Modelling mutational landscapes of human cancers in vitro

Experimental models that recapitulate mutational landscapes of human cancers are needed to decipher the rapidly expanding data on human somatic mutations. We demonstrate that mutation patterns in immortalised cell lines derived from primary murine embryonic fibroblasts (MEFs) exposed in vitro to carcinogens recapitulate key features of mutational signatures observed in human cancers. In experiments with several cancer-causing agents we obtained high genome-wide concordance between human tumour mutation data and in vitro data with respect to predominant substitution types, strand bias and sequence context. Moreover, we found signature mutations in well-studied human cancer driver genes. To explore endogenous mutagenesis, we used MEFs ectopically expressing activation-induced cytidine deaminase (AID) and observed an excess of AID signature mutations in immortalised cell lines compared to their non-transgenic counterparts. MEF immortalisation is thus a simple and powerful strategy for modelling cancer mutation landscapes that facilitates the interpretation of human tumour genome-wide sequencing data

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFβ signalling and EMT

Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGFß-dependent biological outputs such as epithelial–mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGFß pathway. Interestingly, the WWP2-N isoform interacts with Smad2 and Smad3, whereas WWP2-C interacts only with Smad7. In addition, WWP2-FL and WWP2-C have a preference for Smad7 based on protein turnover and ubiquitination studies. Unexpectedly, we also find that WWP2-N, which lacks the HECT ubiquitin ligase domain, can also interact with WWP2-FL in a TGFß-regulated manner and activate endogenous WWP2 ubiquitin ligase activity causing degradation of unstimulated Smad2 and Smad3. Consistent with our protein interaction data, overexpression and knockdown approaches reveal that WWP2 isoforms differentially modulate TGFß-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGFß-induced EMT. Collectively, our data suggest that WWP2-N can stimulate WWP2-FL leading to increased activity against unstimulated Smad2 and Smad3, and that Smad7 is a preferred substrate for WWP2-FL and WWP2-C following prolonged TGFß stimulation. Significantly, this is the first report of an interdependent biological role for distinct HECT E3 ubiquitin ligase isoforms, and highlights an entirely novel regulatory paradigm that selectively limits the level of inhibitory and activating Smads

Lung Cancer in Pulmonary Fibrosis: Tales of Epithelial Cell Plasticity

Lung epithelial cells exhibit a high degree of plasticity. Alterations to lung epithelial cell function are critically involved in several chronic lung diseases such as pulmonary fibrosis. Pulmonary fibrosis is characterized by repetitive injury and subsequent impaired repair of epithelial cells, which leads to aberrant growth factor activation and fibroblast accumulation. Increased proliferation and hyper- and metaplasia of epithelial cells upon injury have also been observed in pulmonary fibrosis; this epithelial cell activation might represent the basis for lung cancer development. Indeed, several studies have provided histopathological evidence of an increased incidence of lung cancer in pulmonary fibrosis. The mechanisms involved in the development of cancer in pulmonary fibrosis, however, remain poorly understood. This review highlights recently uncovered molecular mechanisms shared between lung cancer and fibrosis, which extend the current evidence of a common trait of cancer and fibrosis, as provided by histopathological observations. Copyright (C) 2011 S. Karger AG, Base

Dynamics of notch pathway expression during mouse testis post-natal development and along the spermatogenic cycle

Articles in International JournalsThe transcription and expression patterns of Notch pathway components (Notch 1–3, Delta1 and 4, Jagged1) and effectors (Hes1, Hes2, Hes5 and Nrarp) were evaluated (through RT-PCR and IHC) in the mouse testis at key moments of post-natal development, and along the adult spermatogenic cycle. Notch pathway components and effectors are transcribed in the testis and expressed in germ, Sertoli and Leydig cells, and each Notch component shows a specific cell-type and timewindow expression pattern. This expression at key testis developmental events prompt for a role of Notch signaling in prepubertal spermatogonia quiescence, onset of spermatogenesis, and regulation of the spermatogenic cycle

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naive cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGF beta/SMAD (transforming growth factor-beta/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naive cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.Fapesp-grant number 2012/04194-1, 2013/05172-4, 2014/24400-0 and 2015/10821-7, CNPq-grant number 150447/2013-2 and 471512/2013-3 and PRODOC-grant no 3193-32/2010. Work in the lab of KS Smalley was supported by the National Institutes of Health grants R01 CA161107, R21 CA198550, and Skin SPORE grant P50 CA168536info:eu-repo/semantics/publishedVersio

Brain abscess and stroke in children and adults with hereditary hemorrhagic telangiectasia: Analysis of a large national claims database

