K. Enfield

An atlas of gastric PIWI-interacting RNA transcriptomes and their utility for identifying signatures of gastric cancer recurrence

The poor survival and recurrence rate in gastric adenocarcinoma highlights the need for cancer gene discovery. Towards this end, we globally assessed the expression of an emerging class of small non-coding RNAs, called PIWI-interacting RNAs (piRNAs). We analysed the transcriptomes of 358 non-malignant stomach tissue and gastric adenocarcinoma samples, and found that nearly half of the expressed piRNAs were overexpressed in tumours. Our gastric piRNA atlas showed that most piRNAs were embedded in protein-coding sequences rather than known piRNA clusters. Furthermore, we identified a three-piRNA signature associated with recurrence-free survival. In this proof-of-principle study, we demonstrate the potential clinical utility of piRNAs in gastric cancer.

HPV status is associated with altered PIWI-interacting RNA expression pattern in head and neck cancer.

OBJECTIVES As HPV-induced cases of oral malignancy increase, it is important to understand the molecular differences between HPV positive and negative head and neck squamous cell carcinoma (HNSCC). PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs aberrantly expressed in cancer. We analyzed global piRNA expression patterns to define the HNSCC piRNA transcriptome and assess whether HPV infection status associates with changes in piRNA levels. MATERIALS AND METHODS A total of 498 HNSCC small RNA sequencing libraries were acquired from the Cancer Genomics Hub (cgHUB) Data Repository and a custom sequence analysis pipeline was developed to deduce piRNA expression from raw sequencing data. Expression matrices were aligned to clinicopathological features in order to analyze piRNA expression patterns across different HNSCC groups. The association of a piRNA signature with HPV-positive patient survival was evaluated using a Cox proportional hazard model. RESULTS Analysis of piRNA levels between HNSCC and non-malignant tissues revealed distinct expression patterns, with 87 piRNAs exclusively expressed in tumor samples. HPV infection status affected the expression of 41 of these piRNAs. Eleven (26.8%) piRNAs were significantly downregulated in HPV16/18 tumors compared to other HPV types. Remarkably, expression of a combination of five-piRNAs in HPV-positive HNSCC tumors was associated with worse overall survival. CONCLUSION The expression of specific piRNAs is deregulated in HNSCC, and changes with both HPV status and type. Importantly, a five-piRNA signature is able to delineate a subset of HPV-positive HNSCC patients with poor outcome, highlighting the potential utility of piRNAs in patient management.

Abstract A21: MiR-106a and miR-106b affect growth and metastasis of lung adenocarcinoma

Introduction: MiR-106a and miR-106b are paralogs of the oncogenic miR-17~92, and have been associated with poor outcome and metastasis in several solid tumors. Their role in lung cancer is relatively unexplored. We characterized the expression of miR-106a and miR-106b in a clinical cohort of lung adenocarcinoma (AC) tumors and assessed their ability to regulate growth and metastasis in cell models. Methods: MicroRNA (miRNA) expression was deduced from small RNA sequencing data derived from clinical lung AC specimens (60 localized, 27 with lymph node invasion) and paired non-malignant tissues. MiR-106a and miR-106b overexpression vectors and controls were stably transfected into immortalized non-malignant Human Bronchial Epithelial Cells (HBECs) and stage I AC cell lines with epithelial expression patterns by lentiviral delivery. Migration and invasion was assessed by Boyden chamber assay, while cell proliferation was assessed by BrdU incorporation assay. Expression of epithelial-to-mesenchymal transition (EMT) markers and other proteins of interest were assessed by Western Blot. Clinical associations in an external cohort were derived using publically available TCGA data. Results: MiR-106a and miR-106b were significantly overexpressed in lung AC with lymph node invasion. Overexpression of miR-106a and miR-106b significantly increased proliferation of lung AC cell lines, and was associated with decreased levels of predicted target, p21. AC cell lines displayed a marked increase in metastatic phenotypes in vitro, and were associated with increased mesenchymal and decreased epithelial markers, characteristic of EMT. Importantly, tumors with high expression of both miR-106a and miR-106b and mesenchymal marker vimentin had significantly poorer outcome. Conclusions: MiR-106a and miR-106b are overexpressed in metastatic lung AC. Lung AC cell models indicate these miRNAs are metastatic agonists, affecting the metastatic potential of cells at least in part via induction of EMT. A deeper characterization of this observation may reveal therapeutic intervention points, or, with the development of miRNA therapeutics, miR-106a/b may be promising targets to prevent or treat metastatic disease. Citation Format: Katey SS Enfield, David A. Rowbotham, Alice Holly, Christine Anderson, Kevin W. Ng, Brenda de Carvalho Minatel, Graham Dellaire, Chiara Pastrello, Igor Jurisica, Calum MacAulay, Stephen Lam, Wan L. Lam. MiR-106a and miR-106b affect growth and metastasis of lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A21.

