Microarray Analysis of Aberrant Splicing in Cancer

PL05-01 Aberrant gene expression plays an enormous role in human cancer by deregulating critical cell processes required for adhesion, proliferation, differentiation, motility and invasion, apoptosis, etc. In addition to the obvious role of transcriptional regulation to insure the proper quantitative levels of expression from each gene, accurate and efficient pre-mRNA splicing is also essential to qualitatively modulate the structure and function of proteins encoded by these transcripts. We know now that the majority of human genes utilize alternative splicing to generate multiple protein isoforms, which frequently have different or even antagonistic functions in regulating a wide range of activities essential for control of cell proliferation and differentiation. Understanding the biology of human cancers, classifying them into biologically distinct subtypes, and designing appropriate therapeutic strategies will all benefit greatly from an improved understanding of alternative splicing. To understand how splicing aberrations alter gene expression, it is useful to recall that proper splicing of an exon requires accurate recognition of relatively degenerate splice site sequences in a sea of long introns that may be full of similar but nonfunctional pseudo splice sites. This process is highly regulated by antagonistic activities of RNA regulatory motifs that enhance or inhibit recognition of real splice sites by interacting with an array of RNA binding protein/ splicing factors. All of these elements are mutational targets whose disruption can lead to aberrant splicing. Indeed, splicing mutations represent a very common cause of inherited disease in humans and have been reported in many cancers as well. For example, others have shown that a number of BRCA mutations lead to inappropriate pre-mRNA splicing. High throughput analysis of cancer genomes and transcriptomes promises more rapid identification of splicing mutations and the resultant aberrant transcripts. As one example, we have recently demonstrated an aberrant splicing phenotype in a p53 allele with a splice site mutation. This mutation, detected in a published breast cancer sequencing study, results in activation of a cryptic splice site in the p53 gene, upstream of the normal 3’ splice site. Use of the cryptic 3’ splice site increases the size of the exon and predicts a translational frameshift that may induce potential nonsense mediated decay of the p53 transcript and/or production of a truncated p53 protein. The advent of microarrays has greatly facilitated our ability to identify global patterns of transcript expression in tumors, allowing identification of critical over-expressed genes and sub-classification of tumors. More recently, global analysis of pre-mRNA alternative splicing patterns has become possible through the use of a new generation of exon arrays with single-exon resolution. In contrast to the tradition arrays used to analyze transcript expression, these arrays have multiple probes to every exon across a transcript. When comparing array patterns between cell types or tumor samples, changes in expression of one exon probeset relative to others in the same transcript is an indication of different splicing between the two samples. As proof of principle, we collaborated with Affymetrix scientists to begin analysis of alternative splicing programs in normal human tissues. Through these studies we were able to identify muscle- and brain-enriched alternative exons that are hypothesized to play critical roles in normal differentiation and development of these tissues. We were also able to identify specific RNA motifs linked to these exons that appear to regulate this tissue-specificity, as an early step in our efforts to understand the molecular regulation of programmed alternative splicing in normal human tissues. We are now using global exon expression profiling with the Affymetrix Human Exon 1.0 ST arrays to analyze aberrant splicing patterns in human cancer. These studies represent part of a larger effort in Dr. Joe Gray’s laboratory to utilize high throughput genome and transcriptome analysis of breast cancer cells to gain insights into the aberrant physiology of these diseases, and ultimately design therapeutics for specific cancer subtypes. For these studies we are using a panel of 51 breast cancer cell lines whose spectrum of genomic aberrations and gene expression generally mirrors that of primary tumors. By profiling these representative breast cancer cell lines on the Affymetrix Human Exon 1.0 ST arrays, we sought to obtain a global analysis of splice variants in breast cancer. However, the complexity of the array design and of the splicing phenomenon itself represents a significant challenge for detection of splicing events in many tumors, where it may not be feasible to perform multiple replicate arrays as one can do with normal tissues. Using a novel computational method termed FIRMA (Finding Isoforms with RMA, in press), in combination with the Affymetrix Splicing Index, we have developed an approach for effective detection of splicing changes in a complex background. This computational analysis identified more than a thousand exons as strong candidates for alternative splicing among these breast cancer cell lines. Interestingly, Gene Ontology (GO analysis reveals that the genes in which these alternatively spliced exons reside are highly significantly enriched for functions such as cell morphogenesis, cell adhesion, cell growth, cell differentiation, etc. Among these are well-known genes such as CD44, TPM1, ENAH, and SH2B. Validation experiments by RT-PCR revealed that CD44 transcripts represent a particularly interesting case. CD44 has previously been shown to exhibit complex alternative splicing in which a series of 10 variable exons is situated in the center of the gene between 5’ and 3’ sets of constitutive exons. Remarkably, we observed three classes of CD44 splicing patterns that correlate very well with known subtypes of breast cancer: basal A, basal B, and luminal. Amplification of basal A cell RNA yielded a complex mix of larger CD44 mRNA isoforms characterized by inclusion of different combinations of variable exons, not unlike normal breast epithelial cells. In contrast, basal B cell lines mostly skipped these exons and instead expressed mainly a smaller transcript including only constitutive exons. These results are consistent with the possibility that differences in CD44 structure and function may play a role in the distinct biology, including cell invasiveness, exhibited by these cancer subtypes. Finally, the exon array data indicated low expression of CD44 transcripts in luminal cells, consistent with previous expression data obtained with analysis by standard expression arrays and by Western blot. Future studies will be aimed at improving computational analysis of the exon array data to identify many more examples of aberrant splicing in cancer. Such efforts should facilitate identification of therapeutic targets in a more biologically meaningful manner than has been possible previously. Detection of cancer subtype-specific splicing events may in some cases also provide insight into useful biomarkers for early detection of some cancers. Finally, in the context of the Berkeley Cancer Genome Center (BCGC) at Lawrence Berkeley National Laboratory, we also are extending the use of exon expression analysis to additional tumor types selected by NIH as part of The Cancer Genome Atlas (TGCA) to accelerate understanding of the molecular basis of human cancer.