Control of TGFß signalling by ubiquitination independent function of E3 ubiquitin ligase TRIP12.

Transforming growth factor ß (TGFß) pathway is a master regulator of cell proliferation, differentiation, and death. Deregulation of TGFß signalling is well established in several human diseases including autoimmune disorders and cancer. Thus, understanding molecular pathways governing TGFß signalling may help better understand the underlying causes of some of those conditions. Here, we show that a HECT domain E3 ubiquitin ligase TRIP12 controls TGFß signalling in multiple models. Interestingly, TRIP12 control of TGFß signalling is completely independent of its E3 ubiquitin ligase activity. Instead, TRIP12 recruits SMURF2 to SMAD4, which is most likely responsible for inhibitory monoubiquitination of SMAD4, since SMAD4 monoubiquitination and its interaction with SMURF2 were dramatically downregulated in TRIP12-/- cells. Additionally, genetic inhibition of TRIP12 in human and murine cells leads to robust activation of TGFß signalling which was rescued by re-introducing wildtype TRIP12 or a catalytically inactive C1959A mutant. Importantly, TRIP12 control of TGFß signalling is evolutionary conserved. Indeed, genetic inhibition of Drosophila TRIP12 orthologue, ctrip, in gut leads to a reduced number of intestinal stem cells which was compensated by the increase in differentiated enteroendocrine cells. These effects were completely normalised in Drosophila strain where ctrip was co-inhibited together with Drosophila SMAD4 orthologue, Medea. Similarly, in murine 3D intestinal organoids, CRISPR/Cas9 mediated genetic targeting of Trip12 enhances TGFß mediated proliferation arrest and cell death. Finally, CRISPR/Cas9 mediated genetic targeting of TRIP12 in MDA-MB-231 breast cancer cells enhances the TGFß induced migratory capacity of these cells which was rescued to the wildtype level by re-introducing wildtype TRIP12. Our work establishes TRIP12 as an evolutionary conserved modulator of TGFß signalling in health and disease.

FBXW7 tumor suppressor regulation by dualspecificity tyrosine-regulated kinase 2.

FBXW7 is a member of the F-box protein family, which functions as the substrate recognition component of the SCF E3 ubiquitin ligase. FBXW7 is a main tumor suppressor due to its ability to control proteasome-mediated degradation of several oncoproteins such as c-Jun, c-Myc, Cyclin E1, mTOR, and Notch1-IC. FBXW7 inactivation in human cancers results from a somatic mutation or downregulation of its protein levels. This work describes a novel regulatory mechanism for FBXW7 dependent on the serine/threonine protein kinase DYRK2. We show that DYRK2 interacts with and phosphorylates FBXW7 resulting in its proteasome-mediated degradation. DYRK2-dependent FBXW7 destabilization is independent of its ubiquitin ligase activity. The functional analysis demonstrates the existence of DYRK2-dependent regulatory mechanisms for key FBXW7 substrates. Finally, we provide evidence indicating that DYRK2-dependent regulation of FBXW7 protein accumulation contributes to cytotoxic effects in response to chemotherapy agents such as Doxorubicin or Paclitaxel in colorectal cancer cell lines and to BET inhibitors in T-cell acute lymphoblastic leukemia cell lines. Altogether, this work reveals a new regulatory axis, DYRK2/FBXW7, which provides an understanding of the role of these two proteins in tumor progression and DNA damage responses.

E3 Ubiquitin Ligase TRIP12 Controls Exit from Mitosis via Positive Regulation of MCL-1 in Response to Taxol.