Abstract B26: OIP5-Antisense 1, a long noncoding RNA deregulated in non-small cell lung cancer

Background: Lung cancer represents an enormous health burden, representing the most common cause of cancer death worldwide. The poor therapeutic outcome is largely due to a complex molecular background as well as late stage diagnosis, with most patients presenting unresectable local tumors, or metastatic disease. While mutations of driver genes is a well-known mechanism, approximately half of all non-small cell lung cancer (NSCLC) tumors harbor no known clinically relevant oncogenic drivers, emphasizing the need to explore alternative mechanisms such as non-coding RNAs (ncRNAs). Natural antisense transcripts (NATS) are ncRNAs that are expressed from the opposite strand of coding mRNAs. These genes overlap with, and are often involved in the regulation of their sense counterparts. NATs can recruit regulatory complexes to their transcriptional locus, leading to silencing of transcription and have recently been described in cancer to silence tumor suppressor genes. NATs are quite prevalent as it is estimated that 25-40% of genes display overlapping transcription, however only a few NATs have been characterized, emphasizing the need to explore these ncRNAs in the context of NSCLC. Hypothesis: We hypothesize NATs of NSCLC-related genes are deregulated in NSCLC. Methods: We performed RNAseq and miRNAseq on a set of 65 NSCLC tumors including 36 adenocarcinomas and 29 squamous cell carcinomas as well as matched non-malignant lung tissue. A sign rank test was used to identify NATs with significantly altered expression between tumor and matched normal. Survival analysis was done using a Cox Proportional hazard model, as well as the Kaplan-Meier method. Results: We have identified a NAT of OIP5, a lung cancer oncogene required for chromatin segregation, to be significantly underexpressed, while its sense counterpart, OIP5 mRNA, is significantly overexpressed. We also find that expression of both OIP5 and OIP5-AS1 has a significant impact on 5 year survival. These findings suggest that deregulation of OIP5 through its antisense RNA may represent a novel mechanism regulating tumor phenotypes in NSCLC. Citation Format: Greg L. Stewart, Katey SS Enfield, David A. Rowbotham, Roland Hubaux, Victor Martinez, Stephen Lam, Wan Lam. OIP5-Antisense 1, a long noncoding RNA deregulated in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B26.

Abstract A4: An expression map of long noncoding RNAs in human lung and non-small cell lung cancer

