No abstract

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR-based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer. [Cancer Res 2008;68(22):9525-31]

Alternative splicing provides a critical and flexible layer of regulation intervening in many biological processes to regulate the diversity of proteins and impact cell phenotype. To identify alternative splicing differences that distinguish epithelial from mesenchymal tissues, we have investigated hundreds of cassette exons using a high-throughput reverse transcription-PCR (RT-PCR) platform. Extensive changes in splicing were noted between epithelial and mesenchymal tissues in both human colon and ovarian tissues, with many changes from mostly one splice variant to predominantly the other. Remarkably, many of the splicing differences that distinguish normal mesenchymal from normal epithelial tissues matched those that differentiate normal ovarian tissues from ovarian cancer. Furthermore, because splicing profiling could classify cancer cell lines according to their epithelial/mesenchymal characteristics, we used these cancer cell lines to identify regulators for these specific splicing signatures. By knocking down 78 potential splicing factors in five cell lines, we provide an extensive view of the complex regulatory landscape associated with the epithelial and mesenchymal states, thus revealing that RBFOX2 is an important driver of mesenchymal tissue-specific splicing.T ransitions from epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) states have important roles not only in normal tissue and organ development but in the pathogenesis of diseases including cancer (1). In normal tissues, epithelial cells display a cuboidal morphology and a polar organization maintained by tight cell-cell interconnections. Mesenchymal cells lack these features and display higher motility and invasiveness. During the process of carcinogenesis, EMT is thought to be crucial to elicit migration, resistance to apoptosis, and ultimately invasion and metastasis (1-5). Conversely, the reverse process, MET, is associated with the colonization of secondary sites by cells that have metastasized. Therefore, profiling the molecular differences between the epithelial and mesenchymal states may help us understand the underlying regulatory programs that establish these states and promote their interconversion.The commonly accepted view is that EMT can be induced by growth factors, such as transcription growth factor ␤1 (TGF-␤1), which trigger signaling pathways that ultimately activate a network of transcription regulators, including Snail, Slug, Twist, and others (6). This transcriptional reprogramming elicits the expression of mesenchymal markers (e.g., vimentin) and represses the expression of epithelial ones (e.g., E-cadherin) to impart distinctive properties such as motility and invasion (7-10). Alternative splicing control provides another layer of regulation that can contribute to EMT (11,12). The tyrosine kinase Ron (MST1R) is alternatively spliced to produce an exon 11-lacking version that can promote invasion (13). Three RNA-binding proteins (RBPs), SRSF1, hnRNP H, and hnRNP A2/B1, affect the alternative splicing of Ro...

Most human genes produce multiple mRNA isoforms through alternative splicing. However, the biological relevance of most splice variants remains unclear. In this study, we evaluated the functional impact of alternative splicing in cancer cells. We modulated the splicing pattern of 41 cancer-associated splicing events and scored the effects on cell growth, viability and apoptosis, identifying three isoforms essential for cell survival. Specifically, changing the splicing pattern of the spleen tyrosine kinase gene (SYK) impaired cell-cycle progression and anchorage-independent growth. Notably, exposure of cancer cells to epithelial growth factor modulated the SYK splicing pattern to promote the pro-survival isoform that is associated with cancer tissues in vivo. The data suggest that splicing of selected genes is specifically modified during tumor development to allow the expression of isoforms that promote cancer cell survival.

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for realtime PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.