PURPOSE: Analyze the functional role of alternative splicing in HPV-related (HPV+) Head and Neck Squamous Cell Carcinoma (HNSCC) biology and oncogenesis.
HPV+ HNSCC incidence is rapidly increasing, while its oncogenic mechanisms, demographic, and clinico-pathological characteristics are largely uncharacterized and discrete from those of HPV- HNSCC. Investigation into the molecular biology of HPV pathogenesis in HNSCC may help with creation of more specific clinical tools for diagnosis and treatment, which are limited and lack accuracy. Alternative Splicing Events (ASEs), post-transcriptional modifications resulting in variant transcripts leading to alternative protein isoforms, have been under-investigated as putative causes of gene expression dysregulation in HPV+ HNSCC. Using RNA-Seq data, a genome-wide ASE discovery analysis was performed on a JHU HPV+ HNSCC cohort, which has expanded HPV+ HNSCC transcriptional characterization by identifying novel ASE biomarkers implicated in carcinogenic progression. Outlier Gene Set Analysis (OGSA) was used to identify genes with high rates of ASE and results were validated in TCGA samples from seven different cancer types. To verify the functional implications of differential isoform expression, vectors for knock-in and knock-down of WT-GSN and ASE-GSN isoforms were developed to evaluate changes in cell proliferation and invasion. ASE-GSN/WT-GSN ratio outliers were identified by OGSA in tumor samples from discovery JHU HPV+ HNSCC cohort, as well as in TCGA public domain data from HNSCC, Lung Squamous Cell Carcinoma, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma, Bladder Urothelial Carcinoma, Breast Invasive Carcinoma, and Colon Adenocarcinoma. The increased ASE-GSN/WT-GSN ratio was also observed in a cohort of HPV+ Head and Neck Cancer Cell lines. SCC61 and UM-SCC-22B HNSCC cell lines were used to evaluate the function GSN ASEs in vitro. Overall expression levels of both GSN isoforms inversely correlated with cell proliferation whereas the high ratio of ASE-GSN to WT-GSN correlated with increased cellular invasion of stroma in vitro. Additionally, a decrease or increase in expression of one isoform is seen to cause a compensatory increase or decrease in the other isoform respectively. ASE-GSN outliers correlated with HPV+ cancers indicate that expression ratio between GSN isoforms is related to tumor behavior, which is reinforced in vitro where WT-GSN is inversely correlated and ASE-GSN is directly correlated to with cell invasion. These relationships suggest that both overall expression and balance between the two GSN isoforms are mediating factors in cell death or proliferation, while shorter ASE-GSN is more likely to indicate progression to cancer. Finally, we propose a mechanism for the role of GSN isoforms in apoptosis and metastasis, in which trends in expression between WT- and ASE-GSN isoforms can alternatively affect rates of apoptosis or metastatic ability.
Citation Format: Dylan Z. Kelley, Emily L. Flam, Theresa Guo, Craig Bohrson, Michael Considine, Ludmila V. Danilova, Justin A. Bishop, Chi Zhang, Wayne M. Koch, David Sidransky, William Westra, Sarah Wheelan, Liliana Florea, Elana J. Fertig, Joseph A. Califano, Daria A. Gaykalova. Functional characterization of alternatively spliced GSN in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4474. doi:10.1158/1538-7445.AM2017-4474