Breast Cancer Research – Dr Raquel Duarte
Department of Internal Medicine, University of the Witwatersrand
Title of the Project
Characterisation of the splicing and transcriptional regulatory system of the Fibroblast Growth Factor Receptor 2 (FGFR2) gene in breast cancer.
Highlights of the Project
Given the role of FGFR2 in breast cancer development and progression and its association with epithelial mesenchynmal events during development a proteomic screen was conducted on two breast cells lines. The cell lines chosen were characterised as being either epithelial or mesenchymal based on the presence of the alternatively spliced FGFR2 gene and pattern of EMT marker gene expression Of the differentially expressed proteins identified three proteins (hnRNPA1, hnRNPA2 and hnRNPA3) were found to be splicing factors previously implicated in alternative splicing events. Another protein identified in the screen was MAP2 RNA transacting protein 1, MARTA1. Upon investigation this 52kDa protein was revealed to be a homologue of KSRP (KH-type splicing regulatory protein), a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing and mRNA decay. KSRP has been shown to co-operate with other splicing factors viz. PTB (polypyrimidine tract binding protein) and its homologue, nPTB, to mediate splicing events.
Western blotting confirmed the differential expression of MARTA1/KSRP in the breast cell line expressing FGFR2IIIc. Furthermore our analysis of the pattern of PTB expression in breast cancer cell lines showing alternative splicing of the FGFR2 gene has identified an nPTB isoform, nPTB7, as being highly preferentially expressed in those cells expressing FGFR2IIIc.
What has been achieved to date?
Tissue specific alternative splicing programmes are believed to be as a result of the fine orchestration of activities of a variety of regulatory factors. Alternative splicing of exons is as a result of the interplay between a series of exonic and intronic splicing enhancer and silencing factors. It is now believed that tissue-specific factors (in this case epithelial vs mesenchymal tissue specific splicing factors) are recruited into the transcriptional machinery at the site of transcriptional initiation so that splicing occurs co-transcriptionally, hence making the elucidation of the transcriptional regulatory mechanism even more critical. There is some evidence to suggest that the FGFR2 transcriptional start site may differ between different tissues. This would suggest that the make up of transcription factors in epithelial vs mesenchymal tissue differs remarkably and therefore directing impacting the transcription of the gene and the subsequent downstream splicing. To investigate this issue we wish to determine the exact sites of transcriptional initiation in the two cell lines using RNA ligase- mediated rapid amplification of cDNA (RLM –RACE) after which the regulatory regions could be finely mapped out using data obtained from the CHiP (Chromatin immunoprecipitation) experiments to establish which transcription factors bind and regulate the FGFR2 gene in the two tissue specific contexts.
Once we have verified the differential expression of the splicing factors (hnRNP A1, A2 and A3) in the two breast cancer cell lines MDA-MB-468 and MDA-MB- 436 we will screen a range of well characterised cell lines to determine whether there is a correlation with the expression of a particular splicing factor with a particular breast cancer phenotype. This would then form the basis of longer term studies to determine if these factors could be used as markers for identifying different stages and types of breast cancer in patient cohorts.
How is the project of value in the struggle against cancer?
Breast cancer, as one of the leading cancers in women in South Africa, is a complex genetic and biochemical disease due to the heterogenous nature of the tumours. Breast tumours consist of many different cell types and are morphologically and genetically diverse all of which have serious implications for diagnosis of the disease and treatment outcome. Recently a number of large-scale, independent genome scans to determine breast cancer associations have identified new loci containing SNPs (single nucleotide polymorphisms) that are strongly associated with breast cancer. Of those loci identified SNPs of the signalling molecule FGFR2 was found to associate strongly with breast cancer in all the studies conducted. Given the importance of these findings, together with the observation that the FGFR2 gene is known to be upregulated in a variety of tumours (including breast) we wish to examine the regulatory mechanisms governing expression of the FGR2 gene and thereby determine the role of this signalling molecule in the development and progression of breast cancer.
Duarte, R., Seedhar, P and Dickson, C. (2009) Differential splicing of the polypyrimidine tract binding protein is associated with alternative splicing of the FGFR2 gene in breast cancer cells (to be submitted).
Poster presentation “New protein biomarkers for Breast Cancer based on alternative splicing of the FGFR2 gene” at the European Biomarkers Summit, 16 – 17 October 2008, Lisbon. Attendance at the conference was sponsored by a CANSA travel grant.