B299: The Role of KSRP in the Alternative Splicing of the Fibroblast Growth Factor Receptor 2 in Triple Negative Breast Cancer

Kiashanee Moodley1,Kurt Lightfoot2,Caroline Dickens1,Therese Dix-Peek1,Juan Valcárcel2,Raque Duarte1,Gwendal Dujardin2

1Department of Internal Medicine, Faculty of Health Sciences, Johannesburg, South Africa,2Gene Regulation, Stem Cells and Cancer, Centre de Regulació Genòmica, Barcelona, Spain

Presenting date: Tuesday 3 November

Background

 

Triple negative breast cancer (TNBC) is characterised by the lack of expression of the oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Therapeutic options for TNBC are limited due to the lack of well defined targets. The fibroblast growth factor receptor 2 (FGFR2), implicated in the development of breast cancer, is alternatively spliced, and the mutually exclusive inclusion of either the IIIb or IIIc isoform has been associated with EMT. A proteomic screen of basal and mesenchymal TNBC cell lines, which include IIIb and IIIc, respectively, found the KH-type splicing regulatory protein (KSRP) overexpressed in the basal cell line. The aim of this study was to determine if KSRP is involved in the alternative splicing of FGFR2.  

Method

 

Two breast cancer cell lines, MDA-MB-468 and MDA-MB-436 expressing alternative FGFR2 isoforms were selected. KSRP was knocked down using siRNA technology. The IIIb and IIIc inclusion pattern was determined using semi quantitative PCR and the band intensities quantified using ImageJ software. UV crosslinking and immunoprecipitation (CLIP) was used to determine if KSRP binds FGFR. Whole genome-expression profiling of KSRP knockdown basal cells was carried out using the Affymetrix GeneChip Human Transcriptome Array 2.0.

Results

KSRP binds FGFR2 pre-mRNA and the IIIb and IIIc inclusion pattern was changed upon KSRP knockdown in the basal TNBC cell line, but not the mesenchymal cell line. Exon arrays data generated from siRNA knockdown of KSRP indicated the upregulation of various transcription factors and members of the Wnt signalling pathway.

Conclusion

 

KSRP has been shown to directly bind the basal cell FGFR2 pre-mRNA and affects the FGFR2 alternative splicing pattern. The microarray data are currently being analysed to determine the impact of KSRP knockdown on the mechanisms governing FGFR2 alternative splicing in TNBC subtypes.