FIGURE 1 from Gigaxonin Suppresses Epithelial-to-Mesenchymal Transition of Human Cancer Through Downregulation of Snail

Abstract

<p>Association of exon 8 SNP to RNA and protein expression. Relative transcript hits are calculated from GAPDH transcript levels in HeLa cells (164,185 transcripts) and differential cell cycle (ΔCt) expression of <i>GAPDH</i> versus gene of interest of the qRT-PCR analysis. <b>A,</b> Normal endometrium (#4N) has higher GAN and vimentin expression, but reduced expression of <i>CDH1</i> (e-cadherin). Two (#17 and #18) of three primary tumors containing a T allele show higher <i>GAN</i> (gigaxonin) and <i>CDH1</i> (e-cadherin) expression. One tumor (#12) with CC alleles has higher gigaxonin, but reduced e-cadherin expression. A recurrence tumor (#8R) is positive for p16 but has low-level expression of gigaxonin and e-cadherin. Although preliminary, the results point to a direct relationship between gigaxonin and e-cadherin in primary tumor samples. Results indicate that gigaxonin expression is not correlating to p16 expression. <b>B,</b> HT3, C33A, HeLa, and ME180 cells express gigaxonin, but e-cadherin expression is seen only in HT3 and ME180 cells. Vimentin expression is observed in HeLa cells and at a reduced level in C33A. <b>C,</b> All cervical cancer cell lines and the head and neck cancer cell line CCL23 express <i>CDKN2A</i> (p16). Expression is absent in other head and neck cancer cell lines. Higher GAN expression is observed in ME180 and C33A cells. Expression of e-cadherin is seen only in ME180 cells. Low level expression of n-cadherin is seen in head and neck cancer cell lines CCL23 and UM-SCC12. While <i>CCND1</i> (cyclin D1) expression is observed in all cancer cell lines except for SiHa cells. Vimentin expression is seen in two each of cervical cancer (HeLa and C33A) and head and neck cancer (C33A and UM-SCC12) cell lines. <b>D,</b> RNA-seq data show 3-fold higher expression of GAN in ME180 in comparison with HeLa cells. Furthermore, RNA-seq data show statistically significant (<i>P</i> < 0.05) upregulation of e-cadherin <i>CDH1</i>, and <i>CD24</i> genes and downregulation of zeb1, and vimentin in ME180 in comparison with HeLa cells. <b>E,</b> Snap-shot view of ME180 versus HeLa shows 11-fold higher RNA transcripts in ME180 (T allele—red) pointing to RNA instability in HeLa cells due to C allele (blue) expression. Although genomic status of HeLa cells shows the presence of both C and T alleles, only C allele expression is seen at exon 8. Both qRT-PCR sequence and RNA-seq data show expression of T allele is absent in HeLa cells. <b>F,</b> Western blots show ME180 cells having highest gigaxonin expression. HT3 cells have higher expression than other cell lines containing C/T or C/C alleles. Reduced expression in CAL27 and absence of expression in UMSCC1 and UM-SCC12 cell lines are observed. Positive control used for gigaxonin protein size represents protein extract of FLAG tag <i>GAN</i> gene overexpressing Cos cells provided by Dr. Pascale Bomont of Lyon, France. While 10 µg of this protein extract was loaded, 40 µg of cell line protein lysates were loaded pointing to reduced β-actin expression in the positive control. Expression of CDKN2A is observed only in cervical cancer cell lines. <b>G,</b> Inverse relationship in the expression of e-cadherin and Zeb1 is seen in cancer cell lines. <b>H,</b> Higher expression of vimentin in HeLa, and UM-SCC12, and reduced expression in C33A cells are seen. Vimentin expression is absent in other cell lines. Same protein lysates were used for all the Western blots and thus a single β-actin blot is presented. UM cell lines refer to University of Michigan cell lines. <i>GAN</i>, gigaxonin; <i>CDKN2A</i>, p16; <i>CCDN1</i>, cyclin D1; <i>CDH1</i>, E-cadherin; <i>CDH2</i>, N-cadherin; <i>VIM</i>, vimentin; G allele, Genomic allele at codon 1293 of <i>GAN</i> gene.</p>