Background: Although still largely unexplored, long non-coding RNAs (lncRNAS) are emerging as an integral functional component of the human transcriptome. LncRNAs are mRNA-like transcripts of at least 200 nucleotides (nts) with no protein-coding capacity. Similar to their protein-coding counterparts, lncRNAs are frequently spliced and polyadenylated, but act at the RNA level. The range of functions described for lncRNAs is extensive, and includes key biological roles in chromatin remodeling, alternative splicing and mRNA degradation. Given their biological functions, dysregulation of lncRNAs is rising as an important feature of many disorders, including malignant transformation. However, the extent of the contribution of differential lncRNA expression to normal lung tissue and lung cancer has not been investigated in a comprehensive manner. Hypothesis: We hypothesized that lncRNAs are expressed in a lung tissue-specific manner and that non-small-cell lung cancer (NSCLC) exhibits aberrant lncRNA expression patterns. Methods: Serial Analysis of Gene Expression (SAGE) libraries were used to characterize polyadenylated transcripts in lung tissue compared to a panel of 25 different normal human tissues, and to a cohort of 12 NSCLCs. To generate lncRNA expression profiles, we developed a lncRNA discovery pipeline to map-tag-to-lncRNA matches. To identify differentially expressed lncRNAs we used a permutation test based statistical analysis. Expression pattern in lung tumors were compared to profiles from a variety of cancer types in order to identify lncRNA changes prominent in lung cancer. Results: Here we show that large-scale expression profiling through SAGE, is an effective resource for investigating the expression pattern of polyadenylated lncRNAs. Applying a novel lncRNA discovery pipeline we reveal extensive, tissue-specific lncRNA expression in normal lung compared to a panel of several different normal human tissues. Importantly, our study reveals that NSCLC demonstrate significantly altered lncRNA expression patterns and identify highly dysregulated transcripts associated with this malignancy as oppose to other types of cancer. Conclusion: Collectively, our findings support an important role for tissue-specific lncRNAs in lung cancer. Characterization of the functional role of these transcripts will have a considerable impact on our understanding of lung cancer development and progression, and may reveal clinically important biomarkers.

Abstract B21: The paralogous microRNA clusters, miR-17-92 and miR-106-25, are specifically overexpressed in metastatic non-small cell lung carcinomas

Background: Lung cancer is the cause of the most cancer-related deaths worldwide, with poor survival being largely attributed to late stage of disease at diagnosis and frequent metastasis. Understanding the mechanisms by which lung tumors metastasize could enable the development of anti-metastatic interventions or more specific therapeutics for metastatic disease. MicroRNAs (miRNAs) are major regulators of gene expression and control a wide range of cellular processes involved in metastasis, including apoptosis and cell cycle progression. Recently, increased expression of two paralogous miRNA clusters, miR-17-92 and miR-106b-25 , was tied to control of these functions through antagonizing transforming growth factor-β (TGFβ) signaling. A tumor suppressive cytokine, TGFβ regulates cell cycle progression and apoptosis through activation of p21 and BIM, respectively. However, evidence suggests this regulation can be repressed by the overexpression of these microRNA clusters to promote tumor progression. To this end, we sought to determine whether or not expression of these clusters was increased in non-small cell lung cancer (NSCLC) cases positive for nodal or distant metastases compared to those with only locally invasive disease. Methods: A panel of 41 non-metastatic NSCLCs and a panel of 28 NSCLCs with nodal or distant metastases were collected along with paired adjacent non-malignant tissues. Expression analysis of miRNAs was conducted in these specimens using Illumina GaXII small RNA sequencing technologies. Matched tumor and normal miRNA normalized read count comparisons were performed for each miRNA in the miR-17-92 ( miR-17 , miR-18a , miR-19a, miR-20a, miR-19b-1, miR-92a-1 ) and miR-106b-25 ( miR-106b, miR-93, miR-25 ) clusters (Wilcoxon Signed-Rank test p<0.05). Only those miRNAs that were significantly overexpressed and displayed a minimum average expression fold change of 2 were further investigated. Results: The significant overexpression of several miRNAs occurred specifically in the metastatic cohort, and included miR-20a , miR-92a-1 , miR-106b and miR-93 . miR-18a and miR-19a were significantly overexpressed in both tumor cohorts as compared to matched normal tissue; however, expression levels were substantially higher in the metastatic cohort and increased by 9.5 and 3 fold, respectively. miR-17 and miR-19b-1 were upregulated in both cohorts to a similar level, while expression of miR-25 was not significantly altered. Conclusion: miRNAs in the miR-17-92 and miR-106b-25 clusters, save miR-25 , were significantly overexpressed in NSCLC cases positive for nodal or distant metastases. There was an overall trend of increasing involvement of miRNAs from these clusters moving from NSCLC cases without metastases to those with metastases, suggesting upregulation of these miRNAs is involved in the metastatic process. Citation Format: Katey SS Enfield, Stephen Lam, Wan L. Lam. The paralogous microRNA clusters, miR-17-92 and miR-106-25 , are specifically overexpressed in metastatic non-small cell lung carcinomas [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 B21.