Chemotherapy resistance is a major hurdle in cancer treatment. Taxol-based chemotherapy is widely used in the treatment of cancers including breast, ovarian, and pancreatic cancer. Loss of function of the tumor suppressor F-box WD-40 domain containing 7 (FBW7) mutations leads to the accumulation of its substrate MCL-1 which is associated with Taxol resistance in human cancers. We recently showed that E3 ubiquitin ligase TRIP12 is a negative regulator of FBW7 protein. In this study, we find that Taxol-induced mitotic block in cancer cells is partly controlled by TRIP12 via its positive regulation of MCL-1 protein. Genetic inhibition of TRIP12 accelerates MCL-1 protein degradation in mitosis. Notably, introducing double-point mutations in lysines 404/412 of FBW7 to arginine which makes it resistant to proteasomal degradation, leads to the sharp reduction of MCL-1 protein levels and sensitizes cancer cells to Taxol-induced cell death. Finally, TRIP12 deletion leads to enhanced mitotic arrest and cell death in an FBW7 and MCL-1 dependent manner in multiple cell lines including colorectal and ovarian cancer but not in breast cancer. Thus, the TRIP12/FBW7/MCL-1 axis may provide a therapeutic target to overcome Taxol-associated chemotherapy resistance in cancer.

A small Rho GTPase RAB25 with a potential role in chemotherapy resistance in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is one of the major human health challenges with minimal therapeutic benefits due to its late detection, and de novo - and acquired chemotherapy resistance. OBJECTIVE: In this work we unravel the potential pro-survival role of RAB25 in pancreatic cancer chemotherapy resistance and aim to identify if RAB25 is a prognostic marker of patients' survival in PDA. METHODS: We used RNA sequencing, shRNA mediated gene knockdown, BioGRID open repository of CRISPR screens (ORCS), GEPIA, kmplot.com, and cBioPortal.org databases to identify the role of RAB25 in PDA cell proliferation, chemotherapy response, expression in tumour versus normal tissues, and overall patients' survival. RESULTS: RNA sequencing show Rab25 to be one of the top upregulated genes in gemcitabine resistance mouse PDA cells. Knockdown of Rab25 in these cells enhanced gemcitabine toxicity. In addition, re-analysis of previously published CRISPR/Cas9 data confirm RAB25 to be responsible for chemotherapy resistance in KRASG12D mutant human pancreatic cancer cell line. Finally, we used publicly available TCGA datasets and identify the upregulation of RAB25 in tumour tissues compared to the adjacent normal tissue, co-occurrence of KRASG12 mutations with RAB25 amplifications, and poor patients' survival in cohorts with higher mRNA expression of RAB25. CONCLUSION: RAB25 expression is a prognostic marker for patient's survival and gemcitabine resistance in PDA.

Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12.

The tumour suppressor FBW7 is a substrate adaptor for the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), that targets several oncoproteins for proteasomal degradation. FBW7 is widely mutated and FBW7 protein levels are commonly downregulated in cancer. Here, using an shRNA library screen, we identify the HECT-domain E3 ubiquitin ligase TRIP12 as a negative regulator of FBW7 stability. We find that SCFFBW7-mediated ubiquitylation of FBW7 occurs preferentially on K404 and K412, but is not sufficient for its proteasomal degradation, and in addition requires TRIP12-mediated branched K11-linked ubiquitylation. TRIP12 inactivation causes FBW7 protein accumulation and increased proteasomal degradation of the SCFFBW7 substrate Myeloid Leukemia 1 (MCL1), and sensitizes cancer cells to anti-tubulin chemotherapy. Concomitant FBW7 inactivation rescues the effects of TRIP12 deficiency, confirming FBW7 as an essential mediator of TRIP12 function. This work reveals an unexpected complexity of FBW7 ubiquitylation, and highlights branched ubiquitylation as an important signalling mechanism regulating protein stability.

Protein prenylation restrains innate immunity by inhibiting Rac1 effector interactions.

Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1, but not Rhoa and Cdc42, reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.

The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer.

The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations.

Repeated Quantitative Urine Toxicology Analysis May Improve Chronic Pain Patient Compliance with Opioid Therapy.

