7 research outputs found
Author Correction: c-Met activation leads to the establishment of a TGFβ-receptor regulatory network in bladder cancer progression (Nature Communications, (2019), 10, 1, (4349), 10.1038/s41467-019-12241-2)
No full text© 2019, The Author(s). The original version of this Article contained an error in the spelling of the author Azad Saei, which was incorrectly given as Azad Saie. This has now been corrected in both the PDF and HTML versions of the Article
OTUD4 enhances TGFβ signalling through regulation of the TGFβ receptor complex
No full text© 2020, The Author(s). Systematic control of the transforming growth factor-β (TGFβ) pathway is essential to keep the amplitude and the intensity of downstream signalling at appropriate levels. Ubiquitination plays a crucial role in the general regulation of this pathway. Here we identify the deubiquitinating enzyme OTUD4 as a transcriptional target of the TGFβ pathway that functions through a positive feedback loop to enhance overall TGFβ activity. Interestingly we demonstrate that OTUD4 functions through both catalytically dependent and independent mechanisms to regulate TGFβ activity. Specifically, we find that OTUD4 enhances TGFβ signalling by promoting the membrane presence of TGFβ receptor I. Furthermore, we demonstrate that OTUD4 inactivates the TGFβ negative regulator SMURF2 suggesting that OTUD4 regulates multiple nodes of the TGFβ pathway to enhance TGFβ activity
The association of comorbidity with Parkinson's disease-related hospitalizations
No full textIntroduction: Unplanned hospital admissions associated with Parkinson's disease could be partly attributable to comorbidities. Methods: We studied nationwide claims databases and registries. Persons with newly diagnosed Parkinson's disease were identified based on the first Parkinson's disease-related reimbursement claim by a medical specialist. Comorbidities were classified based on the Charlson Comorbidity Index. We studied hospitalization admissions because of falls, psychiatric diseases, pneumonia and urinary tract infections, PD-related hospitalizations-not otherwise specified. The association between comorbidities and time-to-hospitalization was estimated using Cox proportional hazard modelling. To better understand pathways leading to hospitalizations, we performed multiple analyses on causes for hospitalizations. Results: We identified 18 586 people with newly diagnosed Parkinson's disease. The hazard of hospitalization was increased in persons with peptic ulcer disease (HR 2.20, p = 0.009), chronic obstructive pulmonary disease (HR 1.61, p < 0.001), stroke (HR 1.37, p = 0.002) and peripheral vascular disease (HR 1.31, p = 0.02). In the secondary analyses, the hazard of PD-related hospitalizations-not otherwise specified (HR 3.24, p = 0.02) and pneumonia-related hospitalization (HR 2.90, p = 0.03) was increased for those with comorbid peptic ulcer disease. The hazard of fall-related hospitalization (HR 1.57, p = 0.003) and pneumonia-related hospitalization (HR 2.91, p < 0.001) was increased in persons with chronic obstructive pulmonary disease. The hazard of pneumonia-related hospitalization was increased in those with stroke (HR 1.54, p = 0.03) or peripheral vascular disease (HR 1.60, p = 0.02). The population attributable risk of comorbidity was 8.4%. Conclusion: Several comorbidities increase the risk of Parkinson's disease related-hospitalization indicating a need for intervention strategies targeting these comorbid disorders.Pattern Recognition and Bioinformatic
Environmental Risk Factors for Parkinson's Disease: A Critical Review and Policy Implications.
