Multi-cancer V-ATPase molecular signatures: A distinctive balance of subunit C isoforms in esophageal carcinoma

Figures

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  Fig. 1. V-ATPase expression profile in ESCC by microarray database. (a) Unsupervised hierarchical clustering analysis of 15 esophageal samples (tumors and respective surrounding tissues) showing the expression of the 25 genes of V-ATPase. Green indicates decreased gene expression and red increased genes expression. N and gray bar correspond to normal surrounding tissues; T and black bar correspond to tumor. (b) ATP6V1C1 and ATP6V1C2 expression levels in tumor, normal-appearing surrounding tissues and healthy esophageal mucosa from individuals without cancer. *p = 0.0146; ***p = 0.0003; ****p < 0.0001 [ATP6V1C1 - one-way ANOVA with Tukey's post-test. ATP6V1C2 - Kruskal-Wallis test with Dunn's post-test].

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  Fig. 2. The expression pattern of V-ATPase C isoforms in ESCC. Left images: RT-PCR analysis of (a) ATP6V1C1, (b) ATP6V1C2-v1 and (c) ATP6V1C2-v1,2 expression in paired ESCC samples organized by tumor stage. Values are expressed as relative to those obtained in tumors respective normal surrounding tissue (= 1 or −1), represented by the red dashed line. Right images: qRT-PCR evaluation of (d) ATP6V1C1, (e) ATP6V1C2-v1 and (f) ATP6V1C2-v1, 2 mRNA levels distribution in the groups of normal surrounding and their paired ESCC tissues. Values are shown in relative units. mRNA levels were normalized by those of GAPDH, used as the housekeeping gene. NA – no information available about tumor stages. ****p < 0.0001 [Wilcoxon matched pair test].

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  Fig. 3. ROC curves of C isoforms mRNA expression for the discrimination of ESCC from normal surrounding mucosa samples. ROC curves are relative to mRNA expression of: (a) ATP6V1C1, (b) ATP6V1C2-v1, (c) ATP6V1C2-v1,2, (d) C1/C2-v1 ratio and (e) C1/C2-v1,2 ratio. The area under curve (AUC) indicates the accuracy of the test in discriminating tumors from normal surrounding tissues. Numbers in a box correspond to a cut-off used for the determination of the sensitivity and specificity. ROC, receiver operating characteristic. p < 0.0001 [ROC curve].

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  Fig. 4. Prediction of three-dimensional structure of C subunit isoforms and putative actin-binding sites. (a) hC1 isoform, (b) hC2b isoform, (c) most promising model of hC2a isoform and (d) model of hC2a isoform validated by protein docking. C1 and C2b were modeled by homology using as template the resolved structure from yeast V-ATPase (3J9V.pdb) and C2a was obtained by ab initio approach. e) Multiple sequence alignment comparing “profilin-like” domain in B subunit (blue box) and similar sequence in C subunit isoforms. (f) 3D structure of hC2a isoform highlighting in green the putative "profilin-like" domain for actin-binding. The additional peptide stretch of 46 aa present in the hC2a isoform is shown in red.

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  Fig. 5. V-ATPase mRNA expression in esophageal cancer histological subtypes. (a) Expression profile of all V-ATPase genes in esophageal adenocarcinoma (EAC) and ESCC. (b) Respective ATP6V1C1 and ATP6V1C2 mRNA levels in EAC and ESCC assessed by RNA-sequencing data from TCGA and by qRT-PCR in the current study.

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  Fig. 6. mRNA expression pattern of V-ATPase genes in multiple cancer types. (a) Comparative table indicating the number of the analyses that meet the threshold with mRNA over-expression (left column, red) and under-expression (right column, blue) in cancer versus corresponding normal tissues. Each column refers to a V-ATPase gene and each line refers to a cancer site (some tumor sites include different histological types). The threshold was designed with the following parameters: p-value of 1E-4, fold-change of 2, and gene ranking of 10%. (b) Proposed combinatorial assemblies of V-ATPase subunits/isoforms with dominantly expressed isoforms (on the right) in multiple tumor types based on Oncomine analysis. Accessory subunits are shown in dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)