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Androgen deprivation therapy (ADT) is the standard of care for treatment of nonresectable prostate cancer. Despite high treatment efficiency, most patients ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we performed a comparative proteomic analysis of three in vivo, androgen receptor (AR)-responsive orthograft models of matched hormone-naïve prostate cancer and CRPC. Differential proteomic analysis revealed that distinct molecular mechanisms, including amino acid (AA) and fatty acid metabolism, are involved in the response to ADT in the different models. Despite this heterogeneity, Schlafen family member 5 (SLFN5) was identified as an AR-regulated protein in CRPC. SLFN5 expression was high in CRPC tumors and correlated with poor patient outcome. In vivo, SLFN5 depletion strongly impaired tumor growth in castrated conditions. Mechanistically, SLFN5 interacted with ATF4 and regulated the expression of LAT1, an essential AA transporter. Consequently, SLFN5 depletion in CRPC cells decreased intracellular levels of essential AA and impaired mTORC1 signaling in a LAT1-dependent manner. These results confirm that these orthograft models recapitulate the high degree of heterogeneity observed in patients with CRPC and further highlight SLFN5 as a clinically relevant target for CRPC. Significance: This study identifies SLFN5 as a novel regulator of the LAT1 amino acid transporter and an essential contributor to mTORC1 activity in castration-resistant prostate cancer.

Casitas B-lineage lymphoma (CBL) is a ubiquitin ligase (E3) that becomes activated upon Tyr371-phosphorylation and targets receptor protein tyrosine kinases for ubiquitin-mediated degradation. Deregulation of CBL and its E3 activity is observed in myeloproliferative neoplasms and other cancers, including breast, colon, and prostate cancer. Here, we explore the oncogenic mechanism of E3-inactive CBL mutants identified in myeloproliferative neoplasms. We show that these mutants bind strongly to CIN85 under normal growth conditions and alter the CBL interactome. Lack of E3 activity deregulates CIN85 endosomal trafficking, leading to an altered transcriptome that amplifies signaling events to promote oncogenesis. Disruption of CBL mutant interactions with EGFR or CIN85 reduces oncogenic transformation. Given the importance of the CBL–CIN85 interaction in breast cancers, we examined the expression levels of CIN85, CBL, and the status of Tyr371-phosphorylated CBL (pCBL) in human breast cancer tissue microarrays. Interestingly, pCBL shows an inverse correlation with both CIN85 and CBL, suggesting that high expression of inactivated CBL could coordinate with CIN85 for breast cancer progression. Inhibition of the CBL–CIN85 interaction with a proline-rich peptide of CBL that binds CIN85 reduced the proliferation of MDA-MB-231 cells. Together, these results provide a rationale for exploring the potential of targeting the EGFR–CBL–CIN85 axis in CBL-inactivated mutant cancers.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

Malignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no ‘druggable’ driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease., Treating malignant pleural mesothelioma (MpM) is challenging due to a lack of druggable genes, but other molecular features could be clinically useful. Here the authors profile mRNA translation dysregulation in MpM cell lines using polysome profiling, and identify mTORC1 and 2 as potential pharmacological targets.

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. In patients, THEM6 expression correlates with progressive disease and is associated with poor survival. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, THEM6 is located at the endoplasmic reticulum (ER) membrane and controls lipid homeostasis by regulating intracellular levels of ether lipids. Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated induction of ATF4. Finally, we show that THEM6 is required for the establishment of the MYC-induced stress response. Thus, similar to PCa, THEM6 loss significantly impairs tumorigenesis in the MYC-dependent subtype of triple negative breast cancer. Altogether, our results highlight THEM6 as a novel component of the treatment-induced stress response and a promising target for the treatment of CRPC and MYC-driven cancer.