BACKGROUND: Even though serious efforts have been undertaken by different medical societies to reduce opioid use for treating chronic benign pain, many Americans continue to seek pain relief through opioid consumption. Assuring compliance of these patients may be a difficult aspect of proper management even with regular behavioral monitoring. OBJECTIVE: The purpose of this study was to accurately assess the compliance of chronic opioid-consuming patients in an outpatient setting and evaluate if utilizing repeated urine drug testing (UDT) could improve compliance. STUDY DESIGN: Retrospective analysis of prospectively collected data. SETTING: Outpatient pain management clinic. METHODS: After Institutional Review Board (IRB) approval, a retrospective analysis of data for 500 patients was conducted. We included patients who were aged 18 years and older who were treated with opioid analgesic medication for chronic pain. Patients were asked to provide supervised urine toxicology specimens during their regular clinic visits, and were asked to do so without prior notification. The specimens were sent to an external laboratory for quantitative testing using liquid chromatography-tandem mass spectrometry. RESULTS: Three hundred and eighty-six (77.2%) patients were compliant with prescribed medications and did not use any illicit drugs or undeclared medications. Forty-one (8.2%) patients tested positive for opioid medication(s) that were not prescribed in our clinic; 8 (1.6%) of the patients were positive for medication that was not prescribed by any physician and was not present in the Illinois Prescription Monitoring Program; 5 (1%) patients tested negative for prescribed opioids; and 60 (12%) patients were positive for illicit drugs (8.6% marijuana, 3.2% cocaine, 0.2% heroin). Repeated UDTs following education and disclosure, showed 49 of the 77 patients (63.6%) had improved compliance. LIMITATIONS: This was a single-site study and we normalized concentrations of opioids in urine with creatinine levels while specific gravity normalization was not used. CONCLUSIONS: Our results showed that repeated UDT can improve compliance of patients on opioid medications and can improve overall pain management. We believe UDT testing should be used as an important adjunctive tool to help guide clinical decision-making regarding opioid therapy, potentially increasing future quality of care.Key words: Urine toxicology analysis, chronic pain, opioids, compliance, pain management, urine drug testing, urine drug screening.

Loss of one copy of Zfp148 reduces lesional macrophage proliferation and atherosclerosis in mice by activating p53.

RATIONALE: Cell proliferation and cell cycle control mechanisms are thought to play central roles in the pathogenesis of atherosclerosis. The transcription factor Zinc finger protein 148 (Zfp148) was shown recently to maintain cell proliferation under oxidative conditions by suppressing p53, a checkpoint protein that arrests proliferation in response to various stressors. It is established that inactivation of p53 accelerates atherosclerosis, but whether increased p53 activation confers protection against the disease remains to be determined. OBJECTIVE: We aimed to test the hypothesis that Zfp148 deficiency reduces atherosclerosis by unleashing p53 activity. METHODS AND RESULTS: Mice harboring a gene-trap mutation in the Zfp148 locus (Zfp148(gt/+)) were bred onto the apolipoprotein E (Apoe)(-/-) genetic background and fed a high-fat or chow diet. Loss of 1 copy of Zfp148 markedly reduced atherosclerosis without affecting lipid metabolism. Bone marrow transplantation experiments revealed that the effector cell is of hematopoietic origin. Peritoneal macrophages and atherosclerotic lesions from Zfp148(gt/+)Apoe(-/-) mice showed increased levels of phosphorylated p53 compared with controls, and atherosclerotic lesions contained fewer proliferating macrophages. Zfp148(gt/+)Apoe(-/-) mice were further crossed with p53-null mice (Trp53(-/-) [the gene encoding p53]). There was no difference in atherosclerosis between Zfp148(gt/+)Apoe(-/-) mice and controls on a Trp53(+/-) genetic background, and there was no difference in levels of phosphorylated p53 or cell proliferation. CONCLUSIONS: Zfp148 deficiency increases p53 activity and protects against atherosclerosis by causing proliferation arrest of lesional macrophages, suggesting that drugs targeting macrophage proliferation may be useful in the treatment of atherosclerosis.

Targeting GGTase-I activates RHOA, increases macrophage reverse cholesterol transport, and reduces atherosclerosis in mice.

