Loss of ATM/Chk2/p53 Pathway Components Accelerates Tumor Development and Contributes to Radiation Resistance in Gliomas

SummaryMaintenance of genomic integrity is essential for adult tissue homeostasis and defects in the DNA-damage response (DDR) machinery are linked to numerous pathologies including cancer. Here, we present evidence that the DDR exerts tumor suppressor activity in gliomas. We show that genes encoding components of the DDR pathway are frequently altered in human gliomas and that loss of elements of the ATM/Chk2/p53 cascade accelerates tumor formation in a glioma mouse model. We demonstrate that Chk2 is required for glioma response to ionizing radiation in vivo and is necessary for DNA-damage checkpoints in the neuronal stem cell compartment. Finally, we observed that the DDR is constitutively activated in a subset of human GBMs, and such activation correlates with regions of hypoxia

Glioblastoma and glioblastoma stem cells are dependent on functional MTH1

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease

Somatic genome editing with the RCAS-TVA-CRISPR-Cas9 system for precision tumor modeling

To accurately recapitulate the heterogeneity of human diseases, animal models require to recreate multiple complex genetic alterations. Here, we combine the RCAS-TVA system with the CRISPR-Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells of a variety of known tumor suppressor genes (Trp53, Cdkn2a, and Pten) leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs we generate different gene fusions with oncogenic potential, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk). Lastly, using homology-directed-repair, we also produce tumors carrying the homologous mutation to human BRAF V600E, frequently identified in a variety of tumors, including different types of gliomas. In summary, we have developed an extremely versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing.A.C.-G is recipient of a Severo-Ochoa PhD fellowship. C.M. and V.M. are recipients of a "La Caixa "PhD fellowship. We thank A.J. Schuhmacher for the initial assistance with the intracranial injections in adult mice and C.S. Clemente-Troncone for the technical support. We thank Carmen Blanco, David Olmeda, and Marisol Soengas for sharing reagents and Orlando Dominguez for the help with the design of the BRAF high- throughput sequencing. We sincerely thank Dr. José Luis Rodríguez Peralto (Hospital U. 12 de Octubre Madrid) for the BRAF V600 IHCs staining. This research was supported by funds from the Acción Estratégica en Salud Spanish National Research and Development Plan, Instituto de Salud Carlos III (ISCIII), cofounder by FEDER (ERDF) (PI14/01884) to S.R.-P., by a 017 Leonardo Grant for Researchers and Cultural Creators from the BBVA Foundation and a grant from the Seve Ballesteros Foundation to M.S.S

CLINICAL OUTCOMES OF SELF-EXPAMDABLE METALLIC STENTS IN PALLIATION OF MALIGNANT ANASTOMOTIC STRICTURES: A SINGLE CENTER EXPERIENCE

Background: self-expandable metallic stents (SEMS) are employed as the preferred non surgical palliative treatment for gastric outlet obstruction due to malignancies. Metallic stents are often employed to treat malignant anastomotic obstructions after surgicsl interventions as esophagojejunostomy, gastrojejunostomy and esophagogastrojejunostomy. Methods: this case series reports prospectively the clinical outcomes of SEMS in the palliative care of malignant anastomotic strictures caused by the recurrence gastric cancer follwing gastric surgery as oncological curative treatment, in a series of nine consecutive patients, treated between January 2009 and december 2012 in our center. Results: Nine patients (M:F=8:1) were enrolled in the study. The operation was a total gastrectomy with esophagogastrojejunostomy (n=4), subtotal gastrectomy with Bilroth-II reconstruction (n=4), subtotal gastrectomy with Billroth-II reconstruction (n=3), and subtotal gastrectomy with esophagogastrostomy (n=2). The technical success rate was 88,9%, and the clinical success rate was 88.9%. The reostruction of the stent, due to the ingrowth of the tumor, occurred in 1 patient (11,1%) within 1 month after stent placement. the migration of the stent occurred after the placement of a covered stent in 1 patient who underwent a subtotal gastrectomy (with Billroth-II reconstruction). A case o partial stent dislodgement was treated with the placement of a second stent. The median survival period was 180 days (range, 30-240 days) and the median stent patency was 45 days 8range, 30-90 days). Conclusions: Although the number of the patients treated with SEMS results, in this series, almost small to certainly judge the safety and feasibility of SEMS, we believe that the endoscopic insertion of SEMS seems to be a safe, easily feasible, and effective treatment in the palliative care of malignant anastomotic strictures caused by the recurrence of gastric cancer following gastric surgery. The technical and clinical success, and the onset of complications of this procedure are influenced by several factors, such as the type of anastomosis, the technical features of the stent, and the extent of the underlying tumor