Background Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzyme activity is crucial for the generation of reducing potential in normal cells, yet the impact of the mutation on the cellular antioxidant system in glioma is not understood. The aim of this study was to determine how glutathione (GSH), the main antioxidant in the brain, is maintained in IDH1-mutant gliomas, despite an altered NADPH/NADP balance. Methods Proteomics, metabolomics, metabolic tracer studies, genetic silencing, and drug targeting approaches in vitro and in vivo were applied. Analyses were done in clinical specimen of different glioma subtypes, in glioma patient-derived cell lines carrying the endogenous IDH1 mutation and corresponding orthotopic xenografts in mice. Results We find that cystathionine-γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis, is specifically upregulated in IDH1-mutant astrocytomas. CSE inhibition sensitized these cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with an increase in reactive oxygen species and reduced GSH synthesis. Propargylglycine (PAG), a brain-penetrant drug specifically targeting CSE, led to delayed tumor growth in mice. Conclusions We show that IDH1-mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis via CSE to maintain the antioxidant defense, which highlights a novel metabolic vulnerability that may be therapeutically exploited.

Prostate cancer is highly prevalent, being the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of PTEN and SPRY2 (Sprouty 2) tumour suppressors, we identified enhanced creatine metabolism within the phosphagen system in progressive disease. Altered creatine metabolism was validated in in vitro and in vivo prostate cancer models and in clinical cases. Upregulated creatine levels were due to increased uptake through the SLC6A8 creatine transporter and de novo synthesis, resulting in enhanced cellular basal respiration. Treatment with cyclocreatine (a creatine analogue that potently and specifically blocks the phosphagen system) dramatically reduces creatine and phosphocreatine levels. Blockade of creatine biosynthesis by cyclocreatine leads to cellular accumulation of S-adenosyl methionine (SAM), an intermediary of creatine biosynthesis, and suppresses prostate cancer growth in vitro. Furthermore, cyclocreatine treatment impairs cancer progression in our GEMM and in a xenograft liver metastasis model. Hence, by targeting the phosphagen system, cyclocreatine results in anti-tumourigenic effects from both SAM accumulation and suppressed phosphagen system.

Castration-resistant prostate cancer (CRPC) is incurable and remains a significant worldwide challenge. Matched untargeted multi-level omic datasets may reveal biological changes driving CRPC, identifying novel biomarkers and/or therapeutic targets. Untargeted RNA sequencing, proteomics, and metabolomics was performed on xenografts derived from three independent sets of hormone naive and matched CRPC human cell line models of local, lymph node and bone metastasis grown as murine orthografts. Untargeted metabolomics revealed NAA and NAAG commonly accumulating in CRPC across three independent models and proteomics showed upregulation of related enzymes, namely N-Acetylated Alpha-Linked Acidic Dipeptidases (FOLH1/NAALADL2). Based on pathway analysis integrating multiple omic levels we hypothesise that increased NAA in CRPC may be due to upregulation of NAAG hydrolysis via NAALADLases providing a pool of Acetyl Co-A for upregulated sphingolipid metabolism and a pool of glutamate and aspartate for nucleotide synthesis during tumour growth.

Androgen-deprivation therapy (ADT) is the standard of care for the treatment of non-resectable prostate cancer (PCa). Despite high treatment efficiency, most patients ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we perform a comparative proteomic analysis of three in vivo, androgen receptor (AR)–driven, orthograft models of CRPC. Differential proteomic analysis reveals that distinct molecular mechanisms, including amino acid (AA) and fatty acid (FA) metabolism, are involved in the response to ADT between the different models. Despite this heterogeneity, we identify SLFN5 as an AR-regulated biomarker in CRPC. SLFN5 expression is high in CRPC tumours and correlates with poor patient outcome. In vivo, SLFN5 depletion strongly impairs tumour growth in castrated condition. Mechanistically, SLFN5 interacts with ATF4 and regulates the expression of LAT1, an essential AA transporter. Consequently, SLFN5 depletion in CRPC cells decreases intracellular levels of essential AA and impairs mTORC1 signalling in a LAT1-dependent manner.

