Greg L. Stewart

Human Cancer Long Non-Coding RNA Transcriptomes

Once thought to be a part of the ‘dark matter’ of the genome, long non-coding RNAs (lncRNAs) are emerging as an integral functional component of the mammalian transcriptome. LncRNAs are a novel class of mRNA-like transcripts which, despite no known protein-coding potential, demonstrate a wide range of structural and functional roles in cellular biology. However, the magnitude of the contribution of lncRNA expression to normal human tissues and cancers has not been investigated in a comprehensive manner. In this study, we compiled 272 human serial analysis of gene expression (SAGE) libraries to delineate lncRNA transcription patterns across a broad spectrum of normal human tissues and cancers. Using a novel lncRNA discovery pipeline we parsed over 24 million SAGE tags and report lncRNA expression profiles across a panel of 26 different normal human tissues and 19 human cancers. Our findings show extensive, tissue-specific lncRNA expression in normal tissues and highly aberrant lncRNA expression in human cancers. Here, we present a first generation atlas for lncRNA profiling in cancer.

Long non-coding RNAs are expressed in oral mucosa and altered in oral premalignant lesions

Oral epithelial dysplasias are believed to progress through a series of histopathological stages; from mild to severe dysplasia, to carcinoma in situ, and finally to invasive OSCC. Underlying this change in histopathological grade are gross chromosome alterations and changes in gene expression of both protein-coding genes and non-coding RNAs. Recent papers have described associations of aberrant expression of microRNAs, one class of non-coding RNAs, with oral cancer. However, expression profiling of long non-coding RNAs (lncRNAs) has not been reported. Long non-coding RNAs are a novel class of mRNA-like transcripts with no protein coding capacity, but with a variety of functions including roles in epigenetics and gene regulation. In recent reports, the aberrant expression of lncRNAs has been associated with human cancers, suggesting a critical role in tumorigenesis. Here, we present the first long non-coding RNA expression map for the human oral mucosa. We describe the expression of 325 long non-coding RNAs, suggesting lncRNA expression contributes significantly to the oral transcriptome. Intriguingly, ∼60% of the detected lncRNAs show aberrant expression in oral premalignant lesions. A number of these lncRNAs have been previously associated with other human cancers.

MicroRNA Gene Dosage Alterations and Drug Response in Lung Cancer

Chemotherapy resistance is a key contributor to the dismal prognoses for lung cancer patients. While the majority of studies have focused on sequence mutations and expression changes in protein-coding genes, recent reports have suggested that microRNA (miRNA) expression changes also play an influential role in chemotherapy response. However, the role of genetic alterations at miRNA loci in the context of chemotherapy response has yet to be investigated. In this study, we demonstrate the application of an integrative, multidimensional approach in order to identify miRNAs that are associated with chemotherapeutic resistance and sensitivity utilizing publicly available drug response, miRNA loci copy number, miRNA expression, and mRNA expression data from independent resources. By instigating a logical stepwise strategy, we have identified specific miRNAs that are associated with resistance to several chemotherapeutic agents and provide a proof of principle demonstration of how these various databases may be exploited to derive relevant pharmacogenomic results.

Deregulation of small non-coding RNAs at the DLK1-DIO3 imprinted locus predicts lung cancer patient outcome

Deregulation of the imprinted DLK1-DIO3 locus at chromosome 14q32.1-14q32.31 has been associated with developmental and respiratory disorders, including cancer. In lung cancer, deregulation of imprinting at DLK1-DIO3 was recently described in smokers. Deregulated expression of a microRNA (miRNA) cluster mapping to this locus was also associated with patient outcome, suggesting the importance of this locus to lung cancer disease phenotypes. The DLK1-DIO3 locus is complex, and encodes several protein-coding genes, in addition to long and short non-coding RNAs. While the role of miRNAs is established, the biological importance of another relevant class of small RNAs, PIWI-interacting RNAs (piRNAs), has not been investigated. When somatically expressed, piRNAs regulate gene transcription through DNA methylation. Interestingly, their expression patterns have been observed to be altered in cancer and correlated with patient outcome. Here, we characterize the somatic expression of piRNAs encoded at DLK1-DIO3 in two independent cohorts of lung adenocarcinoma and lung squamous cell carcinoma and investigate their associations with patient outcome. We find that the expression of piRNAs encoded at DLK1-DIO3 enhances the prognostic potential of small non-coding RNAs specific to this locus in predicting patient outcome, further emphasizing the importance of regulation at this locus in lung cancer.

The Novel Ubiquitin Ligase Complex, SCFFbxw4, Interacts with the COP9 Signalosome in an F-Box Dependent Manner, Is Mutated, Lost and Under-Expressed in Human Cancers

Identification of novel proteins that can potentially contribute to carcinogenesis is a requisite venture. Herein, we report the first biochemical characterization of the novel F-box and WD40 containing protein, FBXW4. We have identified interacting protein partners and demonstrated that FBXW4 is part of a ubiquitin ligase complex. Furthermore, the Fbxw4 locus is a common site of proviral insertion in a variety of retroviral insertional mutagenesis murine cancer models and Fbxw4 mRNA is highly expressed in the involuting murine mammary gland. To begin to characterize the biochemical function of Fbxw4, we used proteomic analysis to demonstrate that Fbxw4 interacts with Skp1 (SKP1), Cullin1 (CUL1), Ring-box1 (RBX1) and all components of the COP9 signalosome. All of these interactions are dependent on an intact F-box domain of Fbxw4. Furthermore, Fbxw4 is capable of interacting with ubiquitinated proteins within cells in an F-box dependent manner. Finally, we demonstrate that FBXW4 is mutated, lost and under-expressed in a variety of human cancer cell lines and clinical patient samples. Importantly, expression of FBXW4 correlates with survival of patients with non-small cell lung cancer. Taken together, we suggest that FBXW4 may be a novel tumor suppressor that regulates important cellular processes.

