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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.

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Purpose: Bone is the most predominant site of distant metastasis in prostate cancer, and patients have limited therapeutic options at this stage.Experimental Design: We performed a system-wide quantitative proteomic analysis of bone metastatic prostate tumors from 22 patients operated to relieve spinal cord compression. At the time of surgery, most patients had relapsed after androgen-deprivation therapy, while 5 were previously untreated. An extended cohort of prostate cancer bone metastases (n = 65) was used for immunohistochemical validation.Results: On average, 5,067 proteins were identified and quantified per tumor. Compared with primary tumors (n = 26), bone metastases were more heterogeneous and showed increased levels of proteins involved in cell-cycle progression, DNA damage response, RNA processing, and fatty acid β-oxidation; and reduced levels of proteins were related to cell adhesion and carbohydrate metabolism. Within bone metastases, we identified two phenotypic subgroups: BM1, expressing higher levels of AR canonical targets, and mitochondrial and Golgi apparatus resident proteins; and BM2, with increased expression of proliferation and DNA repair–related proteins. The two subgroups, validated by the inverse correlation between MCM3 and prostate specific antigen immunoreactivity, were related to disease prognosis, suggesting that this molecular heterogeneity should be considered when developing personalized therapies.Conclusions: This work is the first system-wide quantitative characterization of the proteome of prostate cancer bone metastases and a valuable resource for understanding the etiology of prostate cancer progression. Clin Cancer Res; 24(21); 5433–44. ©2018 AACR.

Purpose:An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood.Experimental Design: We applied mass spectrometry–based proteomics to a unique set of 17 prostate cancer patient–derived xenografts (PDX) to characterize the effects of castration in vivo, and the proteome differences between NEPC and prostate adenocarcinomas. Genome-wide profiling of REST-occupied regions in prostate cancer cells was correlated to the expression changes in vivo to investigate the role of the transcriptional repressor REST in castration-induced NEPC differentiation.Results:An average of 4,881 proteins were identified and quantified from each PDX. Proteins related to neurogenesis, cell-cycle regulation, and DNA repair were found upregulated and elevated in NEPC, while the reduced levels of proteins involved in mitochondrial functions suggested a prevalent glycolytic metabolism of NEPC tumors. Integration of the REST chromatin bound regions with expression changes indicated a direct role of REST in regulating neuronal gene expression in prostate cancer cells. Mechanistically, depletion of REST led to cell-cycle arrest in G1, which could be rescued by p53 knockdown. Finally, the expression of the REST-regulated gene secretagogin (SCGN) correlated with an increased risk of suffering disease relapse after radical prostatectomy.Conclusions:This study presents the first deep characterization of the proteome of NEPC and suggests that concomitant inhibition of REST and the p53 pathway would promote NEPC. We also identify SCGN as a novel prognostic marker in prostate cancer.

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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.

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.

Understanding disease biology at a cellular level from disease specific tissues is imperative for effective drug development for complex neurodegenerative diseases. We profiled 87,086 nuclei from putamen tissue of healthy controls, Parkinson’s Disease (PD), and Multiple System Atrophy (MSA) subjects to construct a comprehensive single cell atlas. Although both PD and MSA are manifestations of α-synuclein protein aggregation, we observed that both the diseases have distinct cell-type specific changes. We see a possible expansion and activation of microglia and astrocytes in PD compared to MSA and controls. Contrary to PD microglia, we found absence of upregulated unfolded protein response in MSA microglia compared to controls. Differentially expressed genes in major cell types are enriched for genes associated with PD-GWAS loci. We found altered expression of major neurodegeneration associated genes — SNCA, MAPT, LRRK2, and APP — at cell-type resolution. We also identified disease associated gene modules using a network biology approach. Overall, this study creates an interactive atlas from synucleinopathies and provides major cell-type specific disease insights.Link to interactive atlas will be made available at the time of publication.

The role of oligodendrocytes in neurodegenerative diseases remains incompletely understood and largely unexplored at the single cell level. We profiled 87,086 single nuclei from human brain putamen region for healthy control, Parkinson’s Disease (PD), and Multiple System Atrophy (MSA). Oligodendrocyte lineage cells were the dominant cell-type in the putamen with oligodendrocyte subpopulations clustered by transcriptomic variation found to exhibit diverse functional enrichment patterns, and this oligodendrocyte heterogeneity was altered in a disease-specific way. Among profiled oligodendrocyte subpopulations, differences in expression of SNCA, HAPLN2, MAPT, APP, and OPALIN were observed for PD and MSA compared with healthy controls. Intriguingly, greater activation of unfolded protein response pathway gene expression was observed in PD nuclei versus MSA. Using network analysis, we then identified specific PD- and MSA-correlated gene co-expression modules enriched with disease relevant pathways; the PD-correlated module was significantly enriched for Parkinson’s Disease GWAS loci (p = 0.01046). Our analysis provides a broader understanding of oligodendrocyte heterogeneity and reveals distinctive oligodendrocyte pathological alterations associated with PD and MSA which may suggest potential novel therapeutic targets and new strategies for disease modification.

Background: The development of CRPCa was driven by complex genetic and epigenetic mechanisms that remained poorly understood. TNIK (Traf2 and Nck-interacting kinase) has been reported to be a serine/threonine kinase and associated with tumor cell proliferation or unfavorable cancer behavior. The present study was conducted to investigate the TNIK gene expressions in CRPCa. Methods: Using a microarray approach, we identified higher expression of TNIK in CRPCa. The interaction between AR and H3K27me3 upon TNIK depression was determined through molecular and cell biological methods. Co-immunoprecipitations assays were Performed to confirm that TNIK interacted and phosphorylated with β-catenin in CRPCa cell. Results: Specifically we found AR repressed TNIK gene transcription via forming complex with H3K27me3. TNIK was recruited to promote transcription of Wnt target genes in a β-catenin-dependent manner in C4-2 cells. In vitro binding showed that TNIK directly band and phosphorylated β-catenin. Depletion or mutant of TNIK kinase abrogated β-catenin transcription, highlighting the essential function of TNIK kinase activity in Wnt target gene activation. Conclusions: Our findings revealed a regulatory role of AR in TNIK repressor, TNIK interacted with β-catenin and phosphorylated activaing Wnt pathway to promote CRPCa progression. TNIK may present an attractive candidate for drug targeting in CRPCa.

Purpose: Bone is the most predominant site of distant metastasis in prostate cancer, and patients have limited therapeutic options at this stage. Experimental Design: We performed a system-wide quantitative proteomic analysis of bone metastatic prostate tumors from 22 patients operated to relieve spinal cord compression. At the time of surgery, most patients had relapsed after androgen-deprivation therapy, while 5 were previously untreated. An extended cohort of prostate cancer bone metastases (n = 65) was used for immunohistochemical validation. Results: On average, 5,067 proteins were identified and quantified per tumor. Compared with primary tumors (n = 26), bone metastases were more heterogeneous and showed increased levels of proteins involved in cell-cycle progression, DNA damage response, RNA processing, and fatty acid β-oxidation; and reduced levels of proteins were related to cell adhesion and carbohydrate metabolism. Within bone metastases, we identified two phenotypic subgroups: BM1, expressing higher levels of AR canonical targets, and mitochondrial and Golgi apparatus resident proteins; and BM2, with increased expression of proliferation and DNA repair–related proteins. The two subgroups, validated by the inverse correlation between MCM3 and prostate specific antigen immunoreactivity, were related to disease prognosis, suggesting that this molecular heterogeneity should be considered when developing personalized therapies. Conclusions: This work is the first system-wide quantitative characterization of the proteome of prostate cancer bone metastases and a valuable resource for understanding the etiology of prostate cancer progression. Clin Cancer Res; 24(21); 5433–44. ©2018 AACR.

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.

