Rad6B acts downstream of Wnt signaling to stabilize β-catenin: Implications for a novel Wnt/β-catenin target

Abstract Background Aberrant Wnt/β-catenin signaling is associated with breast cancer even though genetic mutations in Wnt signaling components are rare. We have previously demonstrated that Rad6B, an ubiquitin conjugating enzyme, stabilizes β-catenin via polyubiqutin modifications that render β-catenin insensitive to proteasomal degradation. Rad6B is a transcriptional target of β-catenin, creating a positive feedback loop between Rad6B expression and β-catenin activation. Methods To isolate subpopulations expressing high or low Rad6B levels, we transfected MDA-MB-231 or WS-15 human breast cancer cells with ZsGreen fluorescent reporter vector in which the expression of ZsGreen was placed under the control of Rad6B promoter. ZsGreenhigh and ZsGreenlow subpopulations, reflective of high and low Rad6B promoter activity, respectively, were isolated by FACS. To determine the relevance of Wnt signaling in Rad6B-mediated β-catenin stabilization/activation, the ZsGreenhigh cells were transfected with signaling-defective Wnt coreceptor LRP6Δ173. Rad6B expression and promoter activity were determined by RT-PCR, Western blot and Rad6B promoter-mediated luciferase assays. β-catenin levels and transcriptional activity were determined by Western blot and TOP/FOP Flash reporter assays. Tumor formation and morphologies of ZsGreenlow, ZsGreenhigh, and ZsGreenhigh/LRP6Δ173 cells compared to unsorted vector controls were evaluated in nude mice. Expression of Wnt signaling related genes was profiled using the Wnt signaling pathway RT2 Profiler PCR arrays. Results ZsGreenhigh subpopulations showed high Rad6B expression and Rad6B promoter activity as compared to ZsGreenlow cells. ZsGreenhigh (high Rad6B expressors) also showed elevated β-catenin levels and TOP/Flash activity. Inhibiting Wnt signaling in the high Rad6B expressors decreased ZsGreen fluorescence, Rad6B gene expression, β-catenin levels and TOP/Flash activity. Tumors derived from high Rad6B expressors were predominantly composed of cells with epithelial mesenchymal transition (EMT) phenotype as compared to control tumors that were composed of both cuboidal and EMT-type cells. Tumors derived from low Rad6B expressors lacked EMT phenotype. Inhibition of LRP6 function in the high Rad6B expressors abrogated the EMT phenotype. Gene expression profiling showed upregulation of several Wnt signaling pathway regulators in high Rad6B expressors that were downregulated by interference of Wnt signaling with mutant LRP6 or by Rad6B silencing. Conclusions These data reveal a functional link between the canonical Wnt pathway and Rad6B in β-catenin activation and breast cancer progression

Nontransgenic models of breast cancer

Numerous models have been developed to address key elements in the biology of breast cancer development and progression. No model is ideal, but the most useful are those that reflect the natural history and histopathology of human disease, and allow for basic investigations into underlying cellular and molecular mechanisms. We describe two types of models: those that are directed toward early events in breast cancer development (hyperplastic alveolar nodules [HAN] murine model, MCF10AT human xenograft model); and those that seek to reflect the spectrum of metastatic disease (murine sister cell lines 67, 168, 4T07, 4T1). Collectively, these models provide cell lines that represent all of the sequential stages of progression in breast disease, which can be modified to test the effect of genetic changes

Deep generative modeling for single-cell transcriptomics.

Single-cell transcriptome measurements can reveal unexplored biological diversity, but they suffer from technical noise and bias that must be modeled to account for the resulting uncertainty in downstream analyses. Here we introduce single-cell variational inference (scVI), a ready-to-use scalable framework for the probabilistic representation and analysis of gene expression in single cells ( https://github.com/YosefLab/scVI ). scVI uses stochastic optimization and deep neural networks to aggregate information across similar cells and genes and to approximate the distributions that underlie observed expression values, while accounting for batch effects and limited sensitivity. We used scVI for a range of fundamental analysis tasks including batch correction, visualization, clustering, and differential expression, and achieved high accuracy for each task

Sensitivity of the Himalayan orography representation in simulation of winter precipitation using Regional Climate Model (RegCM) nested in a GCM

This document is the Accepted Manuscript version of the following article: Tiwari, P.R., Kar, S.C., Mohanty, U.C., Climate Dynamics (2017). The final publication is available at Springer via https://link.springer.com/article/10.1007%2Fs00382-017-3567-3. The Accepted Manuscript is under embargo. Embargo end date: 24 February 2018.The role of the Himalayan orography representationin a Regional Climate Model (RegCM4) nested inNCMRWF global spectral model is examined in simulatingthe winter circulation and associated precipitation over theNorthwest India (NWI; 23°–37.5°N and 69°–85°E) region.For this purpose, nine different set of orography representationsfor nine distinct precipitation years (three years eachfor wet, normal and dry) have been considered by increasing(decreasing) 5, 10, 15, and 20% from the mean height(CNTRL) of the Himalaya in RegCM4 model. Validationwith various observations revealed a good improvementin reproducing the precipitation intensity and distributionwith increased model height compared to the resultsobtained from CNTRL and reduced orography experiments.Further it has been found that, increase in heightby 10% (P10) increases seasonal precipitation about 20%,while decrease in height by 10% (M10) results around 28%reduction in seasonal precipitation as compared to CNTRLexperiment over NWI region. This improvement in precipitationsimulation comes due to better representation ofvertical pressure velocity and moisture transport as thesefactors play an important role in wintertime precipitationprocesses over NWI region. Furthermore, a comparison of model-simulated precipitation with observed precipitationat 17 station locations has been also carried out. Overall,the results suggest that when the orographic increment of10% (P10) is applied on RegCM4 model, it has better skillin simulating the precipitation over the NWI region andthis model is a useful tool for further regional downscalingstudies.Peer reviewe

A synergistic antiproliferation effect of curcumin and docosahexaenoic acid in SK-BR-3 breast cancer cells: unique signaling not explained by the effects of either compound alone

<p>Abstract</p> <p>Background</p> <p>Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.</p> <p>Methods</p> <p>Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED₅₀. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.</p> <p>Results</p> <p>CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ER^-PR^-Her2⁺) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.</p> <p>Conclusions</p> <p>The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds.</p