Gigaxonin as a prognistic marker of human cancer

Gigaxonin, the GAN gene product, is a E3 ubiquitin ligase adaptor and is involved in neural cell and fibroblast intermediate filament processing. We have previously shown that gigaxonin in association with p16 ubiquitinates NF-κB in head and neck cancer. The cancer genomic database search has shown higher frequency of GAN gene exon 8 single nucleotide polymorphism (SNP, rs2608555), c.1293 C>T, p.Y431Y, in the normal Caucasian population (44.25% vs <20% in other populations). To determine whether this SNP is related to tumor development, we analyzed normal and tumor DNAs of cervical and head and neck cancer subjects and have found higher frequency of the SNP (Tumor 47% vs 25% normal, p=0.0017) in tumor samples. Although there was no relationship to HPV status, an inverse relationship to tumor recurrence and metastasis was observed. Two cervical cancer cell lines, ME180 and HT3, containing the SNP (T/T alleles) showed higher gigaxonin expression, reduced in vitro tumor cell growth and enhanced cisplatin sensitivity in comparison to 5 other cervical and 5 head and neck cancer cell lines containing the C allele (C/C or C/T alleles). CRISPR-Cas9 mediated conversion of T/T to C/C alleles of the ME180 cell line led to decreased gigaxonin expression, enhanced expression of snail, and twist (Epithelial to Mesenchymal transition - EMT markers) and increased in vitro tumor cell growth including invasion through Matrigel. Re-expression of gigaxonin in CRISPR-Cas9 cells using a lentiviral system resulted in increased expression of gigaxonin and reduced expression of snail accompanied by decreased in vitro cell growth. Mouse tail vein injection studies showed lung metastasis of CRISP-Cas9 cells which was inhibited in gigaxonin overexpressing cells. Our results strongly suggest that GAN gene exon 8 SNP correlates with gigaxonin expression, and inhibition of EMT phenotype in human tumors.