Castration resistant prostate cancer (CRPC) is a stage of relapse that arises after various forms of androgen ablation therapy (ADT) and causes significant morbidity and mortality. However, the mechanism underlying progression to CRPC remains poorly understood. Here, we report that YAP1, which is negatively regulated by AR, influences prostate cancer (PCa) cell self-renewal and CRPC development. Specifically, we found that AR directly regulates the methylation of YAP1 gene promoter via the formation of a complex with Polycomb group protein EZH2 and DNMT3a. In normal conditions, AR recruits EZH2 and DNMT3a to YAP1 promoter, thereby promoting DNA methylation and the repression of YAP1 gene transcription. Following ADT treatment or when AR activity is antagonized by Bicalutamide or Enzalutamide, YAP1 gene expression is switched on. In turn, YAP1 promotes SOX2 and Nanog expression and the de-differentiation of PCa cells to stem/progenitor-like cells (PCSC), which potentially contribute to disease recurrence. Finally, the knock down of YAP1 expression or the inhibition of YAP1 function by Verteporfin in TRAMP prostate cancer mice significantly suppresses tumor recurrence following castration. In conclusion, our data reveals that AR suppresses YAP1 gene expression through a novel epigenetic mechanism, which is critical for PCa cells self-renewal and the development of CRPC.

Bone metastasis is the lethal end‐stage of prostate cancer (PC), but the biology of bone metastases is poorly understood. The overall aim of this study was therefore to explore molecular variability in PC bone metastases of potential importance for therapy. Specifically, genome‐wide expression profiles of bone metastases from untreated patients (n = 12) and patients treated with androgen‐deprivation therapy (ADT, n = 60) were analyzed in relation to patient outcome and to morphological characteristics in metastases and paired primary tumors. Principal component analysis and unsupervised classification were used to identify sample clusters based on mRNA profiles. Clusters were characterized by gene set enrichment analysis and related to histological and clinical parameters using univariate and multivariate statistics. Selected proteins were analyzed by immunohistochemistry in metastases and matched primary tumors (n = 52) and in transurethral resected prostate (TUR‐P) tissue of a separate cohort (n = 59). Three molecular subtypes of bone metastases (MetA‐C) characterized by differences in gene expression pattern, morphology, and clinical behavior were identified. MetA (71% of the cases) showed increased expression of androgen receptor‐regulated genes, including prostate‐specific antigen (PSA), and glandular structures indicating a luminal cell phenotype. MetB (17%) showed expression profiles related to cell cycle activity and DNA damage, and a pronounced cellular atypia. MetC (12%) exhibited enriched stroma–epithelial cell interactions. MetB patients had the lowest serum PSA levels and the poorest prognosis after ADT. Combined analysis of PSA and Ki67 immunoreactivity (proliferation) in bone metastases, paired primary tumors, and TUR‐P samples was able to differentiate MetA‐like (high PSA, low Ki67) from MetB‐like (low PSA, high Ki67) tumors and demonstrate their different prognosis. In conclusion, bone metastases from PC patients are separated based on gene expression profiles into molecular subtypes with different morphology, biology, and clinical outcome. These findings deserve further exploration with the purpose of improving treatment of metastatic PC.

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.

Purpose: An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood. Experimental Design: We applied mass spectrometry–based proteomics to a unique set of 17 prostate cancer patient–derived xenografts (PDX) to characterize the effects of castration in vivo, and the proteome differences between NEPC and prostate adenocarcinomas. Genome-wide profiling of REST-occupied regions in prostate cancer cells was correlated to the expression changes in vivo to investigate the role of the transcriptional repressor REST in castration-induced NEPC differentiation. Results: An average of 4,881 proteins were identified and quantified from each PDX. Proteins related to neurogenesis, cell-cycle regulation, and DNA repair were found upregulated and elevated in NEPC, while the reduced levels of proteins involved in mitochondrial functions suggested a prevalent glycolytic metabolism of NEPC tumors. Integration of the REST chromatin bound regions with expression changes indicated a direct role of REST in regulating neuronal gene expression in prostate cancer cells. Mechanistically, depletion of REST led to cell-cycle arrest in G1, which could be rescued by p53 knockdown. Finally, the expression of the REST-regulated gene secretagogin (SCGN) correlated with an increased risk of suffering disease relapse after radical prostatectomy. Conclusions: This study presents the first deep characterization of the proteome of NEPC and suggests that concomitant inhibition of REST and the p53 pathway would promote NEPC. We also identify SCGN as a novel prognostic marker in prostate cancer.