Oncogenomic disruptions in arsenic-induced carcinogenesis

Chronic exposure to arsenic affects more than 200 million people worldwide, and has been associated with many adverse health effects, including cancer in several organs. There is accumulating evidence that arsenic biotransformation, a step in the elimination of arsenic from the human body, can induce changes at a genetic and epigenetic level, leading to carcinogenesis. At the genetic level, arsenic interferes with key cellular processes such as DNA damage-repair and chromosomal structure, leading to genomic instability. At the epigenetic level, arsenic places a high demand on the cellular methyl pool, leading to global hypomethylation and hypermethylation of specific gene promoters. These arsenic-associated DNA alterations result in the deregulation of both oncogenic and tumour-suppressive genes. Furthermore, recent reports have implicated aberrant expression of non-coding RNAs and the consequential disruption of signaling pathways in the context of arsenic-induced carcinogenesis. This article provides an overview of the oncogenomic anomalies associated with arsenic exposure and conveys the importance of non-coding RNAs in the arsenic-induced carcinogenic process.

Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma

Pheochromocytomas (PCC) are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL) tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A) for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL) of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation.

Large-scale discovery of previously undetected microRNAs specific to human liver

MicroRNAs (miRNAs) are crucial regulators of gene expression in normal development and cellular homeostasis. While miRNA repositories contain thousands of unique sequences, they primarily contain molecules that are conserved across several tissues, largely excluding lineage and tissue-specific miRNAs. By analyzing small non-coding RNA sequencing data for abundance and secondary RNA structure, we discovered 103 miRNA candidates previously undescribed in liver tissue. While expression of some of these unannotated sequences is restricted to non-malignant tissue, downregulation of most of the sequences was detected in liver tumors, indicating their importance in the maintenance of liver homeostasis. Furthermore, target prediction revealed the involvement of the unannotated miRNA candidates in fatty-acid metabolism and tissue regeneration, which are key pathways in liver biology. Here, we provide a comprehensive analysis of the undiscovered liver miRNA transcriptome, providing new resources for a deeper exploration of organ-specific biology and disease.

Expanding the miRNA Transcriptome of Human Kidney and Renal Cell Carcinoma

Despite advancements in therapeutic strategies, diagnostic and prognostic molecular markers of kidney cancer remain scarce, particularly in patients who do not harbour well-defined driver mutations. Recent evidence suggests that a large proportion of the human noncoding transcriptome has escaped detection in early genomic explorations. Here, we undertake a large-scale analysis of small RNA-sequencing data from both clear cell renal cell carcinoma (ccRCC) and nonmalignant samples to generate a robust set of miRNAs that remain unannotated in kidney tissues. We find that these novel kidney miRNAs are also expressed in renal cancer cell lines. Moreover, these sequences are differentially expressed between ccRCC and matched nonmalignant tissues, implicating their involvement in ccRCC biology and potential utility as tumour-specific markers of disease. Indeed, we find some of these miRNAs to be significantly associated with patient survival. Finally, target prediction and subsequent pathway analysis reveals that miRNAs previously unannotated in kidney tissues may target genes involved in ccRCC tumourigenesis and disease biology. Taken together, our results represent a new resource for the study of kidney cancer and underscore the need to characterize the unexplored areas of the transcriptome.

Abstract A26: DNA copy number alterations deregulate expression of miRNAs in lung adenocarcinoma

Background: Lung cancer represents an enormous health burden, representing the most common cause of cancer death worldwide, with a five-year survival of less than 15%. microRNAs (miRNAs) have emerged as major players in lung cancer oncogenesis, displaying both oncogenic and tumor suppressive functions. DNA copy number (CN) amplification of oncogenes is a major molecular mechanism driving cancer, and like protein coding genes, CN alterations can influence miRNA expression levels. We hypothesize that DNA copy number gain modulates the expression of miRNAs important to cancer cell growth, and that integrative analysis can identify these cancer driving miRNAs. Methods: Global gene dosage profiles for 46 lung adenocarcinoma and paired adjacent non-malignant tissues were generated by array comparative genomic hybridization. miRNA sequencing analysis was performed on this same panel of tumors and matched non-malignant tissues using Illumina GAXII small RNA sequencing technologies. CN and expression were correlated for each miRNA (Spearmans correlation >0.3, p<0.05) and expression of each significant miRNA was compared between tumors with and without CN gain (U-test p<0.05). To select for miRNAs that most likely play a role in cancer we compared the expression between tumor and matched normal and selected for miRNAs with a fold change of at least 2 in tumors as compared to normal tissues. Results: We identified a panel of miRNAs in lung adenocarcinoma whose expression deregulation was associated with CN alteration. These miRNAs were found to be gained in >15% of tumors and underwent at least a 2 fold greater increase in expression in tumors compared to matched normal tissues. This study identified several miRNAs known to play a role in cancer including miR-141, known to be highly upregulated in lung and ovarian cancer, and miR-301a, upregulated in invasive early cervical cancer. In addition we identified several novel miRNAs that have not yet been implicated in cancer. Conclusion: Here we identify a panel of miRNAs that are selectively gained at the genomic level to alter expression in lung adenocarcinoma. Several of these miRNAs have been previously shown to have oncogenic properties in many cancer types including lung cancer and may be potentially useful as therapeutic targets. In addition we identify several miRNAs that have not been previously reported in lung cancer. Future characterization of these miRNAs may lead to increased knowledge of adenocarcinoma oncogenesis. Citation Format: Greg L. Stewart, Katey SS Enfield, Stephen Lam, Wan Lam. DNA copy number alterations deregulate expression of miRNAs in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A26.