Current nutritional recommendations are focused on energy, fat, carbohydrate, protein and vitamins. Less attention has been paid to the nutritional demand of one-carbon units for nucleotide and methionine synthesis. Here, we investigated the impact of sodium formate supplementation as a nutritional intervention to increase the dietary intake of one-carbon units. A cohort of six female and six male mice received 125 mM of sodium formate in the drinking water for three months. A control group of another six female and six male mice was also followed up for the same period of time. Tail vein blood samples were collected once a month and profiled with a haematology analyser. At the end of the study, blood and tissues were collected for metabolomics analysis and immune cell profiling. Formate supplementation had no significant physiological effect on male mice, except for a small decrease in body weight. Formate supplementation had no significant effect on the immune cell counts during the intervention or at the end of the study in either gender. In female mice, however, the body weight and spleen wet weight were significantly increased by formate supplementation, while the blood plasma levels of amino acids were decreased. Formate supplementation also increased the frequency of bifidobacteria, a probiotic bacterium, in the stools of female mice. We conclude that formate supplementation induces physiological changes in a gender-specific manner.

TElevated production of collagen-rich extracellular matrix (ECM) is a hallmark of cancer associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. How to target ECM production to oppose cancer is yet unclear, since targeting CAFs has been shown to restrain but also promote cancer progression. Metabolic rewiring is a hallmark of CAFs. Here we find that proline, which is a highly abundant amino acid in collagen proteins, is newly synthesised from glutamine to make tumour collagen in breast cancer xenografts, and that its production is elevated in breast cancer CAFs. PYCR1 is the rate-limiting enzyme for proline synthesis and is highly expressed in the tumour stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. PYCR1 and COL1A1 are overexpressed in patients with invasive ductal carcinoma with poor prognosis. Both collagen and glutamine-derived proline synthesis in CAFs are enhanced by increased pyruvate dehydrogenase-derived acetyl-CoA levels, via gene expression regulation through the epigenetic regulator histone acetyl transferase EP300. Altogether, our work unveils unprecedented roles of CAF metabolism to support pro-tumorigenic collagen production. PYCR1 is a recognised cancer cell vulnerability and potential target for therapy, hence, our work provides evidence that targeting PYCR1 in tumours may have the additional benefit of halting the production of pro-tumorigenic ECM.

Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance., Androgen receptor (AR) signalling regulates cellular metabolism in prostate cancer. Here, the authors perform a proteomics and metabolomics characterisation of prostate cancer cells adapted to long-term resistance to AR inhibition and show rewiring of glucose and lipid metabolism, and further identify a signature associated with resistance to AR inhibition.

When chromatin bridges form in cytokinesis, cells display actin patches at the intercellular canal. Dandoulaki et al. show that Chk1 phosphorylates Src at serine 51 to fully induce Src catalytic activity. In turn, phosphorylated Src-S51 promotes the formation of actin patches and prevents chromatin bridges from breaking., In cytokinesis with chromatin bridges, cells delay abscission and retain actin patches at the intercellular canal to prevent chromosome breakage. In this study, we show that inhibition of Src, a protein-tyrosine kinase that regulates actin dynamics, or Chk1 kinase correlates with chromatin breakage and impaired formation of actin patches but not with abscission in the presence of chromatin bridges. Chk1 is required for optimal localization and complete activation of Src. Furthermore, Chk1 phosphorylates human Src at serine 51, and phosphorylated Src localizes to actin patches, the cell membrane, or the nucleus. Nonphosphorylatable mutation of S51 to alanine reduces Src catalytic activity and impairs formation of actin patches, whereas expression of a phosphomimicking Src-S51D protein rescues actin patches and prevents chromatin breakage in Chk1-deficient cells. We propose that Chk1 phosphorylates Src-S51 to fully induce Src kinase activity and that phosphorylated Src promotes formation of actin patches and stabilizes chromatin bridges. These results identify proteins that regulate formation of actin patches in cytokinesis., Graphical Abstract