[Background]: Diabetes type 1 is characterized by the failure of beta cells to produce insulin. Suppressor of cytokine signaling (SOCS) proteins are important regulators of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. Previous studies have shown that GH can prevent the development of type I diabetes in mice and that SOCS2 deficiency mimics a state of increased GH sensitivity. [Methodology]: The elevated sensitivity of SOCS2 mice to GH and possibly to PRL was the rationale to analyze the effects of multiple low dose streptozotocin (MLDSTZ)-induced diabetes in SOCS2 mice. [Results]: We show that 6-month-old SOCS2 mice, but not 2-month-old mice, were less sensitive to MLDSTZ-induced diabetes, compared to controls. MLDSTZ treatment induced glucose intolerance in both SOCS2 and SOCS2 mice, as shown by glucose tolerance tests, with SOCS2 mice showing a more marked intolerance, compared to SOCS2 mice. Furthermore, insulin tolerance tests showed that the SOCS2 mice have an improved hypoglycemic response to exogenous insulin, compared to SOCS2 mice. Moreover, in isolated islets, lipotoxic effects on insulin release could partly be overcome by ligands, which bind to GH or PRL receptors. [Conclusion]: Knockdown of SOCS2 makes mice less sensitive to MLDSTZ. These results are consistent with the proposal that elimination of SOCS2 in pancreatic islets creates a state of β-cell hypersensitivity to GH/PRL that mimics events in pregnancy, and which is protective against MLDSTZ-induced type I diabetes in mice. SOCS2-dependent control of β-cell survival may be of relevance to islet regeneration and survival in transplantation., The Spanish Ministry of Science and Innovation, with the European Regional Development Fund, supported this research by grants-in-aid to L.F.-P. (SAF2012– 37344). L.F.-P. was also supported by grants-in-aid from ACIISI (PI2010/0110) and Alfredo Martin-Reyes Foundation (Arehucas)-FICIC. The Danish Council for Independent Research and the Novo Nordisk Foundation supports A.F-M. G.N. is supported by a grant from Swedish Heart and Lung Foundation. A.A is supported by a fellowship from Sultan Qaboos University, Muscat, Oman. M.M-G. is a recipient of predoctoral fellowship from ULPGCMEC (AP2001–3499) and Fundación Universitaria de Las Palmas (INNOVA).

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2−/− mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway.

Prostate cancer remains a leading cause of cancer-related mortality worldwide owing to our inability to treat effectively castration-resistant tumors. To understand the signaling mechanisms sustaining castration-resistant growth, we implemented a mass spectrometry-based quantitative proteomic approach and use it to compare protein phosphorylation in orthotopic xenograft tumors grown in either intact or castrated mice. This investigation identified changes in phosphorylation of signaling proteins such as MEK, LYN, PRAS40, YAP1 and PAK2, indicating the concomitant activation of several oncogenic pathways in castration-resistant tumors, a notion that was confirmed by tumor transcriptome analysis. Further analysis demonstrated that the activation of mTORC1, PAK2 and the increased levels of YAP1 in castration-resistant tumors can be explained by the loss of androgen inhibitory actions. The analysis of clinical samples demonstrated elevated levels of PAK2 and YAP1 in castration-resistant tumors, whereas knockdown experiments in androgen-independent cells demonstrated that both YAP1 and PAK2 regulate cell colony formation and cell invasion activity. PAK2 also influenced cell proliferation and mitotic timing. Interestingly, these phenotypic changes occur in the absence of obvious alterations in the activity of AKT, MAPK or mTORC1 pathways, suggesting that PAK2 and YAP1 may represent novel targets for the treatment of castration-resistant prostate cancer. Pharmacologic inhibitors of PAK2 (PF-3758309) and YAP1 (Verteporfin) were able to inhibit the growth of androgen-independent PC3 xenografts. This work demonstrates the power of applying high-resolution mass spectrometry in the proteomic profiling of tumors grown in vivo for the identification of novel and clinically relevant regulatory proteins.

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

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 β-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.

Purpose To investigate the genomic signaling that defines sensitive lymphocytes to radiation and if such molecular profiles are consistent with clinical toxicity; trying to disclose the radiobiology mechanisms behind these cellular processes. Patients and methods Twelve consecutive patients suffering from locally advanced breast cancer and treated with high-dose hyperfractionated radiotherapy were recruited. Initial DNA damage was measured by pulsed-field gel electrophoresis and radiation-induced apoptosis was measured by flow cytometry. Gene expression was assessed by DNA microarray. Results Thirty-four constitutive genes segregated patients with lower DNA-double strand break from those patients with higher DNA-double strand break (p Conclusion We introduced new data in the field of molecular genomics regarding to the relation established between radiation toxicity and these predictive factors to radiation injury.

