David A. Rowbotham

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.