Nicole M. White

Transcriptome sequencing reveals altered long intergenic non-coding RNAs in lung cancer

BackgroundLong intergenic non-coding RNAs (lncRNAs) represent an emerging and under-studied class of transcripts that play a significant role in human cancers. Due to the tissue- and cancer-specific expression patterns observed for many lncRNAs it is believed that they could serve as ideal diagnostic biomarkers. However, until each tumor type is examined more closely, many of these lncRNAs will remain elusive.ResultsHere we characterize the lncRNA landscape in lung cancer using publicly available transcriptome sequencing data from a cohort of 567 adenocarcinoma and squamous cell carcinoma tumors. Through this compendium we identify over 3,000 unannotated intergenic transcripts representing novel lncRNAs. Through comparison of both adenocarcinoma and squamous cell carcinomas with matched controls we discover 111 differentially expressed lncRNAs, which we term lung cancer-associated lncRNAs (LCALs). A pan-cancer analysis of 324 additional tumor and adjacent normal pairs enable us to identify a subset of lncRNAs that display enriched expression specific to lung cancer as well as a subset that appear to be broadly deregulated across human cancers. Integration of exome sequencing data reveals that expression levels of many LCALs have significant associations with the mutational status of key oncogenes in lung cancer. Functional validation, using both knockdown and overexpression, shows that the most differentially expressed lncRNA, LCAL1, plays a role in cellular proliferation.ConclusionsOur systematic characterization of publicly available transcriptome data provides the foundation for future efforts to understand the role of LCALs, develop novel biomarkers, and improve knowledge of lung tumor biology.

INTEGRATE: gene fusion discovery using whole genome and transcriptome data

While next-generation sequencing (NGS) has become the primary technology for discovering gene fusions, we are still faced with the challenge of ensuring that causative mutations are not missed while minimizing false positives. Currently, there are many computational tools that predict structural variations (SV) and gene fusions using whole genome (WGS) and transcriptome sequencing (RNA-seq) data separately. However, as both WGS and RNA-seq have their limitations when used independently, we hypothesize that the orthogonal validation from integrating both data could generate a sensitive and specific approach for detecting high-confidence gene fusion predictions. Fortunately, decreasing NGS costs have resulted in a growing quantity of patients with both data available. Therefore, we developed a gene fusion discovery tool, INTEGRATE, that leverages both RNA-seq and WGS data to reconstruct gene fusion junctions and genomic breakpoints by split-read mapping. To evaluate INTEGRATE, we compared it with eight additional gene fusion discovery tools using the well-characterized breast cell line HCC1395 and peripheral blood lymphocytes derived from the same patient (HCC1395BL). The predictions subsequently underwent a targeted validation leading to the discovery of 131 novel fusions in addition to the seven previously reported fusions. Overall, INTEGRATE only missed six out of the 138 validated fusions and had the highest accuracy of the nine tools evaluated. Additionally, we applied INTEGRATE to 62 breast cancer patients from The Cancer Genome Atlas (TCGA) and found multiple recurrent gene fusions including a subset involving estrogen receptor. Taken together, INTEGRATE is a highly sensitive and accurate tool that is freely available for academic use.

Multi-institutional Analysis Shows that Low PCAT-14 Expression Associates with Poor Outcomes in Prostate Cancer

BACKGROUND Long noncoding RNAs (lncRNAs) are an emerging class of relatively underexplored oncogenic molecules with biological and clinical significance. Current inadequacies for stratifying patients with aggressive disease presents a strong rationale to systematically identify lncRNAs as clinical predictors in localized prostate cancer. OBJECTIVE To identify RNA biomarkers associated with aggressive prostate cancer. DESIGN, SETTING, AND PARTICIPANTS Radical prostatectomy microarray and clinical data was obtained from 910 patients in three published institutional cohorts: Mayo Clinic I (N=545, median follow-up 13.8 yr), Mayo Clinic II (N=235, median follow-up 6.7 yr), and Thomas Jefferson University (N=130, median follow-up 9.6 yr). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The primary clinical endpoint was distant metastasis-free survival. Secondary endpoints include prostate cancer-specific survival and overall survival. Univariate and multivariate Cox regression were used to evaluate the association of lncRNA expression and these endpoints. RESULTS AND LIMITATIONS An integrative analysis revealed Prostate Cancer Associated Transcript-14 (PCAT-14) as the most prevalent lncRNA that is aberrantly expressed in prostate cancer patients. Down-regulation of PCAT-14 expression significantly associated with Gleason score and a greater probability of metastatic progression, overall survival, and prostate cancer-specific mortality across multiple independent datasets and ethnicities. Low PCAT-14 expression was implicated with genes involved in biological processes promoting aggressive disease. In-vitro analysis confirmed that low PCAT-14 expression increased migration while overexpressing PCAT-14 reduced cellular growth, migration, and invasion. CONCLUSIONS We discovered that androgen-regulated PCAT-14 is overexpressed in prostate cancer, suppresses invasive phenotypes, and lower expression is significantly prognostic for multiple clinical endpoints supporting its significance for predicting metastatic disease that could be used to improve patient management. PATIENT SUMMARY We discovered that aberrant prostate cancer associated transcript-14 expression during prostate cancer progression is prevalent across cancer patients. Prostate cancer associated transcript-14 is also prognostic for metastatic disease and survival highlighting its importance for stratifying patients that could benefit from treatment intensification.