Growth Hormone is essential for the regulation of growth and the homeostatic control of intermediary metabolism. GH actions are mediated by the Growth Hormone Receptor; a member of the cytokine receptor super family that signals chiefly through the JAK2/STAT5 pathway. Target tissue responsiveness to GH is under regulatory control to avoid excessive and off-target effects upon GHR activation. The suppressor of cytokine signalling 2 (SOCS) is a key regulator of GHR sensitivity. This is clearly shown in mice where the SOCS2 gene has been inactivated, which show 30-40% increase in body length, a phenotype that is dependent on endogenous GH secretion. SOCS2 is a GH-stimulated, STAT5b-regulated gene that acts in a negative feedback loop to downregulate GHR signalling. Since the biochemical basis for these actions is poorly understood, we studied the molecular function of SOCS2. We demonstrated that SOCS2 is part of a multimeric complex with intrinsic ubiquitin ligase activity. Mutational analysis shows that the interaction with Elongin B/C controls SOCS2 protein turnover and affects its molecular activity. Increased GHR levels were observed in livers from SOCS2 -/- mice and in the absence of SOCS2 in in vitro experiments. We showed that SOCS2 regulates cellular GHR levels through direct ubiquitination and in a proteasomally dependent manner. We also confirmed the importance of the SOCS-box for the proper function of SOCS2. Finally, we identified two phosphotyrosine residues in the GHR to be responsible for the interaction with SOCS2, but only Y487 to account for the effects of SOCS2. The demonstration that SOCS2 is an ubiquitin ligase for the GHR unveils the molecular basis for its physiological actions. © 2011 Vesterlund et al.

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.

Bone is the most predominant site of distant metastasis in prostate cancer. Patients who develop bone metastases have limited therapeutic options and poor prognosis. Understanding the cellular and molecular changes occurring at this stage is critical to find new treatments and to stratify patients for personalized therapies. In the current study, we used a system-wide quantitative proteomic approach in order to identify (i) cellular alterations occurring during the transition from localized to metastasized prostate cancer and (ii) phenotypic subgroups and potential novel targets for treatment of metastatic prostate cancer. Mass spectrometry-based quantitative proteomic profiling was applied to paraffin-embedded, formalin-fixed bone metastasis tissue from prostate cancer patients (n=22) operated to relieve spinal cord compression. At the time of surgery, 5 patients were previously untreated, 16 had relapsed after androgen-deprivation therapy (ADT), and 1 was under early ADT. Mechanisms behind disease progression were identified through comparative analysis with proteomic profiles of localized prostate cancer (n=26) and adjacent normal tissue (n=8) obtained from radical prostatectomies. Expression levels of selected proteins were verified by immunohistochemical analysis of metastases from our extended bone-metastasis cohort. A mean of 5,067 proteins were identified and quantified per tumor. Compared to primary prostate tumors (1) bone metastases showed increased levels of proteins involved in cell cycle progression, DNA damage response, RNA processing, lipid transport, and fatty acid β-oxidation as well as reduced levels of proteins involved in cell adhesion and carbohydrate metabolism. Based on their proteomes, prostate cancer metastases were more heterogeneous (ρ:0.64±0.077) than localized tumors (ρ:0.8±0.037). We identified two phenotypically different subgroups of metastatic tumors: (i) those expressing higher levels of androgen receptor canonical targets together with elevated levels of mitochondrial and Golgi apparatus resident proteins and (ii) those with increased expression of proliferation and DNA repair-related proteins. Importantly, the two subgroups were seen among both treatment-naïve and castration-resistant cases and thus unrelated to previous treatment. The inverse correlation between tumor cell proliferation and metabolically/secretory active tumor cells was verified by assessment of Ki67 and PSA immunoreactivity in 78 bone metastases (Rs=-0.36, P=0.001). Identification of novel therapeutic targets/strategies for the two subgroups is ongoing. In conclusion, this work describes the first system-wide quantitative characterization of the proteome in prostate cancer bone metastases. By deep proteomic analysis we revealed the existence of at least two different subtypes of prostate cancer bone metastasis according to proliferative and metabolic features, suggesting that personalized therapies could be considered based on molecular signatures in individual cases. Reference: 1. Iglesias-Gato D, et al. The proteome of primary prostate cancer. Eur Urol 2016:69:942-52. Citation Format: Diego Iglesias-Gato, Elin Thysell, Sead Crnalic, Matthias Mann, Anders Widmark, Anders Bergh, Amilcar Flores-Morales, Pernilla Wikström. The proteome of prostate cancer bone metastases [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B025.