Yeast two-hybrid junk sequences contain selected linear motifs

Yeast two-hybrid (Y2H) screenings result in identification of many out-of-frame (OOF) clones that code for short (2-100 amino acids) peptides with no sequence homology to known proteins. We hypothesize that these peptides can reveal common short linear motifs (SLiMs) responsible for their selection. We present a new protocol to address this issue, using an existing SLIM detector (TEIRESIAS) as a base method, and applying filters derived from a mathematical model of SLiM selection in OOF clones. The model allows for initial analysis of likely presence of SLiM(s) in a collection of OOF sequences, assisting investigators with the decision of whether to invest resources in further analysis. If SLiM presence is detected, it estimates the length and number of amino acid residues involved in binding specificity and the amount of noise in the Y2H screen. We demonstrate that our model can double the prediction sensitivity of TEIRESIAS and improve its specificity from 0 to 1.0 on simulated data and apply the model to seven sets of experimentally derived OOF clones. Finally, we experimentally validate one SLiM found by our method, demonstrating its utility

Structural insights into the transcriptional and translational roles of Ebp1.

Accepted versio

Polyphenol intake and cardiovascular risk factors in a population with\ua0type 2 diabetes: The TOSCA.IT study.

Summary Background The role of polyphenol intake on cardiovascular risk factors is little explored, particularly in people with diabetes. Aim: To evaluate the association between the intake of total polyphenols and polyphenol classes with the major cardiovascular risk factors in a population with type 2 diabetes. Methods Dietary habits were investigated in 2573 males and females participants of the TOSCA.IT study. The European Prospective Investigation on Cancer and Nutrition (EPIC) questionnaire was used to assess dietary habits. In all participants, among others, we assessed anthropometry, plasma lipids, blood pressure, C-reactive protein and HbA1c following a standard protocol. The USDA and Phenol-Explorer databases were used to estimate the polyphenol content of the habitual diet. Results Average intake of polyphenols was 683.3 \ub1 5.8 mg/day. Flavonoids and phenolic acids were the predominant classes (47.5% and 47.4%, respectively). After adjusting for potential confounders, people with the highest intake of energy-adjusted polyphenols (upper tertile) had a more favorable cardiovascular risk factors profile as compared to people with the lowest intake (lower tertile) (BMI was 30.7 vs 29.9 kg/m2, HDL-cholesterol was 45.1 vs 46.9 mg/dl, LDL-cholesterol was 103.2 vs 102.1 mg/dl, triglycerides were 153.4 vs 148.0 mg/dl, systolic and diastolic blood pressure were respectively 135.3 vs 134.3 and 80.5 vs 79.6 mm/Hg, HbA1c was 7.70 vs 7.67%, and C-reactive Protein was 1.29 vs 1.25 mg/dl, p < .001 for all). The findings were very similar when the analysis was conducted separately for flavonoids or phenolic acids, the two main classes of polyphenols consumed in this population. Conclusions Polyphenol intake is associated with a more favorable cardiovascular risk factors profile, independent of major confounders. These findings support the consumption of foods and beverages rich in different classes of polyphenols particularly in people with diabetes. Clinical trial http://www.clinicaltrials.gov; Study ID number: NCT00700856

Evidence that Proteasome-Dependent Degradation of the Retinoblastoma Protein in Cells Lacking A-Type Lamins Occurs Independently of Gankyrin and MDM2

A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB) stabilization through unknown mechanism(s). Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna-/- fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna-/- fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16(ink4a)-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna-/- cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna-/- cells with p14(arf). p14(arf) expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna-/- cells.Our findings suggest that pRB degradation in Lmna-/- cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna-/- fibroblasts. Second, Lmna-/- cells are refractory to p14(arf)-mediated cell cycle arrest, as was previously shown with p16(ink4a). Potential roles of lamin A/C in the suppression of tumorigenesis are discussed

Impact of COVID-19 pandemic on mental health: An international study

Background The COVID-19 pandemic triggered vast governmental lockdowns. The impact of these lockdowns on mental health is inadequately understood. On the one hand such drastic changes in daily routines could be detrimental to mental health. On the other hand, it might not be experienced negatively, especially because the entire population was affected. Methods The aim of this study was to determine mental health outcomes during pandemic induced lockdowns and to examine known predictors of mental health outcomes. We therefore surveyed n = 9,565 people from 78 countries and 18 languages. Outcomes assessed were stress, depression, affect, and wellbeing. Predictors included country, sociodemographic factors, lockdown characteristics, social factors, and psychological factors. Results Results indicated that on average about 10% of the sample was languishing from low levels of mental health and about 50% had only moderate mental health. Importantly, three consistent predictors of mental health emerged: social support, education level, and psychologically flexible (vs. rigid) responding. Poorer outcomes were most strongly predicted by a worsening of finances and not having access to basic supplies. Conclusions These results suggest that on whole, respondents were moderately mentally healthy at the time of a population-wide lockdown. The highest level of mental health difficulties were found in approximately 10% of the population. Findings suggest that public health initiatives should target people without social support and those whose finances worsen as a result of the lockdown. Interventions that promote psychological flexibility may mitigate the impact of the pandemic