FIGURE 8 from Gigaxonin Suppresses Epithelial-to-Mesenchymal Transition of Human Cancer Through Downregulation of Snail

Absence of T allele expression is associated with absence of multimeric protein formation and mRNA instability. A, Western blot analysis of ME180, HeLa, HT3, and SiHa cells post actinomycin D treatment (10 µg/mL, 8 µmol/L) shows enhanced γH2AX expression in all four cell lines pointing to positive drug effect. Higher expression of 60 kD gigaxonin protein is seen in ME180, HT3, and SiHa cells in comparison with that of HeLa cells. Multimeric forms of the protein are absent in SiHa cells pointing to the association between loss of T allelic expression and absence of multimeric protein formation. B, RNA stability assays show half-life of HeLa cells to be 3.22 hours and that of ME180 and SiHa cell lines to be 5.09 and 7.88 hours, respectively. GAN RNA is more stable in HT3 cells. These results therefore indicate instability of GAN mRNA in HeLa cells showing C allele expression. C, Half-live of p16 (CDKN2A) is 5.49 hours and cyclin D1 mRNA is more stable in HeLa cells, matching the half-lives reported in the literature (34, 35). These results therefore indicate that actinomycin D treatment was effective in the cancer cell lines. D, qRT-PCR studies of ME180 cells transfected with RelA cDNA (3-fold) or treated with TNFα (5-fold after 15 minutes) show activation of NFκB compared with control cDNA or untreated control cells, respectively. There is activation of Snail in RelA transfected cells (2-fold) and in 30 and 60 minutes treatment of TNFα (1.6-fold) confirming transcription activation of Snail by NFκB. The time lag to NFκB may be related to time delay in the activation of NFκB signaling pathway. Downregulation of CDH1 (e-cadherin) and GAN (gigaxonin) coinciding with Snail upregulation points to transcriptional downregulation of these two genes. While inhibition of e-cadherin transcription by Snail is known (58–60), we for the first time show transcriptional regulation of GAN with NFκB activation indicating a feedback regulation. E, A mechanism is proposed where GAN gene exon 8 SNP (T allele) expression is associated with enhanced gigaxonin expression (upward arrow) leading to ubiquitination of NFκB resulting in Snail downregulation, increased expression of e-cadherin and maintenance of epithelial phenotype. Loss of gigaxonin through C allele expression (downward arrow) leads to enhanced NFκB expression, activation of Snail, downregulation of e-cadherin and development of mesenchymal (EMT) phenotype. Our results extend the interrelationship between these genes reported earlier by the other investigators (52–55). → Activation, ┤ Inhibition.