No full textContains fulltext :
316836.pdf (Publisher’s version ) (Open Access)The age-standardized prevalence of Parkinson's disease (PD) has increased substantially over the years and is expected to increase further. This emphasizes the need to identify modifiable risk factors of PD, which could form a logical entry point for the prevention of PD. The World Health Organization (WHO) has recommended reducing exposure to specific environmental factors that have been reported to be associated with PD, in particular pesticides, trichloroethylene (TCE), and air pollution. In this review we critically evaluate the epidemiological and biological evidence on the associations of these factors with PD and review evidence on whether these putative associations are causal. We conclude that when considered in isolation, it is difficult to determine whether these associations are causal, in large part because of the decades-long lag between relevant exposures and the incidence of manifest PD. However, when considered in tandem with evidence from complementary research lines (such as animal models), it is increasingly likely that these associations reflect harmful causal effects. Fundamentally, whilst we highlight some evidence gaps that require further attention, we believe the current evidence base is sufficiently strong enough to support our call for stronger policy action. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.01 februari 202
Integrin αvβ3/c-src "Oncogenic Unit" Promotes Anchorage- independence and Tumor Progression HHS Public Access Author manuscript Supplementary Material Refer to Web version on PubMed Central for supplementary material
No full textAbstract Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin αvβ3 is associated with progression of a variety of human tumors. Here, we reveal a novel adhesion-independent role for integrin αvβ3 in pancreatic cancer and other carcinomas. Specifically, αvβ3 expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. This required recruitment of c-src to the β3 integrin cytoplasmic tail, leading to c-src activation, crk-associated substrate (CAS) phosphorylation and tumor cell survival that, surprisingly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Reduced expression of endogenous αvβ3 or c-src not only suppressed anchorage-independent growth, but also decreased metastasis in vivo, yet did not affect migration/invasion. These data define an unexpected role for an integrin as a mediator of anchorage-independence suggesting that an αvβ3/c-src signaling module may account for the aggressive behavior of αvβ3-expressing tumors in man. While anchorage-independent growth is a hallmark of transformed cells, tumor growth and metastasis depend on tumor cell interactions with the extracellular matrix, mediated by the integrin family of adhesion receptors. Integrins promote a wide range of adhesion-dependent effects in tumor cells including proliferation, survival, migration/invasion and chemotherapeutic resistance1 attributed to activation of FAK2,3-5 which recruits other signaling molecules including c-src6, a kinase whose activity is associated with enhanced malignancy7. Following adhesion, c-src phosphorylates CAS, a large adaptor protein implicated in cell invasion and survival8-10. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms * Correspondence should be addressed to: [email protected]. AUTHOR CONTRIBUTIONS J.S.D designed the project, performed most of the experiments, analyzed the data and wrote the manuscript. L.A.B. helped design and conduct the orthotopic tumor experiments. D.J.S. designed and conducted the dasatinib treatment study. M.H. initiated and performed the CAS experiments while S.K.L planned, conducted and analyzed many of the experiments with breast cancer cell lines. N.P. planned and analyzed the experiments involving the src/β3 interaction. D.T. analyzed and interpreted the immunohistochemistry and histology experiments. S.J.S. helped conceive the study and analyzed the data. D.A.C. initiated the study, analyzed the data, supervised the overall project and wrote the manuscript. HHS Public Access Author Manuscript Author Manuscript Author Manuscript Author Manuscript Integrin αvβ3 is expressed in some of the most aggressive tumor cells in a variety of cancers, including: melanoma and carcinomas of the prostate, breast, cervix and pancreas. In melanoma, αvβ3 expression initiates the transition from the benign radial growth phase to the malignant vertical growth phase11,12. In both breast and prostate carcinomas αvβ3 mediates bone metastasis through enhanced tumor cell adhesion13-16. Expression of αvβ3 correlates with disease progression and shorter survival in patients with cervical carcinoma17. In pancreatic ductal adenocarcinoma αvβ3 expression occurs in approximately 58% of human tumors and is associated with increased lymph node metastasis18. Integrins provide context-dependent cues to both normal and transformed cells that paradoxically promote both cell survival and initiate apoptosis. While expression of some integrins enhances malignancy, others inhibit malignant progression19,20. We recently demonstrated that in some tumors the expression of an unligated integrin