Statins are widely prescribed inhibitors of the mevalonate pathway, acting to lower systemic cholesterol levels. The mevalonate pathway is critical for tumorigenesis and is frequently upregulated in cancer. Nonetheless, reported effects of statins on tumor progression are ambiguous, making it unclear whether statins, alone or in combination, can be used for chemotherapy. Here, using advanced mass spectrometry and isotope tracing, we showed that statins only modestly affected cancer cholesterol homeostasis. Instead, they significantly reduced synthesis and levels of another downstream product, the mitochondrial electron carrier coenzyme Q, both in cultured cancer cells and tumors. This compromised oxidative phosphorylation, causing severe oxidative stress. To compensate, cancer cells upregulated antioxidant metabolic pathways, including reductive carboxylation, proline synthesis, and cystine import. Targeting cystine import with an xCT transporter–lowering MEK inhibitor, in combination with statins, caused profound tumor cell death. Thus, statin-induced ROS production in cancer cells can be exploited in a combinatorial regimen. Significance: Cancer cells induce specific metabolic pathways to alleviate the increased oxidative stress caused by statin treatment, and targeting one of these pathways synergizes with statins to produce a robust antitumor response. See related commentary by Cordes and Metallo, p. 151

Plasmax, a physiological cell culture medium, prevents metabolic artifacts imposed on cancer cells by commonly used media., Currently available cell culture media may not reproduce the in vivo metabolic environment of tumors. To demonstrate this, we compared the effects of a new physiological medium, Plasmax, with commercial media. We prove that the disproportionate nutrient composition of commercial media imposes metabolic artifacts on cancer cells. Their supraphysiological concentrations of pyruvate stabilize hypoxia-inducible factor 1α in normoxia, thereby inducing a pseudohypoxic transcriptional program. In addition, their arginine concentrations reverse the urea cycle reaction catalyzed by argininosuccinate lyase, an effect not observed in vivo, and prevented by Plasmax in vitro. The capacity of cancer cells to form colonies in commercial media was impaired by lipid peroxidation and ferroptosis and was rescued by selenium present in Plasmax. Last, an untargeted metabolic comparison revealed that breast cancer spheroids grown in Plasmax approximate the metabolic profile of mammary tumors better. In conclusion, a physiological medium improves the metabolic fidelity and biological relevance of in vitro cancer models.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries. Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321–36. ©2018 AACR.

Exploiting oxidative stress has recently emerged as a plausible strategy for treatment of human cancer, and antioxidant defenses are implicated in resistance to chemotherapy and radiotherapy. Targeted suppression of antioxidant defenses could thus broadly improve therapeutic outcomes. Here, we identify the AMPK-related kinase NUAK1 as a key component of the antioxidant stress response pathway and reveal a specific requirement for this role of NUAK1 in colorectal cancer. We show that NUAK1 is activated by oxidative stress and that this activation is required to facilitate nuclear import of the antioxidant master regulator NRF2: Activation of NUAK1 coordinates PP1β inhibition with AKT activation in order to suppress GSK3β-dependent inhibition of NRF2 nuclear import. Deletion of NUAK1 suppresses formation of colorectal tumors, whereas acute depletion of NUAK1 induces regression of preexisting autochthonous tumors. Importantly, elevated expression of NUAK1 in human colorectal cancer is associated with more aggressive disease and reduced overall survival. Significance: This work identifies NUAK1 as a key facilitator of the adaptive antioxidant response that is associated with aggressive disease and worse outcome in human colorectal cancer. Our data suggest that transient NUAK1 inhibition may provide a safe and effective means for treatment of human colorectal cancer via disruption of intrinsic antioxidant defenses. Cancer Discov; 8(5); 632–47. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 517