Recurrent rearrangements in prostate cancer: causes and therapeutic potential.

DNA damage and genetic rearrangements are hallmarks of cancer. However, gene fusions as driver mutations in cancer have classically been a distinction in leukemia and other rare instances until recently with the discovery of gene fusion events occurring in 50 to 75% of prostate cancer patients. The discovery of the TMPRSS2-ERG fusion sparked an onslaught of discovery and innovation resulting in a delineation of prostate cancer via a molecular signature of gene fusion events. The increased commonality of high-throughput sequencing data coupled with improved bioinformatics approaches not only elucidated the molecular underpinnings of prostate cancer progression, but the mechanisms of gene fusion biogenesis. Interestingly, the androgen receptor (AR), already known to play a significant role in prostate cancer tumorigenesis, has recently been implicated in the processes resulting in gene fusions by inducing the spatial proximity of genes involved in rearrangements, promoting the formation of double-strand DNA breaks (DSB), and facilitating the recruitment of proteins for non-homologous end-joining (NHEJ). Our increased understanding of the mechanisms inducing genomic instability may lead to improved diagnostic and therapeutic strategies. To date, the majority of prostate cancer patients can be molecularly stratified based on their gene fusion status thereby increasing the potential for tailoring more specific and effective therapies.

Abstract 971: A novel long noncoding RNA, onco-lncRNA 230, induces apoptosis and invasion in lung squamous cell carcinoma

Long non-coding RNAs (lncRNAs) are RNAs that are longer than 200 base pairs and do not translate into proteins but have been shown to play roles in all aspects of biological regulation. LncRNAs have also been shown to regulate gene expression including chromatin organization, transcriptional regulation, and post-transcriptional control. In addition, they are now emerging to be important players in our understanding of cancer and have been shown to promote tumorigenesis. However, the mechanisms driving the advancement of tumor progression to metastasis is poorly understood. To identify lncRNAs critical to tumorigenesis, our lab conducted a pan-cancer analysis of normal and primary tumor tissues from The Cancer Genome Atlas to discover lncRNAs that are commonly altered across cancer types, which we termed ‘onco-lncRNAs’. During our analysis, we discovered a novel unannotated onco-lncRNA, onco-lncRNA-230, that was significantly up regulated in head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) tumors relative to matched adjacent normal tissues, when available. We chose to focus on onco-lncRNA-230 as it displayed more than seven fold increase in LUAD and more than thirty seven fold change in LUSC tumors relative to adjacent normal. First, we validated this finding by quantitative PCR which revealed greater than a two fold enrichment of onco-lncRNA-230 expression in two out of seven LUAD and all six LUSC cells lines compared to a normal lung cell line. Because we observed the greatest increase in expression in HCC95 LUSC cells, we next sought to determine if modulating the expression of onco-lncRNA-230 promotes oncogenic phenotypes. Silencing of onco-lncRNA-230 in HCC95 cells did not change cellular proliferation but significantly increased apoptosis. Furthermore, silencing onco-lncRNA-230 resulted in a decrease in cellular invasion by transwell assay. Taken together, this represents the first study reporting that onco-lncRNA-230 is dysregulated in multiple cancer types, significantly up-regulated in lung cancer patient samples and lung cancer cell lines, and confers oncogenic phenotypes. Moving forward, we intend to explore how onco-lncRNA-230 mechanistically promotes lung cancer with the objective of utilizing onco-lncRNA-230 as a novel cancer diagnostic and therapy. Citation Format: Cynthia Y. Tang, Jessica M. Silva-Fisher, Ha X. Dang, Nicole M. White, Christopher A. Maher. A novel long noncoding RNA, onco-lncRNA 230, induces apoptosis and invasion in lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 971.