BACKGROUND AND OBJECTIVES: Hereditary hemorrhagic telangiectasia (HHT) is an inherited disease associated with pathogenic variants in transforming growth factor-β signaling pathway-related genes, resulting in abnormal vascular development in various organs. Brain arteriovenous malformations (AVMs) may lead to intracranial hemorrhage, and brain abscess or ischemic stroke may result from right to left shunting via pulmonary AVMs. We aimed to investigate the risk for these severe complications in both adults and children with HHT. METHODS: We conducted a case-control study among participants aged 1-64 years in the MarketScan Commercial (2006-2019) and Multistate Medicaid Databases (2011-2019). We identified cases with HHT using RESULTS: A total of 5,796 patients with HHT, of whom 588 were children (age younger than 16 years), were matched with 57,960 controls. There was an increased incidence of brain abscesses in HHT cases compared with controls, with an RR of 35.6 (95% CI 15.4-82.5). No brain abscesses were recorded in children aged 15 years or younger. Hemorrhagic strokes/subarachnoid hemorrhages were more common in HHT cases, with an RR of 4.01 (95% CI 2.8-5.7) in adults and 60.2 (95% CI 7.2-500.4) in children. Ischemic strokes were also more common in cases, with an RR of 3.7 (95% CI, 3.0-4.5) in adults and 70.4 (95% CI 8.7-572.3) in children. DISCUSSION: We observed a much higher incidence of severe CNS vascular complications in patients with HHT, particularly in children. Although a higher incidence of brain abscesses was noted in adult patients with HHT, no brain abscesses were recorded in children, a result that may be considered when surveillance recommendations for this population are revisited

Estrogen Receptor Alpha Is Expressed in Mesenteric Mesothelial Cells and Is Internalized in Caveolae upon Freund's Adjuvant Treatment

Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-beta) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-alpha) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-alpha showed an inverse correlation with the secretion of TGF-beta. At the cellular and subcellular levels ER-alpha was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-alpha is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-alpha and its caveola-mediated endocytosis might play role in TGF-beta induced type II EMT in vivo

Eastern Wind Integration and Transmission Study -- Preliminary Findings: Preprint

This paper reviews the Eastern Wind Integration and Transmission Study, the development of wind datasets, the transmission analysis, and the results of wind integration analysis for four scenarios

Evaluation of the porous silicon capacitor as a moisture sensor for vacuum applications

A growing demand exists for inexpensive and reliable sensors for moisture detection in reduced pressure processing environments. Sandia`s Porous Silicon Capacitor (PSC) sensor appears to be an ideal candidate for this application. This sensor is a solid state device that detects moisture through changes in dielectric constant with water adsorption. Standard microelectronic fabrication techniques are used in its production affording low cost production and ready integration into complex sensor and electronic arrays. This sensor has previously been investigated for moisture detection in fluid streams, however, little effort has been placed on its behavior in a vacuum environment. Sandia`s Sensors in Vacuum (SIV) test facility has been employed to evaluate the performance characteristics of this sensor in vacuum. In addition, a vacuum-based study allows for a more controlled environment in which the intrinsic lower limit for moisture detection and response times to moisture changes can be easily determined quantitatively. This report describes the performance characteristics of a series of sensors from a single production lot. Calibration of these sensors to moisture levels from part per billion to part per hundred concentrations has been performed. The concentration-dependent sensitivity of these sensors is documented. The response time and drift characteristics of these sensors are also discussed. The investigation of a preliminary method for increasing the recovery time of the sensor after moisture exposure is presented. The role of hydrocarbon contamination, a potential problem in some vacuum schemes, is also evaluated. Specific recommendations are made on how to implement this sensor for vacuum applications

Low Resistivity Ohmic Contacts to Moderately Doped N-Gaas With Low Temperature Processing

A low-temperature process for forming ohmic contacts to moderately doped GaAs has been optimized using a PdGe metallization scheme. Minimum specific contact resistivity of 1.5 {times} 10{sup {minus}6} {minus}cm{sup 2} has been obtained with a low anneal temperature of 250 C. Results for optimizing both time and temperature are reported and compared to GeAu n-GaAs contacts. Material compositions was analyzed by x-ray photoelectron spectroscopy and circuit metal interconnect contact resisitivity to the low-temperature processed PdGe contacts is reported. For the lowest temperature anneals considered, excess Ge on the ohmic contact layer is suspected of degrading interconnect metal contacts, while higher temperature anneals permitted interconnect metal formation with negligible contact resistivity. Atomic force microscopy measurements showed that the PdGe surface morphology is much more uniform than standard GeAu contacts