Ewan A. Gibb

The functional role of long non-coding RNA in human carcinomas

Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer paradigm demonstrating potential roles in both oncogenic and tumor suppressive pathways. These novel genes are frequently aberrantly expressed in a variety of human cancers, however the biological functions of the vast majority remain unknown. Recently, evidence has begun to accumulate describing the molecular mechanisms by which these RNA species function, providing insight into the functional roles they may play in tumorigenesis. In this review, we highlight the emerging functional role of lncRNAs in human cancer.

Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival

Somatic missense mutations can initiate tumorogenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. Tumor genome analysis has revealed extreme heterogeneity among tumor missense mutation profiles, but their relevance to tumor immunology and patient outcomes has awaited comprehensive evaluation. Here, for 515 patients from six tumor sites, we used RNA-seq data from The Cancer Genome Atlas to identify mutations that are predicted to be immunogenic in that they yielded mutational epitopes presented by the MHC proteins encoded by each patients autologous HLA-A alleles. Mutational epitopes were associated with increased patient survival. Moreover, the corresponding tumors had higher CTL content, inferred from CD8A gene expression, and elevated expression of the CTL exhaustion markers PDCD1 and CTLA4. Mutational epitopes were very scarce in tumors without evidence of CTL infiltration. These findings suggest that the abundance of predicted immunogenic mutations may be useful for identifying patients likely to benefit from checkpoint blockade and related immunotherapies.

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.

Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy

BACKGROUND An early report on the molecular subtyping of muscle-invasive bladder cancer (MIBC) by gene expression suggested that response to neoadjuvant chemotherapy (NAC) varies by subtype. OBJECTIVE To investigate the ability of molecular subtypes to predict pathological downstaging and survival after NAC. DESIGN, SETTING, AND PARTICIPANTS Whole transcriptome profiling was performed on pre-NAC transurethral resection specimens from 343 patients with MIBC. Samples were classified according to four published molecular subtyping methods. We developed a single-sample genomic subtyping classifier (GSC) to predict consensus subtypes (claudin-low, basal, luminal-infiltrated and luminal) with highest clinical impact in the context of NAC. Overall survival (OS) according to subtype was analyzed and compared with OS in 476 non-NAC cases (published datasets). INTERVENTION Gene expression analysis was used to assign subtypes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Receiver-operating characteristics were used to determine the accuracy of GSC. The effect of GSC on survival was estimated by Cox proportional hazard regression models. RESULTS AND LIMITATIONS The models generated subtype calls in expected ratios with high concordance across subtyping methods. GSC was able to predict four consensus molecular subtypes with high accuracy (73%), and clinical significance of the predicted consensus subtypes could be validated in independent NAC and non-NAC datasets. Luminal tumors had the best OS with and without NAC. Claudin-low tumors were associated with poor OS irrespective of treatment regimen. Basal tumors showed the most improvement in OS with NAC compared with surgery alone. The main limitations of our study are its retrospective design and comparison across datasets. CONCLUSIONS Molecular subtyping may have an impact on patient benefit to NAC. If validated in additional studies, our results suggest that patients with basal tumors should be prioritized for NAC. We discovered the first single-sample classifier to subtype MIBC, which may be suitable for integration into routine clinical practice. PATIENT SUMMARY Different molecular subtypes can be identified in muscle-invasive bladder cancer. Although cisplatin-based neoadjuvant chemotherapy improves patient outcomes, we identified that the benefit is highest in patients with basal tumors. Our newly discovered classifier can identify these molecular subtypes in a single patient and could be integrated into routine clinical practice after further validation.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

Summary Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

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.

Activation of an endogenous retrovirus-associated long non-coding RNA in human adenocarcinoma

BackgroundLong non-coding RNAs (lncRNAs) are emerging as molecules that significantly impact many cellular processes and have been associated with almost every human cancer. Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs). ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency. The aberrant regulation of ERVs associated with lncRNAs (ERV-lncRNAs), coupled with the diverse range of lncRNA functions, creates significant potential for ERV-lncRNAs to impact cancer biology.MethodsWe used RNA-seq analysis to identify and profile the expression of a novel lncRNA in six large cohorts, including over 7,500 samples from The Cancer Genome Atlas (TCGA).ResultsWe identified the tumor-specific expression of a novel lncRNA that we have named Endogenous retroViral-associated ADenocarcinoma RNA or ‘EVADR’, by analyzing RNA-seq data derived from colorectal tumors and matched normal control tissues. Subsequent analysis of TCGA RNA-seq data revealed the striking association of EVADR with adenocarcinomas, which are tumors of glandular origin. Moderate to high levels of EVADR were detected in 25 to 53% of colon, rectal, lung, pancreas and stomach adenocarcinomas (mean = 30 to 144 FPKM), and EVADR expression correlated with decreased patient survival (Cox regression; hazard ratio = 1.47, 95% confidence interval = 1.06 to 2.04, P = 0.02). In tumor sites of non-glandular origin, EVADR expression was detectable at only very low levels and in less than 10% of patients. For EVADR, a MER48 ERV element provides an active promoter to drive its transcription. Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR. The sequence and structure of the EVADR locus is highly conserved among Old World monkeys and apes but not New World monkeys or prosimians, where the MER48 insertion is absent. Conservation of the EVADR locus suggests a functional role for this novel lncRNA in humans and our closest primate relatives.ConclusionsOur results describe the specific activation of a highly conserved ERV-lncRNA in numerous cancers of glandular origin, a finding with diagnostic, prognostic and therapeutic implications.

