The cancer gene WWOX behaves as an inhibitor of SMAD3 transcriptional activity via direct binding

Background: The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor in breast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us to investigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells. Methods: Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX (shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptional regulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate a direct interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyses were employed to determine the effect of WWOX silencing on TGFβ-signaling. Results: WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genes involved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated by the SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts. Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1. Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFβ responsive reporter. Additionally, WWOX expression leads to redistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFβ target ANGPTL4 plays a key role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX in microarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX and ANGPTL4. Furthermore, the WWOX lo/ANGPTL4hi cluster of breast tumors is enriched in triple-negative and basal-like sub-types. Tumors with this gene expression signature could represent candidates for anti-TGFβ targeted therapies. Conclusions: We show for the first time that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain mediated binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFβ signaling in advanced breast cancer.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

The cancer gene WWOX behaves as an inhibitor of SMAD3 transcriptional activity via direct binding

Background: The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor in breast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us to investigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells. Methods: Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX (shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptional regulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate a direct interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyses were employed to determine the effect of WWOX silencing on TGFβ-signaling. Results: WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genes involved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated by the SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts. Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1. Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFβ responsive reporter. Additionally, WWOX expression leads to redistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFβ target ANGPTL4 plays a key role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX in microarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX and ANGPTL4. Furthermore, the WWOX lo/ANGPTL4hi cluster of breast tumors is enriched in triple-negative and basal-like sub-types. Tumors with this gene expression signature could represent candidates for anti-TGFβ targeted therapies. Conclusions: We show for the first time that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain mediated binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFβ signaling in advanced breast cancer.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

Third-party policing: A theoretical analysis of an emerging trend

“Third-party policing” describes police efforts to persuade or coerce nonoffending persons to take actions which are outside the scope of their routine activities, and which are designed to indirectly minimize disorder caused by other persons or to reduce the possibility that crime may occur. The practice applies formal, noncriminal controls found in civil law as coercive tools against an intermediate class of nonoffending persons who are thought to have some power over offenders' primary environments. The police use coercion to create place guardianship that previously was absent, so as to decrease opportunities for crime and disorder. We link the theoretical bases of crime prevention to the theory of third-party policing, and examine gaps in traditional policing that have led to a formalization of policing through third parties. We examine third-party policing in two location-specific programs: the drug-abatement Beat Health Program in Oakland and the problem-solving RECAP Unit in Minneapolis. We conclude by discussing the potential ramifications of the third-party trend

Induced pluripotent stem cell-related genes influence biological behavior and 5-fluorouracil sensitivity of colorectal cancer cells*

Objective: We aimed to perform a preliminary study of the association between induced pluripotent stem cell (iPS)-related genes and biological behavior of human colorectal cancer (CRC) cells, and the potential for developing anti-cancer drugs targeting these genes. Methods: We used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the transcript levels of iPS-related genes NANOG, OCT4, SOX2, C-MYC and KLF4 in CRC cell lines and cancer stem cells (CSCs)-enriched tumor spheres. NANOG was knockdowned in CRC cell line SW620 by lentiviral transduction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, plate colony formation, and a mouse xenograft model were used to evaluate alterations in biological behavior in NANOG-knockdown SW620 cells. Also, mock-knockdown and NANOG-knockdown cells were treated with 5-fluorouracil (5-FU) and survival rate was measured by MTT assay to evaluate drug sensitivity. Results: A significant difference in the transcript levels of iPS-related genes between tumor spheres and their parental bulky cells was observed. NANOG knockdown suppressed proliferation, colony formation, and in vivo tumorigenicity but increased the sensitivity to 5-FU of SW620 cells. 5-FU treatment greatly inhibited the expression of the major stemness-associated genes NANOG, OCT4, and SOX2. Conclusions: These results collectively suggest an overlap between iPS-related genes and CSCs in CRC. Quenching a certain gene NANOG may truncate the aggressiveness of CRC cells

Vorapaxar in the secondary prevention of atherothrombotic events

Item does not contain fulltextBACKGROUND: Thrombin potently activates platelets through the protease-activated receptor PAR-1. Vorapaxar is a novel antiplatelet agent that selectively inhibits the cellular actions of thrombin through antagonism of PAR-1. METHODS: We randomly assigned 26,449 patients who had a history of myocardial infarction, ischemic stroke, or peripheral arterial disease to receive vorapaxar (2.5 mg daily) or matching placebo and followed them for a median of 30 months. The primary efficacy end point was the composite of death from cardiovascular causes, myocardial infarction, or stroke. After 2 years, the data and safety monitoring board recommended discontinuation of the study treatment in patients with a history of stroke owing to the risk of intracranial hemorrhage. RESULTS: At 3 years, the primary end point had occurred in 1028 patients (9.3%) in the vorapaxar group and in 1176 patients (10.5%) in the placebo group (hazard ratio for the vorapaxar group, 0.87; 95% confidence interval [CI], 0.80 to 0.94; P<0.001). Cardiovascular death, myocardial infarction, stroke, or recurrent ischemia leading to revascularization occurred in 1259 patients (11.2%) in the vorapaxar group and 1417 patients (12.4%) in the placebo group (hazard ratio, 0.88; 95% CI, 0.82 to 0.95; P=0.001). Moderate or severe bleeding occurred in 4.2% of patients who received vorapaxar and 2.5% of those who received placebo (hazard ratio, 1.66; 95% CI, 1.43 to 1.93; P<0.001). There was an increase in the rate of intracranial hemorrhage in the vorapaxar group (1.0%, vs. 0.5% in the placebo group; P<0.001). CONCLUSIONS: Inhibition of PAR-1 with vorapaxar reduced the risk of cardiovascular death or ischemic events in patients with stable atherosclerosis who were receiving standard therapy. However, it increased the risk of moderate or severe bleeding, including intracranial hemorrhage. (Funded by Merck; TRA 2P-TIMI 50 ClinicalTrials.gov number, NCT00526474.)