induces apoptosis through recruitment and activation of caspase-8, a process termed integrin-mediated death (IMD)21. Tumors lacking caspase-8 were resistant to IMD and exhibited increased metastatic potential22. Here, we describe a novel role for an integrin as a mediator of anchorage-independence and suggest this may account for the enhanced malignancy associated with αvβ3 expression in pancreatic carcinoma and a wide array of other tumors. Results Expression of αvβ3 correlates with metastatic potential We compared αvβ3 expression in multiple matched pairs of primary tumor and lymph node metastases from pancreatic and breast cancer patients. Interestingly, in pancreatic cancer specimens, cells in the primary tumor showed heterogeneous staining for αvβ3, however most of the tumor cells in the lymph nodes were αvβ3 positive ( To directly address the role of αvβ3 in tumor malignancy we injected αvβ3 positive or negative GFP-labeled human pancreatic carcinoma cells into the pancreas of nude mice and evaluated primary tumor growth and spontaneous metastasis. Compared to FG cells, which lack αvβ3, FG-β3 cells ectopically expressing αvβ3 ( αvβ3 enhances adhesion-independent activation of c-src Typically, integrins initiate signaling via cell adhesion to the extracellular matrix where they interact with immobilized matrix proteins and cluster in the plane of the membrane. This facilitates the assembly of a focal contact containing the integrin together with tyrosine kinases such as FAK or c-src and adaptor proteins such as CAS23 that mediate downstream signaling leading to a wide array of cellular activities. FG cell adhesion to fibronectin depends on α5β1 whereas FG-β3 adhesion is mediated by either α5β1 or αvβ3 ( To determine which SFK isoform(s) was associated with αvβ3 we examined triton-insoluble lysates for c-src, yes and fyn. This analysis identified c-src as the only isoform associated with αvβ3 ( We further investigated the αvβ3-mediated activation of c-src by analyzing the kinetics of SFK activation in response to cell adhesion. Consistent with our previous findings ( Author Manuscript Author Manuscript Author Manuscript Author Manuscript of c-src may promote anchorage-independent signaling distinct from the response induced by this integrin in adherent cells as measured by FAK activation. αvβ3 promotes anchorage-independence through c-src Growth in anchorage-independent conditions is a hallmark of tumor cell transformation and is suggested to play a role in metastasis25,26. Based on our findings that αvβ3 activates csrc in non-adherent FG-β3 cells, we considered whether this might provide an anchorageindependent growth advantage in soft agar. Strikingly, we found that FG-β3 cells formed approximately twice as many colonies as FG cells To investigate whether αvβ3-mediated anchorage-independent survival was c-srcdependent, cells were placed in suspension culture in the presence or absence of dasatinib, a clinically approved SFK inhibitor. Treatment of FG-β3 cells with dasatinib, reduced colony formation of FG-β3 cells to the level observed for FG cells We next considered whether CAS was required for αvβ3-mediated colony formation in soft agar. Knock-down of CAS with siRNA oligonucleotides specifically reduced colony number in FG-β3 cells compared to FG cells c-src mediates αvβ3 tumor cell survival and metastasis To determine whether αvβ3-mediated c-src activation could lead to increased tumor malignancy, FG and FG-β3 cells expressing control or c-src knockdown shRNA's were injected into the pancreas of nude mice and analyzed after eight weeks. Although c-src knock-down reduced primary tumor mass in both FG and FG-β3 cells To validate the therapeutic relevance of our findings, we compared the SFK/abl inhibitor dasatinib with the abl inhibitor imatinib for their ability to reduce tumor burden and metastasis of αvβ3-expressing tumors. Orthotopically injected FG-β3 tumor cells were established for two weeks prior to dosing with vehicle (b.i.d.), 30 mg kg −1 dasatinib (b.i.d.) or 50 mg kg −1 imatinib (q.d.) by oral gavage for 4 weeks. Dasatinib treatment inhibited primary tumor mass relative to the vehicle control while imatinib had no effect Author Manuscript Author Manuscript Author Manuscript Author Manuscript Importantly, dasatinib appeared to inhibit the enhanced tumor growth associated with αvβ3 expression. While the incidence of metastasis to the hepatic hilar lymph node was relatively unchanged (dasatinib, 7/12; vehicle, 9/12; imatinib, 10/12) the size and extent of the metastatic lesions was significantly reduced Previous studies have linked αvβ3 expression or c-src activation with increased tumor cell migration and invasion27,31. While αvβ3-bearing cells were more migratory on both vitronectin and fibronectin ( Discussion Anchorage-independence is a hallmark of transformed cells and is suggested to play a role in the growth of solid tumors and survival of circulating tumor cells25,26. However, tumor cell adhesion and migration on extracellular matrix proteins, mediated by members of the integrin family, is linked to tumor cell growth and malignancy. Once ligated, integrins activate FAK and other downstream signaling molecules leading to anchorage-dependent