Abstract 848: Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer
Authors: Komor Malgorzata A. , Hiemstra Annemieke C. , Pham Thang V. , Piersma Sander R. , Sebra Robert P. , Han Bo W. , Ashby Meredith , Carvalho Beatriz , Meijer Gerrit A. , Jimenez Connie R. , Fijneman Remond JA 
Published: July 2016

Abstract
Introduction
Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test detecting blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection.
Materials and methods
RNA and proteins were isolated from CRC cell line SW480 before and after siRNA-mediated down-modulation of the splicing machinery: SF3B1, U2AF1, and SRSF1. To identify splice variants, mRNA was sequenced (Illumina HiSeq) and analyzed. RNA-seq analysis included quality checks (FASTQ, RSeQC), reads mapping (STAR), differential gene expression (DESeq2) and differential expression of splicing isoforms between conditions (MATS). Results from the in silico analysis were validated by qRT-PCR. Proteins were analyzed by in-depth tandem mass spectrometry (QExactive). A proteogenomic data analysis pipeline was established to enrich the sequence database, against which the mass spectra are searched, with the predicted protein splice variants and identify protein isoforms. To further extend the splice-variant database, PacBio sequencing of full-length transcripts is being performed for the SW480 siSF3B1- and control-samples.
Results
Differential expression analysis on RNA and protein level proved that the knock-down experiments were performed successfully. The RNA-seq analysis revealed hundreds of mRNA splice variants, including positive controls described in literature. For example, down-modulation of SF3B1 resulted in quantitative mRNA changes of spliced isoforms of ADD3 both in RNA-seq and RT-PCR data. The proteomics experiment yielded over 6000 proteins per sample, among which a number of protein isoforms resulting from alternative splicing.
Conclusions and discussion
We established a proteogenomic pipeline for the analysis of alternative splicing and provided experimental proof of concept.
We expect, however, that the true complexity of RNA variant information remains highly underestimated. We therefore are performing PacBio long-read RNA-seq to validate our approach and to identify additional (novel) splicing events. In future studies we will apply the mRNA-seq based proteogenomic pipeline for detection of protein alterations to a series of adenomas at low- and high-risk of progression, and CRCs to validate the isoforms in a clinically relevant setting. Novel findings will be evaluated for their performance as screening markers for CRC.
This work was financially supported by KWF project nr: VU 2013-6025.
Citation Format: Malgorzata A. Komor, Annemieke C. Hiemstra, Thang V. Pham, Sander R. Piersma, Robert P. Sebra, Bo W. Han, Meredith Ashby, Beatriz Carvalho, Gerrit A. Meijer, Connie R. Jimenez, Remond JA Fijneman. Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal 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 848.