Ras and TGFβ cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling

Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice

[Background and Aims]: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. [Methods]: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/–;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. [Results]: A5+/–;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/–;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/–;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/–;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. [Conclusions]: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.This study was supported in part by the Mildred-Scheel-Professur der Deutschen Krebshilfe 111464, DFG AL 1174/6-1 to H.A., DFG DI 2299/1-1 to K.N.D., DFG SFB1321 (S01) to K.S. and W.W., and the German Federal Ministry of Education and Research to the German Center for Diabetes Research (DZD e.V.) to J.A

Timing Constraints of In Vivo Gag Mutations during Primary HIV-1 Subtype C Infection

Background: Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. Methods: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. Results: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. Conclusions: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI <18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For schoolaged children and adolescents, we report thinness (BMI <2 SD below the median of the WHO growth reference) and obesity (BMI >2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit

Code Ownership and Software Quality: A Replication Study",

Using new and refined code ownership metrics we were able to classify source files that contained at least one bug with a median precision of 0.74 and a median recall of 0.38. On directory level, we achieve a precision of 0.76 and a recall of 0.60

The Art of Testing Less without Sacrificing Quality

Abstract-Testing is a key element of software development processes for the management and assessment of product quality. In most development environments, the software engineers are responsible for ensuring the functional correctness of code. However, for large complex software products, there is an additional need to check that changes do not negatively impact other parts of the software and they comply with system constraints such as backward compatibility, performance, security etc. Ensuring these system constraints may require complex verification infrastructure and test procedures. Although such tests are time consuming and expensive and rarely find defects they act as an insurance process to ensure the software is compliant. However, long lasting tests increasingly conflict with strategic aims to shorten release cycles. To decrease production costs and to improve development agility, we created a generic test selection strategy called THEO that accelerates test processes without sacrificing product quality. THEO is based on a cost model, which dynamically skips tests when the expected cost of running the test exceeds the expected cost of removing it. We replayed past development periods of three major Microsoft products resulting in a reduction of 50% of test executions, saving millions of dollars per year, while maintaining product quality

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2alpha/CK2beta interaction

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2 alpha and CK2 beta in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2 alpha(1-335) and the fluorescent probe CF-Ahx-Pc as a CK2 beta analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2 alpha(1-335). Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2 alpha-binding motif of CK2 beta. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2 alpha(1-335) complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2 alpha(1-335) were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a K-D value of 240 nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2 beta, with an IC50 value of 92 mu M. (C) 2014 Elsevier Inc. All rights reserved