survival and proliferation32,33. However, unligated integrins can negatively influence the malignant properties of tumor cells19-21 by activation of apoptotic pathways inducing a form of death known as IMD. Interestingly, the tumor cells studied here have developed mechanism(s) to escape IMD which contributes to their metastatic behavior22. Integrin αvβ3 expression is linked to metastasis in several cancers including melanoma, as well as breast, prostate, cervical and pancreatic carcinomas11-18 and enhances tumor cell migration, survival and increased growth factor release27,34-38. Here, we present the unexpected result that integrin αvβ3 contributes to tumor progression and metastatic potential by enhancing anchorage-independent growth. This effect requires integrin αvβ3 recruitment and activation of c-src in a manner that is independent of tumor cell adhesion or the activation of FAK. Importantly, αvβ3 expression increases colony formation and cell survival in soft agar, even in the presence of a function-blocking antibody that prevents ligation to either soluble39 or immobilized27 ligands. In addition, expression of a mutant integrin incapable of binding ligand also showed increased anchorage-independence. A similar increase in cell survival was observed in αvβ3-bearing pancreatic tumors grown in mice, suggesting that αvβ3-mediated survival contributes to both anchorage-independence in vitro and tumor malignancy in vivo. Accordingly, knock-down of endogenous β3 decreased the anchorage-independence and metastasis of pancreatic cancer cells. Surprisingly, integrin αvβ3 was found to promote c-src-dependent, but FAK independent, phosphorylation of the CAS substrate domain in non-adherent cells. CAS phosphorylation promotes adhesion-mediated cell survival10,40 through FAK and c-src recruitment to integrin containing focal contacts41. In fibroblasts transformed with v-src or v-crk, CAS Author Manuscript Author Manuscript Author Manuscript Author Manuscript forms complexes with these molecules in a phosphorylation-dependent manner42 and deletion of CAS prevents v-src mediated transformation43, implicating CAS in oncogenesis. Importantly, we demonstrate that both knock-down of CAS or expression of a nonphosphorylated form of CAS abolished the αvβ3/c-src-mediated colony formation in soft agar. Anchorage-independence and tumor progression commonly result from oncogene expression. For example, the v-src oncogene potently stimulates anchorage-independent growth in fibroblasts44 and v-src is associated with enhanced cell invasion8. Expression of an activated mutant of c-src together with integrin αvβ3 promoted the transformation of a mouse pseudo-epithelial cell line, suggesting cooperativity between mutationally activate src and αvβ345,46. However, in some circumstances normal cellular derivatives of oncogenes, such as c-src, also contribute to tumor progression31 by stimulating cell migration and invasion. Here, we define a novel integrin-mediated pathway leading to activation of c-src, promoting increased anchorage-independence and tumor cell malignancy that does not impact cell migration. Previous studies have shown that the platelet integrin αIIbβ3 can recruit and activate c-src in a manner that depends on the C-terminal portion of β3 cytoplasmic tail24. We show that αvβ3 expressing tumor cells also recruit and activate c-src and, similar to the platelet studies, a β3 truncation mutant (759x) prevented c-src recruitment to the integrin. Importantly, cells expressing this truncation mutant failed to increase anchorageindependent growth in vitro or metastasis in vivo. While c-src associates with integrin αvβ3, we could not detect c-src recruitment to other integrins in these cells suggesting that the β3 integrin is unique in this regard. Thus, we conclude that unligated αvβ3 and its ability to recruit c-src contributes to the malignant properties of pancreatic cancer suggesting αvβ3/csrc can function as an oncogenic unit thereby contributing to tumor malignancy. Expression of αvβ3 is associated with the metastatic potential of several cancers 18,47,48. While antagonists of αvβ3 have proven efficacious as angiogenesis inhibitors in mouse tumor models49, and are now in phase III clinical trials in patients with glioblastoma, our studies suggest that direct inhibition of αvβ3 ligation on tumor cells may provide limited clinical benefit given that αvβ3 activates c-src in a ligand-independent manner. As such, we define a novel oncogenic signaling module comprised of unligated integrin αvβ3 and c-src that occurs in a subset of tumors resistant to IMD. Further, our study shows that dasatinib, a clinically approved SFK inhibitor, or c-src knock-down, not only blocked αvβ3-mediated anchorage-independent growth of pancreatic cancer cells in vitro but suppressed their metastatic properties in vivo. This suggests that c-src kinase inhibition may represent a therapeutic approach for those highly malignant tumors known to express integrin αvβ3. Methods Immunohistochemistry We cut 8 μm sections from formalin-fixed, paraffin-embedded primary tumor specimens from eighteen human patients diagnosed with pancreatic ductal adenocarcinoma (7 with matching lymph node