Sanjay Chandriani

Array-Based Transcript Profiling and Limiting-Dilution Reverse Transcription-PCR Analysis Identify Additional Latent Genes in Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is a B-lymphotropic herpesvirus strongly linked to both lymphoproliferative diseases and Kaposis sarcoma. The viral latency program of KSHV is central to persistent infection and plays important roles in the pathogenesis of KSHV-related tumors. Up to six polypeptides and 18 microRNAs are known to be expressed in latency, but it is unclear if all major latency genes have been identified. Here, we have employed array-based transcript profiling and limiting-dilution reverse transcription-PCR (RT-PCR) methodologies to explore this issue in several KSHV-infected cell lines. Our results show that RNAs encoding the K1 protein are found at low levels in most latently infected cell lines. The gene encoding v-IL-6 is also expressed as a latent transcript in some contexts. Both genes encode powerful signaling molecules with particular relevance to B cell biology: K1 mimics signaling through the B cell receptor, and v-IL-6 promotes B cell survival. These data resolve earlier controversies about K1 and v-IL-6 expression and indicate that, in addition to core latency genes, some transcripts can be expressed in KSHV latency in a context-dependent manner.

Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis

Background There is microscopic spatial and temporal heterogeneity of pathological changes in idiopathic pulmonary fibrosis (IPF) lung tissue, which may relate to heterogeneity in pathophysiological mediators of disease and clinical progression. We assessed relationships between gene expression patterns, pathological features, and systemic biomarkers to identify biomarkers that reflect the aggregate disease burden in patients with IPF. Methods Gene expression microarrays (N=40 IPF; 8 controls) and immunohistochemical analyses (N=22 IPF; 8 controls) of lung biopsies. Clinical characterisation and blood biomarker levels of MMP3 and CXCL13 in a separate cohort of patients with IPF (N=80). Results 2940 genes were significantly differentially expressed between IPF and control samples (|fold change| >1.5, p<0.05). Two clusters of co-regulated genes related to bronchiolar epithelium or lymphoid aggregates exhibited substantial heterogeneity within the IPF population. Gene expression in bronchiolar and lymphoid clusters corresponded to the extent of bronchiolisation and lymphoid aggregates determined by immunohistochemistry in adjacent tissue sections. Elevated serum levels of MMP3, encoded in the bronchiolar cluster, and CXCL13, encoded in the lymphoid cluster, corresponded to disease severity and shortened survival time (p<10−7 for MMP3 and p<10−5 for CXCL13; Cox proportional hazards model). Conclusions Microscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns related to bronchiolisation and lymphoid aggregates. MMP3 and CXCL13 are systemic biomarkers that reflect the aggregate burden of these pathological features across total lung tissue. These biomarkers may have clinical utility as prognostic and/or surrogate biomarkers of disease activity in interventional studies in IPF.

Identification of a previously undescribed divergent virus from the Flaviviridae family in an outbreak of equine serum hepatitis

Theilers disease is an acute hepatitis in horses that is associated with the administration of equine blood products; its etiologic agent has remained unknown for nearly a century. Here, we used massively parallel sequencing to explore samples from a recent Theilers disease outbreak. Metatranscriptomic analysis of the short sequence reads identified a 10.5-kb sequence from a previously undescribed virus of the Flaviviridae family, which we designate “Theilers disease-associated virus” (TDAV). Phylogenetic analysis clusters TDAV with GB viruses of the recently proposed Pegivirus genus, although it shares only 35.3% amino acid identity with its closest relative, GB virus D. An epidemiological survey of additional horses from three separate locations supports an association between TDAV infection and acute serum hepatitis. Experimental inoculation of horses with TDAV-positive plasma provides evidence that several weeks of viremia preceded liver injury and that liver disease may not be directly related to the level of viremia. Like hepatitis C virus, the best characterized Flaviviridae species known to cause hepatitis, we find TDAV is capable of efficient parenteral transmission, engendering acute and chronic infections associated with a diversity of clinical presentations ranging from subclinical infection to clinical hepatitis.