The North American bullfrog draft genome provides insight into hormonal regulation of long noncoding RNA

Frogs play important ecological roles, and several species are important model organisms for scientific research. The globally distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Unfortunately, there are currently no genomic resources for the former, important group of amphibians. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines. We report a 5.8 Gbp (NG50 = 69 kbp) genome assembly of a representative North American bullfrog (Rana [Lithobates] catesbeiana). The genome contains over 22,000 predicted protein-coding genes and 6,223 candidate long noncoding RNAs (lncRNAs). RNA-Seq experiments show thyroid hormone causes widespread transcriptional change among protein-coding and putative lncRNA genes. This initial bullfrog draft genome will serve as a key resource with broad utility including amphibian research, developmental biology, and environmental research.The globally-distributed Ranidae (true frogs) are the largest frog family. Here, Hammond et al. present a draft genome of the North American bullfrog, Rana (Lithobates) catesbeiana, as a foundation for future understanding of true frog genetics as amphibian species face difficult environmental challenges.

Aberrant Expression of Long Noncoding RNAs in Cervical Intraepithelial Neoplasia

Objective Long noncoding RNAs (lncRNAs) are a unique class of messenger RNA–like transcripts of at least 200 nucleotides in length with no significant protein-coding capacity. Aberrant lncRNA expression is emerging as a major component of the cancer transcriptome. Here, we sought to determine if differential lncRNA expression is a feature of the human cervical intraepithelial neoplasia (CIN) transcriptome. Methods Sequence data were derived from 16 long serial analyses of gene expression (L-SAGE) libraries constructed from cervical specimens representing mild (CIN1), moderate (CIN2), and severe (CIN3) histopathologic grades of CIN. A novel lncRNA discovery pipeline was developed to query the expression of lncRNAs within the SAGE data sets. Results A total of 2,230,370 sequence tags were delineated from the 16 SAGE libraries, representing the expression of 367,482 unique tags at varying abundance. Using a novel stepwise filtering strategy, we analyzed the cervical SAGE libraries and identified the expression profiles of 1056 lncRNAs in the human cervix. We present the first lncRNA expression profile derived from nonneoplastic cervical tissue and establish that changes in lncRNA expression do occur in cervical intraepithelial lesions. Our analysis also shows statistically significant aberrant expression of lncRNAs in the 3 CIN grades, suggesting that these unique noncoding RNA transcripts may contribute to the development and progression of precursor lesions. Conclusions Through the analysis of L-SAGE libraries constructed from cervical specimens, we provide the first lncRNA expression profile of the cervix and demonstrate aberrant expression in early-stage neoplasia.

Stiffness of pancreatic cancer cells is associated with increased invasive potential

Metastasis is a fundamentally physical process in which cells are required to deform through narrow gaps as they invade surrounding tissues and transit to distant sites. In many cancers, more invasive cells are more deformable than less invasive cells, but the extent to which mechanical phenotype, or mechanotype, can predict disease aggressiveness in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here we investigate the invasive potential and mechanical properties of immortalized PDAC cell lines derived from primary tumors and a secondary metastatic site, as well as noncancerous pancreatic ductal cells. To investigate how invasive behavior is associated with cell mechanotype, we flow cells through micron-scale pores using parallel microfiltration and microfluidic deformability cytometry; these results show that the ability of PDAC cells to passively transit through pores is only weakly correlated with their invasive potential. We also measure the Youngs modulus of pancreatic ductal cells using atomic force microscopy, which reveals that there is a strong association between cell stiffness and invasive potential in PDAC cells. To determine the molecular origins of the variability in mechanotype across our PDAC cell lines, we analyze RNAseq data for genes that are known to regulate cell mechanotype. Our results show that vimentin, actin, and lamin A are among the most differentially expressed mechanoregulating genes across our panel of PDAC cell lines, as well as a cohort of 38 additional PDAC cell lines. We confirm levels of these proteins across our cell panel using immunoblotting, and find that levels of lamin A increase with both invasive potential and Youngs modulus. Taken together, we find that stiffer PDAC cells are more invasive than more compliant cells, which challenges the paradigm that decreased cell stiffness is a hallmark of metastatic potential.