The role of glutaminolysis in providing metabolites to support tumour growth is well-established, but the involvement of glutamine metabolism in invasive processes is yet to be elucidated. Here we show that normal mammary epithelial cells consume glutamine, but do not secrete glutamate. Indeed, low levels of extracellular glutamate are necessary to maintain epithelial homoeostasis, and provision of glutamate drives disruption of epithelial morphology and promotes key characteristics of the invasive phenotype such as lumen-filling and basement membrane disruption. By contrast, primary cultures of invasive breast cancer cells convert glutamine to glutamate which is released from the cell through the system Xc- antiporter to activate a metabotropic glutamate receptor. This contributes to the intrinsic aggressiveness of these cells by upregulating Rab27-dependent recycling of the transmembrane matrix metalloprotease, MT1-MMP to promote invasive behaviour leading to basement membrane disruption. These data indicate that acquisition of the ability to release glutamate is a key watershed in disease aggressiveness., Glutamine metabolism is well known to support tumour growth. Here the authors show that cancer cells also utilize glutamine to promote invasiveness by converting it to glutamate, which upon secretion activates metabotropic glutamate receptors to stimulate matrix metalloproteases recycling to the cell surface.

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.

Summary Mesenchymal cell motility is driven by polarized actin polymerization [1]. Signals at the leading edge recruit actin polymerization machinery to promote membrane protrusion, while matrix adhesion generates tractive force to propel forward movement. To work effectively, cell motility is regulated by a complex network of signaling events that affect protein activity and localization. H2O2 has an important role as a diffusible second messenger [2], and mediates its effects through oxidation of cysteine thiols. One cell activity influenced by H2O2 is motility [3]. However, a lack of sensitive and H2O2-specific probes for measurements in live cells has not allowed for direct observation of H2O2 accumulation in migrating cells or protrusions. In addition, the identities of proteins oxidized by H2O2 that contribute to actin dynamics and cell motility have not been characterized. We now show, as determined by fluorescence lifetime imaging microscopy, that motile cells generate H2O2 at membranes and cell protrusions and that H2O2 inhibits cofilin activity through oxidation of cysteines 139 (C139) and 147 (C147). Molecular modeling suggests that C139 oxidation would sterically hinder actin association, while the increased negative charge of oxidized C147 would lead to electrostatic repulsion of the opposite negatively charged surface. Expression of oxidation-resistant cofilin impairs cell spreading, adhesion, and directional migration. These findings indicate that H2O2 production contributes to polarized cell motility through localized cofilin inhibition and that there are additional proteins oxidized during cell migration that might have similar roles., Highlights • Cell migration leads to localized hydrogen peroxide generation • Motility increases protein oxidation on cysteine residues • Cofilin oxidation on Cys139 and Cys147 reduces actin binding and severing • Oxidation of cofilin, and possibly additional proteins, influences cell motility, Cell migration is regulated by signaling events that regulate protein activity. Here, Cameron et al. imaged highest H2O2 levels in motile cell protrusions, accompanied by cysteine oxidation of proteins, including actin-regulating cofilin. Cofilin oxidation reduced actin binding and severing, linking H2O2-generation with cytoskeleton dynamics.

SOCS3 is an inducible inhibitor of multiple pathways responsible for the neo-intimal hyperplasia (NIH) that results in failure of stenting and coronary artery bypass graft (CABG) reconstructive procedures. However the potential for SOCS3 to limit NIH is compromised by its rapid turnover by the proteasome following ubiquitylation. Our original hypothesis is that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemical analysis of human saphenous vein (HSV) sections derived from CABG patients revealed that while SOCS3 was expressed throughout the media, its expression within the neo-intima was reduced. We have shown that the L189A SOCS box mutant was ubiquitylated despite an inability to interact with the elongin B and C components of the E3 ligase machinery, suggesting that SOCS3 is not auto-ubiquitylated. Moreover, studies using a panel of SOCS3 truncation mutants revealed that SOCS3 is predominantly ubiquitylated within a C-terminal, 44 amino acid region in which Lys173 was the only potential target residue for ubiquitylation. Consistent with a role for this residue, ubiquitylation of a K173R-mutated SOCS3 mutant was markedly reduced versus WT SOCS3. K173 may therefore represent the critical site of ubiquitin attachment important for proteasomal turnover of SOCS3. Finally, we have pursued several approaches to identify the E3 ubiquitin ligase(s) and deubiquitinases that control SOCS3 sensitivity to proteasomal degradation. One of these has involved identification by mass spectrometry of proteins co-immunoprecipitating with SOCS3 and we will present the initial findings of this analysis.