BACKGROUND: Statins have antiinflammatory and antiatherogenic effects that have been attributed to inhibition of RHO protein geranylgeranylation in inflammatory cells. The activity of protein geranylgeranyltransferase type I (GGTase-I) is widely believed to promote membrane association and activation of RHO family proteins. However, we recently showed that knockout of GGTase-I in macrophages activates RHO proteins and proinflammatory signaling pathways, leading to increased cytokine production and rheumatoid arthritis. In this study, we asked whether the increased inflammatory signaling of GGTase-I-deficient macrophages would influence the development of atherosclerosis in low-density lipoprotein receptor-deficient mice. METHODS AND RESULTS: Aortic lesions in mice lacking GGTase-I in macrophages (Pggt1b▵/▵) contained significantly more T lymphocytes than the lesions in controls. Surprisingly, however, mean atherosclerotic lesion area in Pggt1b▵/▵ mice was reduced by ≈60%. GGTase-I deficiency reduced the accumulation of cholesterol esters and phospholipids in macrophages incubated with minimally modified and acetylated low-density lipoprotein. Analyses of GGTase-I-deficient macrophages revealed upregulation of the cyclooxygenase 2-peroxisome proliferator-activated-γ pathway and increased scavenger receptor class B type I- and CD36-mediated basal and high-density lipoprotein-stimulated cholesterol efflux. Lentivirus-mediated knockdown of RHOA, but not RAC1 or CDC42, normalized cholesterol efflux. The increased cholesterol efflux in cultured cells was accompanied by high levels of macrophage reverse cholesterol transport and slightly reduced plasma lipid levels in vivo. CONCLUSIONS: Targeting GGTase-I activates RHOA and leads to increased macrophage reverse cholesterol transport and reduced atherosclerosis development despite a significant increase in inflammation.

Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice.

RHO family proteins are important for the function of inflammatory cells. They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I). Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins. Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders. However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis. The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments. The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane. Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines. The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.

Emergency medical services support for acute ischemic stroke patients receiving thrombolysis at a primary stroke center.

BACKGROUND: Emergency Medical Services (EMS) is a vital link in the overall chain of stroke survival. A Primary Stroke Center (PSC) relies heavily on the 9-1-1 response system along with the ability of EMS personnel to accurately diagnose acute stroke. Other critical elements include identifying time of symptom onset, providing pre-hospital care, selecting a destination PSC, and communicating estimated time of arrival (ETA). PURPOSE: Our purpose was to evaluate the EMS component of thrombolysed acute ischemic stroke patient care at our PSC. METHODS: In a retrospective manner we retrieved electronic copies of the EMS incident reports for every thrombolysed ischemic stroke patient treated at our PSC from September 2001 to August 2005. The following data elements were extracted: location of victim, EMS agency, times of dispatch, scene, departure, emergency department (ED) arrival, recordings of time of stroke onset, blood pressure (BP), heart rate (HR), cardiac rhythm, blood glucose (BG), Glasgow Coma Scale (GCS), Cincinnati Stroke Scale (CSS) elements, emergency medical personnel field assessment, and transport decision making. RESULTS: Eighty acute ischemic stroke patients received thrombolysis during the study interval. Eighty-one percent arrived by EMS. Two EMS agencies transported to our PSC. Mean dispatch-to-scene time was 6 min, on-scene time was 16 min, transport time was 10 min. Stroke onset time was recorded in 68%, BP, HR, and cardiac rhythm each in 100%, BG in 81%, GCS in 100%, CSS in 100%, and acute stroke diagnosis was made in 88%. Various diagnostic terms were employed: cerebrovascular accident in 40%, unilateral weakness or numbness in 20%, loss of consciousness in 16%, stroke in 8%, other stroke terms in 4%. In 87% of incident reports there was documentation of decision-making to transport to the nearest PSC in conjunction with pre-notification. CONCLUSION: The EMS component of thrombolysed acute ischemic stroke patients care at our PSC appeared to be very good overall. Diagnostic accuracy was excellent, field assessment, decision-making, and transport times were very good. There was still room for improvement in documentation of stroke onset and in employment of a common term for acute stroke.