Alternative splice events (ASES) are significant components of potential oncogenic pathways alterations and play a critical role in malignant cell transformation in a variety of solid and liquid tumors, including head and neck squamous cell carcinoma (HNSCC). However, high throughput analyses performed to date have not considered ASEs. Therefore, they have detected a limited number of genetic alterations for HNSCC, which incompletely describe the HNSCC specific pathway alterations. The heterogeneous nature of these alterations has made the discovery of reliable HNSCC biomarkers and therapeutic targets for this disease challenging.
We performed alternative splice events (ASEs) analysis to enhance our understanding of HNSCC biology. To define ASEs specific to HNSCC we designed a novel bioinformatics pipeline from RNA-sequencing data of HNSCC tumors and independent normal samples. Evaluating the top scoring candidates, we have found several highly promising ASE candidates, including GSN, Gelsolin, an actin-binding protein, a key regulator of actin filament assembly and disassembly.
Previously published literature proposes that GSN demonstrates tumor-suppressor properties by reducing cell proliferation in vivo and in vitro via suppression of protein kinase C (PKC, part of the PI3K pathway which is altered in HNSCC). The alternative splicing event involves an insertion of 110 bp from the 14th intron. This insertion contains a stop codon in frame, and the splice variant gives rise to a truncated (562 amino acids(aa)) protein with only 4 Gelsolin domains (instead of the full-length 731 aa protein with 6 Gelsolin domains). Using RNA-Seq data we demonstrated that 40% of tumor samples harbor the GSN-ASE. QRT-PCR confirmed that while total expression of GSN is decreased in HNSCC samples, GSN is expressed in the alternative truncated form only in HNSCC tumors and not normal tissues. Accordingly, total GSN expression is also seen to be downregulated in breast, lung and colon cancers.
Evaluation of TCGA data confirmed the pre-dominant expression of the truncated GSN isoform over the wt GSN in HNSCC, bladder urothelial carcinoma, colon adenocarcinoma, lung SCC, breast invasive carcinoma, cervical SCC and endocervical adenocarcinoma. Moreover, we confirmed that that the expression of the truncated GSN is important for the migration and invasion of the cancer cells in vitro. These data suggest that alternative splicing plays an important role in the GSN gene for multiple tumor types.
Citation Format: Daria A. Gaykalova, Dylan Kelley, Theresa Guo, Craig Bohrson, Ilse Tiscareno, Veronika Zizkova, Michael Considine, Ludmila Danilova, Emily Flam, Justin Bishop, Julie Ahn, Samantha Merritt, Marla Goldsmith, Chi Zhang, Wayne Koch, William Westra, Zubair Khan, Michael Ochs, Sarah Wheelan, Elana Fertig, Joseph Califano. The discovery of novel GSN alternative splicing in head and neck 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 2880.