The Lytic Transcriptome of Kaposi's Sarcoma-Associated Herpesvirus Reveals Extensive Transcription of Noncoding Regions, Including Regions Antisense to Important Genes

ABSTRACT Genomewide analyses of the mammalian transcriptome have revealed that large tracts of sequence previously annotated as noncoding are frequently transcribed and give rise to stable RNA. Although the transcription of individual genes of the Kaposis sarcoma-associated herpesvirus (KSHV) has been well studied, little is known of the architecture of the viral transcriptome on a genomewide scale. Here we have employed a genomewide tiling array to examine the lytic transcriptome of the Kaposis sarcoma-associated herpesvirus, KSHV. Our results reveal that during lytic growth (but not during latency), there is extensive transcription from noncoding regions, including both intergenic regions and, especially, noncoding regions antisense to known open reading frames (ORFs). Several of these transcripts have been characterized in more detail, including (i) a 10-kb RNA antisense to the major latency locus, including many of its microRNAs as well as its ORFs; (ii) a 17-kb RNA antisense to numerous ORFs at the left-hand end of the genome; and (iii) a 0.7-kb RNA antisense to the viral homolog of interleukin-6 (vIL-6). These studies indicate that the lytic herpesviral transcriptome resembles a microcosm of the host transcriptome and provides a useful system for the study of noncoding RNAs.

Host transcript accumulation during lytic KSHV infection reveals several classes of host responses.

Lytic infection by Kaposis sarcoma-associated herpesvirus (KSHV) is associated with an extensive shutoff of host gene expression, mediated chiefly by accelerated mRNA turnover due to expression of the viral SOX protein. We have previously identified a small number of host mRNAs that can escape SOX-mediated degradation. Here we present a detailed, transcriptome-wide analysis of host shutoff, with careful microarray normalization to allow rigorous determination of the magnitude and extent of transcript loss. We find that the extent of transcript reduction represents a continuum of susceptibilities of transcripts to virus-mediated shutoff. Our results affirm that the levels of over 75% of host transcripts are substantially reduced during lytic infection, but also show that another ∼20% of cellular mRNAs declines only slightly (less than 2-fold) during the course of infection. Approximately 2% of examined cellular genes are strongly upregulated during lytic infection, most likely due to transcriptional induction of mRNAs that display intrinsic SOX-resistance.

Endogenously Expressed IL-13Rα2 Attenuates IL-13–Mediated Responses but Does Not Activate Signaling in Human Lung Fibroblasts

IL-13 can bind to two distinct receptors: a heterodimer of IL-13Rα1/IL-4Rα and IL-13Rα2. Whereas IL-13Rα1/IL-4Rα engagement by IL-13 leads to the activation of STAT6, the molecular events triggered by IL-13 binding to IL-13Rα2 remain incompletely understood. IL-4 can bind to and signal through the IL-13Rα1/IL-4Rα complex but does not interact with IL-13Rα2. Idiopathic pulmonary fibrosis is a progressive and generally fatal parenchymal lung disease of unknown etiology with no current pharmacologic treatment options that substantially prolong survival. Preclinical models of fibrotic diseases have implicated IL-13 activity on multiple cell types, including macrophages and fibroblasts, in initiating and perpetuating pathological fibrosis. In this study, we show that IL-13, IL-4, IL-13Rα2, and IL-13–inducible target genes are expressed at significantly elevated levels in lung tissue from patients with idiopathic pulmonary fibrosis compared with control lung tissue. IL-4 and IL-13 induce virtually identical transcriptional responses in human monocytes, macrophages, and lung fibroblasts. IL-13Rα2 expression can be induced in lung fibroblasts by IL-4 or IL-13 via a STAT6-dependent mechanism, or by TNF-α via a STAT6-independent mechanism. Endogenously expressed IL-13Rα2 decreases, but does not abolish, sensitivity of lung fibroblasts to IL-13 and does not affect sensitivity to IL-4. Genome-wide transcriptional analyses of lung fibroblasts stimulated with IL-13 in the presence of Abs that selectively block interactions of IL-13 with IL-13Rα1/IL-4Rα or IL-13Rα2 show that endogenously expressed IL-13Rα2 does not activate any unique IL-13–mediated gene expression patterns, confirming its role as a decoy receptor for IL-13 signaling.