metastases). We also stained a breast cancer tissue microarray Author Manuscript Author Manuscript Author Manuscript Author Manuscript containing 50 matched pairs of primary tumor/lymph node metastases (Cat# BR1004; BioMax). We deparaffinized and digested the sections with proteinase K 15 min at room temperature prior to quenching with 0.3% H 2 O 2 /0.3% normal serum. After washing, we blocked the sections in normal serum and probed with 1:100 primary antibody overnight at 4 °C. We then incubated the sections for 45 min with a biotinylated secondary antibody (1:2,000) followed by 30 min in Vectastain Elite ABC Reagent (Vector Labs). Staining was performed with DAB substrate for 1-2 min prior to counterstaining with hematoxylin and mounting. Orthotopic pancreatic tumors Tumors were generated by injection of GFP-labeled human pancreatic carcinoma cells (1×10 6 tumor cells in 50 μl of sterile PBS) into the tail of the pancreas of 6-8 week old male nude mice. See Supplementary Methods for details regarding the generation of cell lines. After 6 or 8 weeks, we resected both the primary tumors and the hepatic hilar lymph nodes. Primary tumor mass was determined by measuring the wet weight of the resected tumors. We reported metastasis as the incidence of GFP-expressing cells present in the resected lymph nodes. For the dasatinib treatment experiment, 36 mice were injected with GFPlabeled FG-β3 cells and randomized into three groups of twelve. Tumors established for 2 weeks before beginning dosing. Mice were dosed by oral gavage with the citric acid vehicle (b.i.d.), 30 mg kg −1 dasatinib (b.i.d.) or 50 mg kg −1 imatinib (q.d.) for 4 weeks prior to harvest. All research was conducted under protocols approved by the UCSD animal subjects committee and is in accordance with the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals. In vivo apoptosis and proliferation Analysis of both apoptosis and proliferation was performed on OCT-embedded frozen primary tumor sections. We assessed apoptosis in vivo by TUNEL staining using the ApopTag Red kit (Millipore). Proliferation was examined by immunofluorescent staining for Ki-67 using the manufacturer's instructions (Abcam). We measured both TUNEL and Ki-67 by capturing images from four 20× fields per tumor section and quantifying the number of stained cells using metamorph software. All data were normalized to total cell number (by co-staining with TOPRO-3 nuclear dye) and expressed as the percent TUNEL or Ki-67 positive cells per field. Triton-soluble and -insoluble lysates To isolate focal adhesions, serum-starved FG and FG-β3 cells were allowed to specifically adhere and spread for 2 h on dishes coated with 5 μg mL −1 fibronectin. Non-adherent cells are gently washed away with PBS and the remaining adherent cells were lysed in triton lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, and 1% Triton X-100, 50 mM NaF, Protease inhibitor cocktail (Roche), 2 mM PMSF, 2mM sodium orthovanadate) to generate the tritonsoluble lysate. The triton-insoluble lysate was prepared by washing the lysed cells twice with ice-cold PBS before adding RIPA lysis buffer (100 mM Tris pH 7.5, 150 mM sodium chloride, 0.1% deoxycholate, 0.1% SDS, 50 mM NaF, Protease inhibitor cocktail (Roche), 2 mM PMSF, 2mM sodium orthovanadate) and concentrating the lysate in a minimal volume. Soft agar assays We suspended cells in 0.3% agar/complete media and cultured them on a bottom layer of 1% agar/complete media in 48 or 24-well dishes. We then added additional media and cultured cells for 7-10 days prior to counting colonies consisting of at least 5 cells from 10× fields or whole wells. For dasatinib treatment experiments, colonies were grown in vehicle (DMSO), 50 nM, 250 nM or 1 μM dasatinib diluted in DMSO. We replaced the media with fresh inhibitor every other day. To knock-down CAS we transfected 5×10 6 FG or FG-β3 cells with 250 nM of control or CAS siRNA oligonucleotides (Qiagen) in 100 μL of Nucleofector V (Amaxa). We embedded the transfected cells in soft agar 48 h posttransfection. Suspension viability To directly assay for anchorage-independent survival and proliferation we cultured 1×10 6 FG or FG-β3 cells in suspension on 1% agar-coated wells in DMEM/10% FBS for 24 and 48 h prior to trypsinizing, staining with trypan blue and counting viable and non-viable cells on a hemocytometer. Statistical analyses All data, except the metastasis experiments, are presented as the mean±SEM and statistical differences were evaluated by Student's T-test. For metastasis, bars represent the incidence as a percentage of total mice and statistical evaluation was performed using Chi-square analysis. Colony formation in the presence of dasatinib was evaluated using a two-way repeated measure ANOVA to identify a positive interaction between the drug and the cell line. For all analyses, P < 0.05 was considered statistically significant
c-Met activation leads to the establishment of a TGFβ-receptor regulatory network in bladder cancer progression
No full text10.1038/s41467-019-12241-2Nature Communications10
Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence
No full textInternational audienceThe BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease
