flt3 signal transduction through direct binding to phosphorylated tyrosines 591 and 919 of Flt3.

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.

SOCS2 deletion protects against hepatic steatosis but worsens insulin resistance in high-fat-diet-fed mice.

Hepatic steatosis is a prominent feature in patients with growth hormone (GH) deficiency. The ubiquitin ligase SOCS2 attenuates hepatic GH signaling by inhibiting the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) axis. Here, we investigated the role of SOCS2 in the development of diet-induced hepatic steatosis and insulin resistance. SOCS2-knockout (SOCS2(-/-)) mice and wild-type littermates were fed for 4 mo with control or high-fat diet, followed by assessment of insulin sensitivity, hepatic lipid content, and expression of inflammatory cytokines. SOCS2(-/-) mice exhibited increased hepatic TG secretion by 77.6% (P<0.001) as compared with wild-type control mice and were protected from high-fat-diet (HFD)-induced hepatic steatosis, showing 49.3% (P<0.01) reduction in liver TG levels compared to HFD-fed wild-type littermates. In contrast, we found that HFD-triggered attenuation of systemic insulin sensitivity was more marked in SOCS2(-/-) mice. Livers from the HFD-fed SOCS2(-/-) mice showed increased NF-κB activity as well as elevated expression of genes for the inflammatory cytokines IFN-γ and IL-6. An inhibitory role of SOCS2 on Toll-like receptor 4 signaling was demonstrated in macrophages obtained from the SOCS2(-/-) and wild-type mice. This study identified SOCS2 as an important regulator of hepatic homeostasis under conditions of high-fat dietary stress.

Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase.

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564-574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (K(d) = 0.3 µm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.

Parkin-mediated ubiquitination regulates phospholipase C-gamma1.

Mutations in parkin cause autosomal recessive forms of Parkinson's disease (PD), with an early age of onset and similar pathological phenotype to the idiopathic disease. Parkin has been identified as an E3 ubiquitin ligase that mediates different types of ubiquitination, which has made the search for substrates an intriguing possibility to identify pathological mechanisms linked to PD. In this study, we present PLCgamma1 as a novel substrate for parkin. This association was found in non-transfected human neuroblastoma SH-SY5Y cells as well as in stable cell lines expressing parkin WT and familial mutants R42P and G328E. Analysis of cortical, striatal and nigral human brain homogenates revealed that the interaction between parkin and PLCgamma1 is consistent throughout these regions, suggesting that the interaction is likely to have a physiological relevance for humans. Unlike many of the previously identified substrates, we could also show that the steady-state levels of PLCgamma1 is significantly higher in parkin KO mice and lower in parkin WT human neuroblastoma cells, suggesting that parkin ubiquitination of PLCgamma1 is required for proteasomal degradation. In line with this idea, we show that the ability to ubiquitinate PLCgamma1 in vitro differs significantly between WT and familial mutant parkin. In this study, we demonstrate that parkin interacts with PLCgamma1, affecting PLCgamma1 steady state protein levels in human and murine models with manipulated parkin function and expression levels. This finding could be of relevance for finding novel pathogenic mechanisms leading to PD.

Suppressor of cytokine signaling (SOCS) 2, a protein with multiple functions.

Cytokine receptors act through a complex signaling network, involving Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs), to regulate diverse biological processes which control growth, development, homeostasis and immune function, among others. The JAK/STAT signaling pathway is attenuated via three mechanisms controlling the initiation, magnitude, and duration of the signal: the PIAS proteins, which prevent STAT dimerization or DNA interaction, the SHP phosphatases, which dephosphorylate activating tyrosine phosphorylations, and the suppressors of cytokine signaling (SOCS), which are transcribed in response to cytokine stimulation and use several interconnected mechanisms to downregulate the signal. Specific studies targeting the SOCS genes in vivo have unveiled SOCS2 as the main regulator of somatic growth through regulation of GH/IGF-1 signaling. In addition, several studies indicate that SOCS2 also has important actions in the central nervous system, the regulation of metabolism, the immune response, the mammary gland development, cancer, and other cytokine-dependent signaling pathways. Consistent with the role of cytokines in human physiology, any SOCS2 imbalance could result in a broad range of pathologies such as cardiovascular diseases, insulin resistance, cancer, and severe infections, among others. Thus, determining the importance of SOCS2 in health and disease will no doubt aid in the development of novel therapeutic strategies. In this review, we attempt to summarize the available information, including our results, regarding the role of SOCS2 in several biological processes.

Correction: Neurotensin and its receptors mediate neuroendocrine transdifferentiation in prostate cancer.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Neurotensin and its receptors mediate neuroendocrine transdifferentiation in prostate cancer.

Castration-resistant prostate cancer (CRPC) with neuroendocrine differentiation (NED) is a lethal disease for which effective therapies are urgently needed. The mechanism underlying development of CRPC with NED, however, remains largely uncharacterized. In this study, we explored and characterized the functional role of neurotensin (NTS) in cell line and animal models of CRPC with NED. NTS was acutely induced by androgen deprivation in animal models of prostate cancer (PCa) and activated downstream signaling leading to NED through activation of neurotensin receptor 1 (NTSR1) and neurotensin receptor 3 (NTSR3), but not neurotensin receptor 2 (NTSR2). Our findings also revealed the existence of a CK8+/CK14+ subpopulation in the LNCaP cell line that expresses high levels of both NTSR1 and NTSR3, and displays an enhanced susceptibility to develop neuroendocrine-like phenotypes upon treatment with NTS. More importantly, NTSR1 pathway inhibition prevented the development of NED and castration resistance in vivo. We propose a novel role of NTS in the development of CRPC with NED, and a possible strategy to prevent the onset of NED by targeting the NTS signaling pathway.