CXCL14 is a candidate biomarker for Hedgehog signalling in idiopathic pulmonary fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is associated with aberrant expression of developmental pathways, including Hedgehog (Hh). As Hh signalling contributes to multiple pro-fibrotic processes, Hh inhibition may represent a therapeutic option for IPF. However, no non-invasive biomarkers are available to monitor lung Hh activity. Methods We assessed gene and protein expression in IPF and control lung biopsies, mouse lung, fibroblasts stimulated in vitro with sonic hedgehog (SHh), and plasma in IPF patients versus controls, and cancer patients before and after treatment with vismodegib, a Hh inhibitor. Results Lung tissue from IPF patients exhibited significantly greater expression of Hh-related genes versus controls. The gene most significantly upregulated in both IPF lung biopsies and fibroblasts stimulated in vitro with SHh was CXCL14, which encodes a soluble secreted chemokine whose expression is inhibited in vitro by the addition of vismodegib. CXCL14 expression was induced by SHh overexpression in mouse lung. Circulating CXCL14 protein levels were significantly higher in plasma from IPF patients than controls. In cancer patients, circulating CXCL14 levels were significantly reduced upon vismodegib treatment. Conclusions CXCL14 is a systemic biomarker that could be used to identify IPF patients with increased Hh pathway activity and monitor the pharmacodynamic effects of Hh antagonist therapy in IPF. Trial registration number Post-results, NCT00968981.

Abstract B54: Molecular basis of oncogene cooperation in liver cancer

Combinations of genetic alterations in cancer genes lead to aberrant signaling properties and drive cellular transformation. Whether each oncogene gives rise to unique signals or combinations of oncogenes lead to new properties of cancer cells remains unclear. Using cell-based and animal model systems it has been shown that certain oncogenes can cooperate with each other to induce a fully malignant tumor phenotype. In addition, the combination of oncogenes and the extent of cooperation between them to give rise to a tumor may vary in different cancers. Cooperativity between MYC and RAS oncogenes has been observed in different cancer types. MYC is often overexpressed and sometimes amplified in cancer, while, activating RAS mutations occur in about 30% human cancers. Hepatocellular carcinoma (HCC) is among the four leading causes of cancer-related death. Currently, except for early surgical intervention or liver transplantation, no effective cures exist. Overexpression of both MYC and activated HRAS together in primary human liver tumors has been associated with aggressive forms of liver cancer with an especially poor prognosis. However, a systematic approach to understanding MYC and RAS cooperation in liver cancer has not yet been undertaken. We have discovered that transgenic mice overexpressing MYC, RAS or both MYC and RAS in their liver, give rise to distinct liver tumors. MYC and RAS coexpression led to faster tumor development and decreased survival. To gain a comprehensive view of the molecular changes that contribute to MYC and RAS oncogene cooperation in de novo liver tumors, we combined the conditional transgenic models of liver cancer with global gene expression, signaling pathway analyses and functional studies. Comparisons were made to clinically annotated human tumors and key pathways validated in cell lines and animal models. We show that tumors driven by either oncogene or a combination of both are phenotypically and molecularly distinct from each other with differential regulation of multiple genes and pathways. Our work suggests that MYC and RAS cooperate through specific genes, such as CCNE1 and multiple signaling pathways including proliferation, apoptosis, angiogenesis and multiple metabolic pathways, to contribute to liver cancer development. We also show molecular alterations across signaling pathways are comparable between mouse and human liver cancers. Primary human liver tumors can be segregated into distinct subsets based on differentially regulated gene sets from cooperating MYC and RAS tumors. These results indicate that molecular changes underlying MYC and RAS cooperation may serve as ideal molecular markers and therapeutic targets for liver cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B54.