Patricia M. Kessler

A gene expression signature for relapse of primary wilms tumors

Anaplastic histology and metastasis are each associated with higher relapse and mortality rates in Wilms tumor patients. However, not all anaplastic tumors relapse and some nonanaplastic tumors relapse unexpectedly. To identify more accurate early prognostic indicators, we analyzed expression of 4,900 cancer-related genes in 26 primary Wilms tumors. This analysis revealed that expression of a set of four genes predicts future relapse of primary Wilms tumors with high accuracy, independent of anaplasia. Random permutation testing of this prognostic gene expression signature yielded P = 0.003. Real-time reverse transcription-PCR analysis of the four genes in an independent primary tumor set resulted in correct prediction of future relapse with an accuracy of 92%. One of the four genes in the prognostic signature, CCAAT/enhancer binding protein beta (C/EBPB), is expressed at higher levels in both primary relapsing tumors and metastatic tumors than in primary nonrelapsing tumors. Short interfering RNA-mediated down-regulation of C/EBPB expression in WiT49, a cell line derived from a metastatic Wilms tumor, resulted in spontaneous apoptosis. These findings suggest that C/EBPB is a critical survival factor for Wilms tumor cells and that its expression contributes to the prognosis of Wilms tumor patients.

Inhibition of RNase L and RNA-dependent protein kinase (PKR) by sunitinib impairs antiviral innate immunity.

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC50 values of 1.4 and 0.3 μm, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.

Transcript profiling of Wilms tumors reveals connections to kidney morphogenesis and expression patterns associated with anaplasia.

Anaplasia (unfavorable histology) is associated with therapy resistance and poor prognosis of Wilms tumor, but the molecular basis for this phenotype is unclear. Here, we used a cDNA array with 9240 clones relevant to cancer biology and/or kidney development to examine the expression profiles of 54 Wilms tumors, five normal kidneys and fetal kidney. By linking genes differentially expressed between fetal kidney and Wilms tumors to kidney morphogenesis, we found that genes expressed at a higher level in Wilms tumors tend to be expressed more in uninduced metanephrogenic mesenchyme or blastema than in their differentiated structures. Conversely, genes expressed at a lower level in Wilms tumors tend to be expressed less in uninduced metanephrogenic mesenchyme or blastema. We also identified 97 clones representing 76 Unigenes or unclustered ESTs that clearly separate anaplastic Wilms tumors from tumors with favorable histology. Genes in this set provide insight into the nature of the abnormal nuclear morphology of anaplastic tumors and may facilitate identification of molecular targets to improve their responsiveness to treatment.

Reduced expression of autotaxin predicts survival in uveal melanoma.

Aim: In an effort to identify patients with uveal melanoma at high risk of metastasis, the authors undertook correlation of gene expression profiles with histopathology data and tumour-related mortality. Methods: The RNA was isolated from 27 samples of uveal melanoma from patients who had consented to undergo enucleation, and transcripts profiled using a cDNA array comprised of sequence-verified cDNA clones representing approximately 4000 genes implicated in cancer development. Two multivariate data mining techniques—hierarchical cluster analysis and multidimensional scaling—were used to investigate the grouping structure in the gene expression data. Cluster analysis was performed with a subset of 10 000 randomly selected genes and the cumulative contribution of all the genes in making the correct grouping was recorded. Results: Hierarchical cluster analysis and multidimensional scaling revealed two distinct classes. When correlated with the data on metastasis, the two molecular classes corresponded very well to the survival data for the 27 patients. Thirty two discrete genes (corresponding to 44 probe sets) that correctly defined the molecular classes were selected. A single gene (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin) could classify the molecular types. The expression pattern was confirmed using real-time quantitative PCR. Conclusions: Gene expression profiling identifies two distinct prognostic classes of uveal melanoma. Underexpression of autotaxin in class 2 uveal melanoma with a poor prognosis needs to be explored further.

Regulation of CRABP-II expression by MycN in Wilms tumor.