Growing evidence strongly suggests that high fat diet (HFD) has an important role in some neurodegenerative disorders, including Alzheimer's disease (AD). To identify new cellular pathways linking hypercholesterolemia and neurodegeneration, we analyzed the effects of HFD on gene expression in mouse brain. Using cDNA microarrays and real time RT‐PCR, we found that HFD has a mild, but significant effect on the expression of several genes. The altered genes include molecules linked to AD pathology and others of potential interest for neurodegeneration. We further investigated the effect of HFD on the activity‐regulated cytoskeleton‐associated protein (Arc). Expression of Arc was decreased in cerebral cortex and hippocampus of HFD‐fed animals. From the known regulatory mechanisms of Arc expression, HFD reduced N‐methyl‐D‐aspartate receptor (NMDAR) activity, as seen by decreases in tyrosine phosphorylation of NMDAR2A and levels of NMDAR1. Additionally, we demonstrated that 27‐hydroxycholesterol, a cholesterol metabolite that enters the brain from the blood, decreases Arc levels as well as NMDAR and Src kinase activities in rat primary hippocampal neurons. Finally, we showed that Arc levels are decreased in the cortex of AD brains. We propose that one of the mechanisms, by which hypercholesterolemia contributes to neurodegenerative diseases, could be through Arc down‐regulation caused by 27‐hydroxycholesterol.

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.

The GH-activated signal transducer and activator of transcription 5b (STAT5b) is an essential regulator of somatic growth. The transcriptional response to STAT5b in liver is poorly understood. We have combined microarray-based expression profiling and phylogenetic analysis of gene regulatory regions to study the interplay between STAT5b and GH in the regulation of hepatic gene expression. The acute transcriptional response to GH in vivo after a single pulse of GH was studied in the liver of hypophysectomized rats in the presence of either constitutively active or a dominant-negative STAT5b delivered by adenoviral gene transfer. Genes showing differential expression in these two situations were analyzed for the presence of STAT5b binding sites in promoter and intronic regions that are phylogenetically conserved between rats and humans. Using this approach, we showed that most rapid transcriptional effects of GH in the liver are not results of direct actions of STAT5b. In addition, we identified novel STAT5b cis regulatory elements in genes such as Frizzled-4, epithelial membrane protein-1, and the suppressor of cytokine signaling 2 (SOCS2). Detailed analysis of SOCS2 promoter demonstrated its direct transcriptional regulation by STAT5b upon GH stimulation. A novel response element was identified within the first intron of the human SOCS2 gene composed of an E-box followed by tandem STAT5b binding sites, both of which are required for full GH responsiveness. In summary, we demonstrate the power of combining transcript profiling with phylogenetic sequence analysis to define novel regulatory paradigms.

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.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.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.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.

Ezrin is a key signaling molecule that regulates cell survival, adhesion migration, and invasion. We have previously shown that ezrin is regulated by androgen in rat prostate and that its expression is increased in prostate cancer and in prostate intraepithelial neoplasia. We have used the androgen-sensitive cell line LNCaP-FGC to investigate the role of ezrin in androgen-induced cell invasion. We found that androgen treatment of LNCaP-FGC cells induces ezrin expression, an effect that is inhibited by the androgen receptor antagonist, bicalutamide. In addition, androgen treatment induces the phosphorylation of ezrin in Thr-567 and Tyr-353 in a sequential manner. This is mediated through protein kinase C alpha and Src tyrosine kinase, respectively. Androgen treatment induces the translocation of both protein kinase C alpha and ezrin to the cell membrane and their association. Inhibition of ezrin function using short interference RNA or the overexpression of T567A and Y353F-ezrin mutants significantly reduces androgen-induced Matrigel invasion but does not affect cell proliferation or cell adhesion. Matrigel invasion of the androgen-insensitive prostate cancer cell lines PC-3 and LNCaP-R is also dependent on ezrin. In summary, we have shown that androgens regulate ezrin at transcriptional and posttranscriptional levels. Hormonal regulation of ezrin phosphorylation is required for androgen-induced cell invasion.

