Depletion of melanoma fn14 by oligonucleotide-mediated exon skipping

Fibroblast growth factor inducible 14 (Fn14) is the receptor for the TNF family cytokine TNF-like weak inducer of apoptosis (TWEAK). TWEAK binding triggers multiple signaling pathways downstream of Fn14, including the MAPK and NF-kB pathways. Activation of these pathways can promote cell survival, proliferation, and inflammation. Fn14 is poorly expressed in the steady state but is readily induced by cellular stresses and tissue injury. When highly expressed, Fn14 can activate pro-survival and pro-proliferative signaling pathways in a TWEAK-independent manner. Fn14 is highly expressed in melanoma and correlates with poor outcomes. Consequently it is thought that the Fn14 pathway could be a therapeutic target in melanoma. Given that Fn14 can signal in both a TWEAK-dependent and -independent manner, we have developed a strategy to block both signaling mechanisms. We utilized an exon-skipping approach to alter the mRNA encoding Fn14. This alteration in Fn14 mRNA is predicted to encode a receptor that is secreted rather than being expressed at the cell surface and that lacks domains critical for TWEAK-independent signaling. We designed an antisense nucleic acid analog oligonucleotide, morpholino, targeting the predicted exonic splicing enhancer sites in exon 3 of Fn14 pre-mRNA. Here we show that transfection of this morpholino yields efficient skipping of Fn14 exon 3 in human melanoma cell lines. Reverse transcription and cDNA sequencing confirmed that the morpholino prevented splicing of exon 3 and produced mRNA with the predicted sequence. The resulting mRNA, with in-frame splicing of exon 2 to exon 4, encodes a protein lacking a portion of the extracellular domain and entire transmembrane domain of the full-length Fn14 protein. Altered RNA splicing leads to downregulation of Fn14 protein and loss of Fn14 signaling. The truncated Fn14 is predicted to be secreted and may be capable of functioning as a decoy receptor by binding to TWEAK and blocking TWEAK/Fn14 signaling in a paracrine manner. Thus morpholino-based exon skipping represents an efficient and novel means of blocking Fn14 signaling in melanoma.