Cellular retinoic acid binding protein II (CRABP-II) is overexpressed in a wide variety of cancers. Previously we have shown that CRABP-II expression levels are also elevated in neuroblastoma and Wilms tumors. To elucidate the molecular mechanisms underlying the abnormal expression of CRABP-II in Wilms tumor, we studied the expression of MycN and CRABP-II in these tumor samples. Our data revealed that CRABP-II is overexpressed in Wilms tumor compared to normal adjacent non-neoplastic tissue and its levels are even higher in late stage tumors. Its expression correlates with MycN expression in tumors. The tumors that do not express MycN have no CRABP-II expression. The expression of CRABP-II is also regulated by methylation and its promoter is unmethylated in tumors. Knockdown of MycN by small interfering RNA leads to downregulation of CRABP-II. Thus our results suggest that both MycN and DNA methylation are responsible for CRABP-II expression in pediatric tumors and demethylation of CRABP-II may be an early event in tumor development.

IN SITU EXPRESSION OF THE EARLY GROWTH RESPONSE GENE-1 DURING MURINE NEPHROGENESIS

WT1 maps to chromosome 11p13 and encodes a deoxyribonucleic acid (DNA) binding protein whose expression is necessary for normal urogenital development. The WT1 protein binds to some of the same DNA sequences as the early growth response gene-1 (EGR-1) protein, the latter being an immediate-early gene product that activates or represses transcription in a promoter and cell-specific manner. Transient transfection experiments have shown that WT1 can repress EGR-1 activated transcription from the EGR-1 promoter. To determine if WT1 is likely to be a physiologically important repressor of EGR-1 we performed ribonucleic acid (RNA) in situ hybridization of EGR-1 on sequential sagittal sections of murine embryos before and throughout nephrogenesis, and compared the results to our previous study of WT1 expression during murine embryogenesis. Prior to embryological day 9.5 WT1 messenger RNA expression is absent in the embryo proper but is expressed in the maternal uterus. With the initiation of organogenesis on embryological day 10.5 WT1 messenger RNA localizes within the pronephric and mesonephric tissues. By embryological day 11.5 the nephrogenic cord, urogenital ridge and metanephric tissue have WT1 hybridization signals and increasingly centripetal expression of WT1 in the kidney correlates with differentiation from embryological days 11.5 to 16.5. In contrast to previous reports of the tissue restricted expression of WT1, EGR-1 expression by in situ hybridization was apparent in all 3 germ layers and their derivatives throughout embryogenesis. Down-regulation of EGR-1 expression occurred in the maternal uterus as well as the metanephric blastema and its derivatives during renal development. This observation defines a spatial and temporal window during which WT1 competition for EGR-1 DNA binding sites may be involved in regulating EGR-1 expression.

The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development.

The murine double‐stranded RNA‐binding protein termed protein kinase R (PKR)‐associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR‐deficient mice, the phenotype has been attributed to PKR‐independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax−/− mice deficient in PKR, or carrying a kinase‐inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax−/− mice. Generating rax−/− mice with a kinase‐inactive mutant of PKR resulted in similar rescue, confirming that the rax−/− defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax−/− mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax−/− defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR‐regulated cyclin‐dependent kinase inhibitor p21WAF1/CIP1. These results demonstrate that PKR kinase activity is required for onset of the rax−/− phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.

Interferon regulatory factor 8 (IRF8) impairs induction of interferon induced with tetratricopeptide repeat motif (IFIT) gene family members

The chromosomally clustered interferon-induced with tetratricopeptide repeat motif (IFIT) gene family members share structural features at the gene and protein levels. Despite these similarities, different IFIT genes have distinct inducer- and cell type-specific induction patterns. Here, we investigated the mechanism for the observed differential induction of the mouse Ifit1, Ifit2, and Ifit3 genes in B cells and demonstrated that the repressive effect of the transcription factor interferon regulatory factor 8 (IRF8), which is highly expressed in B cells, played an essential role in this regulation. Although IRF8 could impair induction of all three IFIT genes following stimulation of retinoic acid-inducible gene I (RIG-I), it could selectively impair the induction of the Ifit1 gene following IFN stimulation. The above properties could be imparted to IRF8-non-expressing cells by ectopic expression of the protein. Induction of reporter genes, driven by truncated Ifit1 promoters, identified the regions that mediate the repression, and a chromatin immunoprecipitation assay revealed that more IRF8 bound to the IFN-stimulated response element of the Ifit1 gene than to those of the Ifit2 and the Ifit3 genes. Mutational analyses of IRF8 showed that its ability to bind DNA, interact with other proteins, and undergo sumoylation were all necessary to selectively repress Ifit1 gene induction in response to IFN. Our study revealed a new role for IRFs in differentially regulating the induction patterns of closely related IFN-stimulated genes that are located adjacent to one another in the mouse genome.