Mice deficient in SOCS2 display an excessive growth phenotype characterized by a 30–50% increase in mature body size. Here we show that the SOCS2–/– phenotype is dependent upon the presence of endogenous growth hormone (GH) and that treatment with exogenous GH induced excessive growth in mice lacking both endogenous GH and SOCS2. This was reflected in terms of overall body weight, body and bone lengths, and the weight of internal organs and tissues. A heightened response to GH was also measured by examining GH-responsive genes expressed in the liver after exogenous GH administration. To further understand the link between SOCS2 and the GH-signaling cascade, we investigated the nature of these interactions using structure/function and biochemical interaction studies. Analysis of the 3 structural motifs of the SOCS2 molecule revealed that each plays a crucial role in SOCS2 function, with the conserved SOCS-box motif being essential for all inhibitory function. SOCS2 was found to bind 2 phosphorylated tyrosines on the GH receptor, and mutational analysis of these amino acids showed that both were essential for SOCS2 function. Together, the data provide clear evidence that SOCS2 is a negative regulator of GH signaling.

There is growing evidence that the distribution of adipose tissue in the body is of importance in the development of metabolic complications of obesity, such as diabetes, hypertension, and hyperlipidemia. The aim of this study was to identify differentially expressed genes in subcutaneous and omental human adipose tissue in obese men, using a subtractive hybridization strategy. From the obtained set of differentially expressed transcripts, we also aimed to identify genes that have a sex-specific pattern of expression in omental or subcutaneous adipose tissue. Representational difference analysis (RDA) was performed on cDNA from subcutaneous and omental fat tissue from a man with extreme abdominal obesity. Forty-four putatively differentially expressed genes were identified. The obtained RDA products were spotted onto glass slides to screen for differential expression in other obese patients by using a microarray hybridization procedure. Five genes were confirmed to be differentially expressed in subcutaneous or omental adipose tissue from male or female obese patients. One gene was detected only in males and was found to be upregulated in subcutaneous tissue. The findings extend previous knowledge that different fat depots have differential gene expression and indicate that sex differences exist in adipose gene expression patterns. Linder, K., P. Arner, A. Flores-Morales, P. Tollet-Egnell, and G. Norstedt. Differentially expressed genes in visceral or subcutaneous adipose tissue of obese men and women. J. Lipid Res. 2004. 45: 148–154.

The probasin (Pb)-PRL transgenic mice that overexpress the rat PRL gene specifically in the prostate develop a dramatic enlargement of the prostate gland. The objective of this study was to characterize the molecular mechanisms involved in the prostate hyperplasia seen in the Pb-PRL transgenic mice. cDNA microarray analysis was used to identify differentially expressed transcripts in the hyperplastic prostates of 6-month-old transgenic mice compared with age-matched controls. We report the identification of 266 genes (175 upregulated and 91 down-regulated) that were differentially expressed in the enlarged transgenic prostates compared with controls. Subsequential real-time RT-PCR was used to verify a set of differentially regulated transcripts. The hyperplastic prostates of Pb-PRL transgenic mice demonstrate a molecular pattern supporting the importance of reduced degree of apoptosis for the development of the phenotype. Immunohistochemical analysis of apoptotic activity using two different markers of apoptosis (single-stranded DNA and activated caspase-3) were performed, and the results showed diminished apoptosis activity in the prostate of Pb-PRL transgenic mice compared with control prostates. The increased stromal/ epithelial ratio of the Pb-PRL transgenic prostate together with up-regulation of a significant fraction of genes involved in tissue remodeling activity, including the synthesis and degradation of the extracellular matrix and changes in protease activity, suggest that activation of the stroma is involved in the development of prostate hyperplasia. Overall, the differentially expressed transcripts identified in this study show many molecular similarities between the prostate hyperplasia of PRL-transgenic mice and human prostate pathology, including both benign prostatic hyperplasia and prostate cancer. (Endocrinology 144: 4955– 4966, 2003)