Identification of DHX33 as a mediator of rRNA synthesis and cell growth

In this report, we employed a lentiviral RNA interference screen to discover nucleolar DEAD/DEAH-box helicases involved in RNA polymerase I (Pol I)-mediated transcriptional activity. Our screen identified DHX33 as an important modulator of 47S rRNA transcription. We show that DHX33 is a cell cycle-regulated nucleolar protein that associates with ribosomal DNA (rDNA) loci, where it interacts with the RNA Pol I transcription factor upstream binding factor (UBF). DHX33 knockdown decreased the association of Pol I with rDNA and caused a dramatic decrease in levels of rRNA synthesis. Wild-type DHX33 overexpression, but not a DNA binding-defective mutant, enhanced 47S rRNA synthesis by promoting the association of RNA polymerase I with rDNA loci. In addition, an NTPase-defective DHX33 mutant (K94R) acted as a dominant negative mutant, inhibiting endogenous rRNA synthesis. Moreover, DHX33 deficiency in primary human fibroblasts triggered a nucleolar p53 stress response, resulting in an attenuation of proliferation. Thus, we show the mechanistic importance of DHX33 in rRNA transcription and proliferation

ARF and p53 coordinate tumor suppression of an oncogenic IFN-β-STAT1-ISG15 signaling axis

SummaryThe ARF and p53 tumor suppressors are thought to act in a linear pathway to prevent cellular transformation in response to various oncogenic signals. Here, we show that loss of p53 leads to an increase in ARF protein levels, which function to limit the proliferation and tumorigenicity of p53-deficient cells by inhibiting an IFN-β-STAT1-ISG15 signaling axis. Human triple-negative breast cancer (TNBC) tumor samples with coinactivation of p53 and ARF exhibit high expression of both STAT1 and ISG15, and TNBC cell lines are sensitive to STAT1 depletion. We propose that loss of p53 function and subsequent ARF induction creates a selective pressure to inactivate ARF and propose that tumors harboring coinactivation of ARF and p53 would benefit from therapies targeted against STAT1 and ISG15 activation

Analytical performance of an immunoprofiling assay based on RNA models

As immuno-oncology drugs grow more popular in the treatment of cancer, better methods are needed to quantify the tumor immune cell component to determine which patients are most likely to benefit from treatment. Methods such as flow cytometry can accurately assess the composition of infiltrating immune cells; however, they show limited use in formalin-fixed, paraffin-embedded (FFPE) specimens. This article describes a novel hybrid-capture RNA sequencing assay, ImmunoPrism, that estimates the relative percentage abundance of eight immune cell types in FFPE solid tumors. Immune health expression models were generated using machine learning methods and used to uniquely identify each immune cell type using the most discriminatively expressed genes. The analytical performance of the assay was assessed using 101 libraries from 40 FFPE and 32 fresh-frozen samples. With defined samples, ImmunoPrism had a precision of ±2.72%, a total error of 2.75%, and a strong correlation (

PDE7B is a novel, prognostically significant mediator of glioblastoma growth whose expression is regulated by endothelial cells.

Cell-cell interactions between tumor cells and constituents of their microenvironment are critical determinants of tumor tissue biology and therapeutic responses. Interactions between glioblastoma (GBM) cells and endothelial cells (ECs) establish a purported cancer stem cell niche. We hypothesized that genes regulated by these interactions would be important, particularly as therapeutic targets. Using a computational approach, we deconvoluted expression data from a mixed physical co-culture of GBM cells and ECs and identified a previously undescribed upregulation of the cAMP specific phosphodiesterase PDE7B in GBM cells in response to direct contact with ECs. We further found that elevated PDE7B expression occurs in most GBM cases and has a negative effect on survival. PDE7B overexpression resulted in the expansion of a stem-like cell subpopulation in vitro and increased tumor growth and aggressiveness in an in vivo intracranial GBM model. Collectively these studies illustrate a novel approach for studying cell-cell interactions and identifying new therapeutic targets like PDE7B in GBM

Genes differentially expressed by co-culture of U87 and HBMECs.

<p>*All genes with a Benjamini-Hochberg corrected p-value<0.05 and greater than a two-fold change are shown. Fold change was calculated as: (Co-culture dataset/Computational dataset) for genes with increased expression upon co-culture, or as: −(1/(Co-culture dataset/Computational dataset)) for genes downregulated.</p><p>Genes differentially expressed by co-culture of U87 and HBMECs.</p

Expression of PDE7B increases tumor vascularity and invasiveness.

<p>(<b>A</b>) H&E staining of representative U87-GL+PDE7B(WT) tumor, with tumor tissue outlined in black. Scale bar = 1 mm (<b>B</b>) H&E staining of representative U87-GL+PDE7B(H217Q) tumor, with tumor tissue outlined in black. Scale bar = 1 mm (<b>C</b>) Endoglin staining of a representative U87-GL+PDE7B(H217Q) tumor illustrates usual pattern of U87 growth. Black arrows demarcate tumor-surrounding brain boundary. Tumor cells are not detected in the surrounding brain and there is no angiogenic response in the surrounding brain. Scale bar = 300 µm. (<b>D</b>) Immunolabelling for Endoglin of a U87-GL+PDE7B(WT) xenograft revealed a highly invasive tumor leading edge with tumor cells evident in the surrounding brain accompanied by a robust angiogenic response (asterisks). Scale bar = 300 µm.</p

Specification of the cell of origin for expression changes by qRT-PCR validation of differentially expressed genes using conditioned media swap.

<p>Conditioned medias were Serum Free DMEM (SF DMEM), SF DMEM conditioned by HBMECs (HBMEC), SF DMEM conditioned by U87 cells (U87 MG), or SF DMEM conditioned by a co-culture of HBMECs and U87s. HBMECs (left column) or U87s (right column) were grown in one of the respective conditioned media types for 48 hours, RNA was isolated, and qRT-PCR was performed for CXCL6, CXCL1 and THBS1. Expression of CXCL6 and CXCL1 specifically changed in the HBMECs in response to U87 or co-culture CM, while changes in THBS1 expression were limited to the U87 cells and occurred most significantly in response to co-culture CM. Expression is in arbitrary quantification units calculated by performing standard delta-delta C(t) while setting GAPDH expression to an arbitrary level of 10,000. N = 3 for all sets, significance by Student’s t-test.</p

Sample preparation and computational deconvolution of the mixed culture RNA signal.

<p>(<b>A</b>) Flow chart of sample preparation from culture to gathering RNA and computational analysis. (<b>B</b>) The percentage of each monoculture to use for the control dataset was determined by creating one thousand test datasets, each with a different amount of U87 or HBMEC signal, and then calculating the Pearson correlation coefficient between each of these datasets and the measured co-culture dataset.</p

PDE7B expression is correlated with Survival.

<p>(<b>A</b>) Data from NCI’s Rembrandt database indicates that expression of PDE7B at levels two-fold lower than median is strongly correlated with improved survival compared to median levels of expression in all glioma samples. p-value = 2.55E-5 (<b>B</b>) Oncomine’s Murat et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107397#pone.0107397-Murat1" target="_blank">[28]</a> dataset showing that in GBM patients those who were alive at 5 years had very much lower levels of PDE7B than those who had succumb to disease at that point (p-value = 2.56E-9).</p