Constitutively Bound EGFR–Mediated Tyrosine Phosphorylation of TLR9 Is Required for Its Ability To Signal

Mammalian TLRs recognize microbial infection or cell death–associated danger signals and trigger the appropriate cellular response. These responses determine the strength and the outcome of the host–microbe interaction. TLRs are transmembrane proteins located on the plasma or the endosomal membrane. Their ectodomains recognize specific microbial or endogenous ligands, and the cytoplasmic domains interact with specific proteins to activate intracellular signaling pathways. TLR9, an endosomal TLR, is activated by endocytosed DNA. Activated TLR9 recruits the cytoplasmic adapter MyD88 and other signaling proteins to induce the synthesis of inflammatory cytokines and IFN. Uncontrolled activation of TLR9 leads to the undesired overproduction of inflammatory cytokines and consequent pathogenesis. Therefore, appropriate activation and the regulation of TLR9 signaling are critical. Tyrosine (Tyr) phosphorylation of TLR9 is essential for its activation; however, the role of specific Tyr kinases is not clear. In this article, we report that epidermal growth factor receptor (EGFR), a membrane-bound protein Tyr kinase, is essential for TLR9 signaling. Genetic ablation of EGFR or pharmacological inhibition of its kinase activity attenuates TLR9-mediated induction of genes in myeloid and nonmyeloid cell types. EGFR is constitutively bound to TLR9; upon ligand stimulation, it mediates TLR9 Tyr phosphorylation, which leads to the recruitment of MyD88, activation of the signaling kinases and transcription factors, and gene induction. In mice, TLR9-mediated liver injury and death are blocked by an EGFR inhibitor or deletion of the EGFR gene from myeloid cells, which are the major producers of inflammatory cytokines.

Discussion: In situ expression of the early growth response gene-1 during murine nephrogenesis

WT1 maps to chromosome 11p13 and encodes a deoxyribonucleic acid (DNA) binding protein whose expression is necessary for normal urogenital development. The WT1 protein binds to some of the same DNA sequences as the early growth response gene-1 (EGR-1) protein, the latter being an immediate-early gene product that activates or represses transcription in a promoter and cell-specific manner. Transient transfection experiments have shown that WT1 can repress EGR-1 activated transcription from the EGR-1 promoter. To determine if WT1 is likely to be a physiologically important repressor of EGR-1 we performed ribonucleic acid (RNA) in situ hybridization of EGR-1 on sequential sagittal sections of murine embryos before and throughout nephrogenesis, and compared the results to our previous study of WT1 expression during murine embryogenesis. Prior to embryological day 9.5 WT1 messenger RNA expression is absent in the embryo proper but is expressed in the maternal uterus. With the initiation of organogenesis on embryological day 10.5 WT1 messenger RNA localizes within the pronephric and mesonephric tissues. By embryological day 11.5 the nephrogenic cord, urogenital ridge and metanephric tissue have WT1 hybridization signals and increasingly centripetal expression of WT1 in the kidney correlates with differentiation from embryological days 11.5 to 16.5. In contrast to previous reports of the tissue restricted expression of WT1, EGR-1 expression by in situ hybridization was apparent in all 3 germ layers and their derivatives throughout embryogenesis. Down-regulation of EGR-1 expression occurred in the maternal uterus as well as the metanephric blastema and its derivatives during renal development. This observation defines a spatial and temporal window during which WT1 competition for EGR-1 DNA binding sites may be involved in regulating EGR-1 expression.