Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to mediate VEGFR2 expression in breast cancer cells

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. To investigate how mutant p53 impacts transcription, we studied how mutant p53 regulates vascular endothelial growth factor receptor 2 (VEGFR2), one of its strongest target genes that we identified through global gene expression profiling in mutant p53 expressing MDA 468 breast cancer cells. VEGFR2, the primary functional VEGF receptor and clinical target of bevacizumab, mediates endothelial cell neovascularization by promoting increased cellular proliferation, migration, and pro-survival signaling. In breast tumors, VEGFR2 is often aberrantly expressed on the breast tumor epithelia,which correlates with worse overall survival. We identify VEGFR2 as a mutant p53 transcriptional target in multiple breast cancer cell lines. Mutant p53 mediated upregulation of VEGFR2 mediates mutant 53 gain of function including increased cellular growth and migration. In humans, breast tumors with TP53 hotspot mutants have elevated VEGFR2 levels compared to tumors with loss of function mutations. The same class of tumors has significantly upregulated HIF1A and VEGFA compared to TP53 wild type tumors, indicating that mutant p53 containing breast tumors express a neoangiogenic gene signature that may intensify VEGFR2 autocrine signaling. A clinical trial suggests that TP53 mutated breast tumors may specifically respond to anti VEGF therapy, while TP53 wild type tumors may not respond. We suggest that mutant p53 containing breast tumors may be distinctively vulnerable to anti VEGF ntherapies. We investigated how mutant p53 impacts transcription of VEGFR2 using multiple techniques including scanning ChIP, micrococcal nuclease PCR, and in vivo DNase I footprinting by ligation mediated PCR. Mutant p53 was found to bind near the VEGFR2 transcriptional start site, causing the promoter to adopt a transcriptionally active conformation. Using SILAC mass spectrometry, we identified subunits of the SWI/SNF chromatin remodeling complex as mutant p53 interactors. Importantly, re ChIP and immunodepletion ChIP demonstrate that mutant p53 and SWI/SNF co-occupy the VEGFR2 promoter. Depletion of multiple SWI/SNF subunits reduced VEGFR2 RNA expression, and SWI/SNF is required for maximal mutant p53 promoter occupancy. Using RNA sequencing, we report that approximately half of all mutant p53 gene alteration impacts transcription of VEGFR2 as well as myriad other target genes by promoter remodeling through interaction with the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53 expressing tumors be uniquely susceptible to anti VEGF therapies, but restoration of SWI/SNF tumor suppressor function by targeting mutant p53 may have therapeutic potential. Mutant p53 interaction with the SWI/SNF complex may explain how mutant p53 modulates the expression of such a diverse set of genes

EM-Cube: An Architecture for Low-Cost Real-Time Volume Rendering

EM-Cube is a VLSI architecture for low-cost, high quality volume rendering at full video frame rates. Derived from the Cube-4 architecture developed at SUNY at Stony Brook, EM-Cube computes sample points and gradients on-the-fly to project 3-dimensional volume data onto 2-dimensional images with realistic lighting and shading. A modest rendering system based on EM-Cube consists of a PCI card with four rendering chips (ASICs), four 64Mbit SDRAMs to hold the volume data, and four SRAMs to capture the rendered image. The performance target for this configuration is to render images from a 256^3 x 16 bit data set at 30 frames/sec. The EM-Cube architecture can be scaled to larger volume data-sets and/or higher frame rates by adding additional ASICs, SDRAMs, and SRAMs. This paper addresses three major challenges encountered developing EM-Cube into a practical product: exploiting the bandwidth inherent in the SDRAMs containing the volume data, keeping the pin-count between adjacent ASICs at a tractable level, and reducing the on-chip storage required to hold the intermediate results of rendering.Engineering and Applied Science

Safe and Efficient Silencing with a Pol II, but not a Pol lII, Promoter Expressing an Artificial miRNA Targeting Human Huntingtin

Huntington\u27s disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken β-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CβA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CβA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CβA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CβA promoter can provide an effective and safe dose of a human huntingtin miRNA

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington\u27s disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection

HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation

The peripheral immune response is altered in Huntington's disease, but the underlying mechanisms are unclear. Using RNA interference to lower huntingtin levels in leucocytes from patients, Träger et al. reverse disease-associated phenotypes including cytokine elevation and transcriptional dysregulation, and argue for a direct effect of mutant huntingtin on NFκΒ signallin

Artificial miRNAs Reduce Human Mutant Huntingtin Throughout the Striatum in a Transgenic Sheep Model of Huntington's Disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA

Recurrent intragenic rearrangements of EGFR and BRAF in soft tissue tumors of infants.

Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors

A pervasive role for biomass burning in tropical high ozone/low water structures.

Air parcels with mixing ratios of high O3 and low H2O (HOLW) are common features in the tropical western Pacific (TWP) mid-troposphere (300-700 hPa). Here, using data collected during aircraft sampling of the TWP in winter 2014, we find strong, positive correlations of O3 with multiple biomass burning tracers in these HOLW structures. Ozone levels in these structures are about a factor of three larger than background. Models, satellite data and aircraft observations are used to show fires in tropical Africa and Southeast Asia are the dominant source of high O3 and that low H2O results from large-scale descent within the tropical troposphere. Previous explanations that attribute HOLW structures to transport from the stratosphere or mid-latitude troposphere are inconsistent with our observations. This study suggest a larger role for biomass burning in the radiative forcing of climate in the remote TWP than is commonly appreciated.We thank L. Pan for coordinating the CONTRAST flights and her constructive criticism of an early version of the manuscript; S. Schauffler, V. Donets and R. Lueb for collecting and analysing AWAS samples; T. Robinson and O. Shieh for providing meteorology forecasts in the field; and the pilots and crews of the CAST BAe-146 and CONTRAST Gulfstream V aircrafts for their dedication and professionalism. CAST was funded by the Natural Environment Research Council; CONTRAST was funded by the National Science Foundation. Research at the Jet Propulsion Laboratory, California Institute of Technology, is performed under contract with the National Aeronautics and Space Administration (NASA). A number of the US-based investigators also benefitted from the support of NASA as well as the National Oceanic and Atmospheric Administration. The views, opinions, and findings contained in this report are those of the author(s) and should not be construed as an official National Oceanic and Atmospheric Administration or US Government position, policy or decision. We would like to acknowledge high-performance computing support from Yellowstone (ark:/85065/d7wd3xhc) provided by NCAR's Computational and Information Systems Laboratory. NCAR is sponsored by the National Science Foundation.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1026

Single cell derived mRNA signals across human kidney tumors.

Funder: Department of HealthTumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer