Abstract 2973: CXCR3 epigenome switches splice variants in prostate cancer.

Prostate cancer (PCa) is the leading cause of cancer and the second leading cause of cancer death among men in the US, with tumor metastasis being the main reason behind high rates of mortality and morbidity. How these tumors escape the molecular signals that prevent epithelial cell migration from the prostate is being deciphered. A key motility ‘stop’ signaling network is via chemokines that bind to the common receptor CXCR3. CXCR3 is a G protein coupled receptor whose splicing is differentially regulated in prostate cancer. We have found over-expression of the CXCR3A splice isoform in prostate cancers and cell lines whereas normal prostate epithelium expresses by and large only the CXCR3B isoform, representing a possible switch in the expression between the CXCR3 splice variants. As these variants link differentially to pro- and anti-invasion signaling pathways, this may explain the relentless progression of the prostate carcinoma cells. The switch is present at both the RNA and protein level which suggests that the altering factor is at the transcriptional control level. In a series of PCa lines, the CXCR3 gene was scanned for insertions; deletions or substitutions that introduce a splice silencer; enhancer or a premature stop codon. Surprisingly, the gene was free from mutations except for a polymorphism in the intron in one of the cancer cell lines. A mutation is a permanent change whereas an epigenetic change is a reversible change that confers the cell with the ability to wield control over the expression of the isoforms at a given time. This suggests presence of a possible epigenetic change and explains the absence of mutations. To confirm the aforementioned, the CXCR3 epigenome was analyzed to find the intronic region differentially methylated at four different nucleotides while the upstream putative promoter was not. Strikingly, two of the differentially methylated sites were flanking splice sites while two of were adjoining the NK-2 transcription repressor binding site. A bichromatic CXCR3 reporter minigene has been designed to study the cell type specific expression pattern of the variants and the effect of each differentially methylated CpG nucleotide on splicing. Upon transfection of the wildtype minigene, it was found that the cancer cells express both the isoforms and as expected higher levels of CXCR3A when compared to normal prostate epithelial cells. The switch of isoforms was observed as: one subpopulation of cells that express both the isoforms; another that express solely one of the isoforms: either CXCR3A or CXCR3B. The mutant with guanines at the differentially methylated sites would validate the epigenomic link to splicing. The use of this reporter will be presented to dissect the plasticity of this epigenetic change during dissemination in experimental models of prostate cancer. Citation Format: Devi Sharanya Sampath Kumar, Alan Wells. CXCR3 epigenome switches splice variants in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2973. doi:10.1158/1538-7445.AM2013-2973