Abstract 974: Characterization of the novel lncRNA, PCAT14, clinically associated with metastatic prostate cancer

Each year, over 180,000 men are diagnosed with prostate cancer in the United States. Advances in research have established a molecular stratification of prostate cancer disease improving screening and treatment options. However, some patients lack these genetic aberrations, indicating that prostate tumors may harbor disease-associated noncoding RNAs that further characterize molecular subtypes. Long non-coding RNAs (lncRNAs) are largely unexplored and are emerging as a new aspect of cancer biology through advances in sequencing technologies. To discover novel transcripts, and overcome the shortcomings of relying on incomplete or inaccurate annotations, we focused on an approach using a genome-wide annotation-independent method to identify regions of differential expression named SWORD (Sliding WindOw Region Discovery tool). We applied SWORD to recently generated data from aggressive prostate tumors and adjacent normal tissue. We discovered ten novel lncRNAs including the previously annotated Prostate Cancer Associated Transcript-14 (PCAT14). PCAT14 was consistently altered in an integrative analysis performed across three patient cohorts consisting of primary tumors with matched control transcriptome sequencing and Affymetrix gene expression including metastatic tumors. Utilizing the clinical data associated with the Affymetrix cohort, PCAT14 significantly associated with both high (9) and low (6) Gleason scores. Interestingly, PCAT14 is highly upregulated in primary tumors relative to control tissue and its expression is downregulated in metastatic tumors relative to primary tumors. These data suggest that PCAT14 expression promotes a metastatic phenotype. Therefore, we assessed PCAT14 expression within a cohort of 1008 radical prostatectomy specimens from three independent patient cohorts across institutes. We found that patients with high versus low PCAT14 expression showed significantly different rates of distant metastasis free survival, biochemical recurrence free survival, prostate cancer specific survival, and overall survival. Moreover, PCAT14 was implicated with protein-coding genes involved in biological processes promoting aggressive disease. In vitro experiments in prostate cancer cell lines further supported the clinical data associating PCAT14 with aggressive disease. Overall, we discovered that PCAT14 is broadly deregulated, promotes aggressive oncogenic phenotypes, and is significantly prognostic for multiple clinical endpoints supporting its significance for predicting metastatic disease. Due to its tissue-specific expression PCAT14 may serve as a valuable biomarker to define a subgroup of high-grade prostate carcinomas and improve disease management and patient prognosis. Citation Format: Nicole M. White, George Zhao, Jin Zhang, Elias Davicioni, Christopher A. Maher. Characterization of the novel lncRNA, PCAT14, clinically associated with metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 974.

Abstract 169: Metastatic colorectal cancer associated long non-coding RNAs identified by transcriptome sequencing of matched primary and metastatic patient tissues

Colorectal Cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. Approximately 50% of patients with CRC develops metastatic disease (mCRC) with a 5-year survival of less than 20%. To date, colorectal cancer research has primarily focused on the deregulation of protein-coding genes, however due to recent technological advances we can now focus on the under-studied and emerging class of long non-coding RNAs (lncRNAs). Despite advances in our understanding of primary CRC oncogenesis, the mechanisms driving the progression from primary to metastatic colorectal cancer remain poorly characterized. To address this, we performed ab initio transcriptome assembly to detect aberrant transcripts comparing matched normal, primary, and metastatic colon cancer tissues from a cohort of patients. This led us to discover 56 novel lncRNAs solely altered in metastasis when compared to normal and primary tissues, which we refer to as metastatic colorectal cancer associated long non-coding RNAs (mCCAL). To demonstrate functional significance, we characterized the most up-regulated novel lncRNA, mCCAL1. mCCAL1 was highly expressed in multiple colon cancer cell lines with the highest expression in HCT-116 and Lovo cell lines. Silencing mCCAL19s expression caused a decrease in cellular migration in HCT-116 cells and invasion in Lovo cells relative to controls. Furthermore, gene set enrichment analysis of our microarray results and patient cohort data revealed differentially regulated genes enriched in epithelial to mesenchymal transition (EMT), chromatin remodeling, methyltransferase activity, and target genes of the Polycomb Repressive Complex 2 (PRC2). Modulating expression of mCCAL1 also decreased expression of H3K27me3 supporting its role in chromatin remodeling. All together, this is the first study to compare matched patient tissues to discover metastatic lncRNAs in colorectal cancer. Our data revealed that mCCAL1 promotes cellular migration and invasion, EMT, and may function through its association with PRC2. Moving forward, we intend to further understand how mCCALs contribute to metastatic progression of colorectal cancer with the intent of revealing novel cancer diagnostics and therapies. Citation Format: Jessica M. Silva-Fisher, Ha Dang, Julie Grossman, Nicole White, Christopher Cabanski, Simon Goedegebuure, Timothy Fleming, Elizabeth Pittman, Robert Fulton, Matthew Strand, Albert C. Lockhart, Timothy Ley, Richard Wilson, Ryan Fields, Christopher Maher. Metastatic colorectal cancer associated long non-coding RNAs identified by transcriptome sequencing of matched primary and metastatic patient tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 169. doi:10.1158/1538-7445.AM2015-169