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

Some inequalities on generalized entropies

We give several inequalities on generalized entropies involving Tsallis entropies, using some inequalities obtained by improvements of Young's inequality. We also give a generalized Han's inequality.Comment: 15 page

Rhomboid domain containing 2 (RHBDD2): A novel cancer-related gene over-expressed in breast cancer

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapsefree survival (p= 0.0013), and metastasis-free interval (p=0.006) in patients with primary ERnegative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression

A molecular portrait of high-grade ductal carcinoma in situ

Ductal carcinoma in situ (DCIS) is a noninvasive precursor lesion to invasive breast carcinoma. We still have no understanding on why only some DCIS lesions evolve to invasive cancer whereas others appear not to do so during the life span of the patient. Here, we performed full exome (tumor vs. matching normal), transcriptome, and methylome analysis of 30 pure high-grade DCIS (HG-DCIS) and 10 normal breast epithelial samples. Sixty-two percent of HG-DCIS cases displayed mutations affecting cancer driver genes or potential drivers. Mutations were observed affecting PIK3CA (21% of cases), TP53 (17%), GATA3 (7%), MLL3 (7%) and single cases of mutations affecting CDH1, MAP2K4, TBX3, NF1, ATM, and ARID1A. Significantly, 83% of lesions displayed numerous large chromosomal copy number alterations, suggesting they might precede selection of cancer driver mutations. Integrated pathway-based modeling analysis of RNA-seq data allowed us to identify two DCIS subgroups (DCIS-C1 and DCIS-C2) based on their tumor-intrinsic subtypes, proliferative, immune scores, and in the activity of specific signaling pathways. The more aggressive DCIS-C1 (highly proliferative, basal-like, or ERBB2 + ) displayed signatures characteristic of activated Treg cells (CD4 + /CD25 + /FOXP3 + ) and CTLA4 + /CD86 + complexes indicative of a tumor-associated immunosuppressive phenotype. Strikingly, all lesions showed evidence of TP53 pathway inactivation. Similarly, ncRNA and methylation profiles reproduce changes observed postinvasion. Among the most significant findings, we observed upregulation of lncRNA HOTAIR in DCIS-C1 lesions and hypermethylation of HOXA5 and SOX genes. We conclude that most HG-DCIS lesions, in spite of representing a preinvasive stage of tumor progression, displayed molecular profiles indistinguishable from invasive breast cancer.Centro de Investigaciones Inmunológicas Básicas y Aplicada

Effects of dexmedetomidine on subthalamic local field potentials in parkinson's disease

Background: Dexmedetomidine is frequently used for sedation during deep brain stimulator implantation in patients with Parkinson's disease, but its effect on subthalamic nucleus activity is not well known. The aim of this study was to quantify the effect of increasing doses of dexmedetomidine in this population. Methods: Controlled clinical trial assessing changes in subthalamic activity with increasing doses of dexmedetomidine (from 0.2 to 0.6 μg kg-1 h-1) in a non-operating theatre setting. We recorded local field potentials in 12 patients with Parkinson's disease with bilateral deep brain stimulators (24 nuclei) and compared basal activity in the nuclei of each patient and activity recorded with different doses. Plasma levels of dexmedetomidine were obtained and correlated with the dose administered. Results: With dexmedetomidine infusion, patients became clinically sedated, and at higher doses (0.5-0.6 μg kg-1 h-1) a significant decrease in the characteristic Parkinsonian subthalamic activity was observed (P<0.05 in beta activity). All subjects awoke to external stimulus over a median of 1 (range: 0-9) min, showing full restoration of subthalamic activity. Dexmedetomidine dose administered and plasma levels showed a positive correlation (repeated measures correlation coefficient=0.504; P<0.001). Conclusions: Patients needing some degree of sedation throughout subthalamic deep brain stimulator implantation for Parkinson's disease can probably receive dexmedetomidine up to 0.6 μg kg-1 h-1 without significant alteration of their characteristic subthalamic activity. If patients achieve a 'sedated' state, subthalamic activity decreases, but they can be easily awakened with a non-pharmacological external stimulus and recover baseline subthalamic activity patterns in less than 10 min

Mortality by causes in HIV-infected adults: comparison with the general population

<p>Abstract</p> <p>Background</p> <p>We compared mortality by cause of death in HIV-infected adults in the era of combined antiretroviral therapy with mortality in the general population in the same age and sex groups.</p> <p>Methods</p> <p>Mortality by cause of death was analyzed for the period 1999-2006 in the cohort of persons aged 20-59 years diagnosed with HIV infection and residing in Navarre (Spain). This was compared with mortality from the same causes in the general population of the same age and sex using standardized mortality ratios (SMR).</p> <p>Results</p> <p>There were 210 deaths among 1145 persons diagnosed with HIV (29.5 per 1000 person-years). About 50% of these deaths were from AIDS. Persons diagnosed with HIV infection had exceeded all-cause mortality (SMR 14.0, 95% CI 12.2 to 16.1) and non-AIDS mortality (SMR 6.9, 5.7 to 8.5). The analysis showed excess mortality from hepatic disease (SMR 69.0, 48.1 to 78.6), drug overdose or addiction (SMR 46.0, 29.2 to 69.0), suicide (SMR 9.6, 3.8 to 19.7), cancer (SMR 3.2, 1.8 to 5.1) and cardiovascular disease (SMR 3.1, 1.3 to 6.1). Mortality in HIV-infected intravenous drug users did not change significantly between the periods 1999-2002 and 2003-2006, but it declined by 56% in non-injecting drug users (<it>P </it>= 0.007).</p> <p>Conclusions</p> <p>Persons with HIV infection continue to have considerable excess mortality despite the availability of effective antiretroviral treatments. However, excess mortality in the HIV patients has declined since these treatments were introduced, especially in persons without a history of intravenous drug use.</p

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

Wwox Deletion in Mouse B Cells Leads to Genomic Instability, Neoplastic Transformation, and Monoclonal Gammopathies

WWOX (WW domain containing oxidoreductase) expression loss is common in various cancers and characteristic of poor prognosis. Deletions, translocations, and loss of expression affecting the WWOX gene are a common feature of various B cell neoplasms such as certain B cell lymphomas and multiple myeloma. However, the role of this common abnormality in B cell tumor initiation and/or progression has not been defined. In this study, we conditionally deleted Wwox early in B cell development by means of breeding Cd19-Cre transgenic mice crossed to Wwox floxed mice (Cd19 Wwox KO). We observed a significant reduced survival in Cd19 Wwox KO mice and the development of B cell neoplasms including B cell lymphomas, plasma cell neoplasias characterized by increased numbers of CD138+ populations as well as monoclonal gammopathies detected by serum protein electrophoresis. To investigate whether Wwox loss could play a role in genomic instability, we analyzed DNA repair functions during immunoglobulin class switch joining between DNA segments in antibody genes. While class switch recombination (CSR) was only slightly impaired, Wwox deficiency resulted in a dramatic shift of double strand break (DSB) repair from normal classical-NHEJ toward the microhomology-mediated alternative-NHEJ pathway, a pathway associated with chromosome translocations and genome instability. Consistent with this, Wwox deficiency resulted in a marked increase of spontaneous translocations during CSR. This work defines for the first time a role for Wwox for maintaining B cell genome stability during a process that can promote neoplastic transformation and monoclonal gammopathies.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada