WIPI1, BAG1, and PEX3 Autophagy-Related Genes Are Relevant Melanoma Markers

Abstract

ROS and oxidative stress may promote autophagy; on the other hand, autophagy may help reduce oxidative damages. According to the known interplay of ROS, autophagy, and melanoma onset, we hypothesized that autophagy-related genes (ARGs) may represent useful melanoma biomarkers. We therefore analyzed the gene and protein expression of 222 ARGs in human melanoma samples, from 5 independent expression databases (overall 572 patients). Gene expression was first evaluated in the GEO database. Forty-two genes showed extremely high ability to discriminate melanoma from nevi (63 samples) according to ROC (AUC ≥ 0.85) and Mann-Whitney (p < 0.0001) analyses. The 9 genes never related to melanoma before were then in silico validated in the IST online database. BAG1, CHMP2B, PEX3, and WIPI1 confirmed a strong differential gene expression, in 355 samples. A second-round validation performed on the Human Protein Atlas database showed strong differential protein expression for BAG1, PEX3, and WIPI1 in melanoma vs control samples, according to the image analysis of 80 human histological sections. WIPI1 gene expression also showed a significant prognostic value (p < 0.0001) according to 102 melanoma patients' survival data. We finally addressed in Oncomine database whether WIPI1 overexpression is melanoma-specific. Within more than 20 cancer types, the most relevant WIPI1 expression change (p = 0.00002; fold change = 3.1) was observed in melanoma. Molecular/functional relationships of the investigated molecules with melanoma and their molecular/functional network were analyzed via Chilibot software, STRING analysis, and gene ontology enrichment analysis. We conclude that WIPI1 (AUC = 0.99), BAG1 (AUC = 1), and PEX3 (AUC = 0.93) are relevant novel melanoma markers at both gene and protein levels.

Figure 1

ROC analysis on the expression data of 9 genes never related to melanoma diagnosis or prognosis. The area under the curve (AUC) is plotted as sensitivity% vs 100-specificity%. The calculated AUC is reported in each case. The p value is <0.0001 in all cases.

Figure 2

Gene expression according to the IST online database. The four reported genes show different expression levels in melanoma vs healthy skin. The expression level of each gene is reported in 208 melanoma biopsies and 147 healthy skin biopsies, according to the IST Online database. Gating indicated with dashed lines include 90% of melanoma and 90% of ctrl skin samples. PEX3, BAG1, and CHMP2B expression in melanoma is clearly lower than healthy skin. WIPI1 expression in melanoma is clearly higher than healthy controls.

Figure 3

Protein expression according to the Human Protein Atlas. The plot reports the distribution of pixel as function of the expression level. Positions at the right end of the graph indicate higher protein expression. Median level in melanoma samples (dashed curve) shows a clear right shift as compared to healthy skin (gray curve), for BAG1, PEX3, and WIPI1 proteins.

Figure 4

Functional relations reported in Pubmed abstracts, according to Chilibot analysis. Only strong interactive relationships are reported (i.e., interactive relationships reported by at least 5 Pubmed abstracts). Green dotted lines indicate stimulatory relationships; yellow dashed/dotted lines indicate both stimulatory and inhibitory relationships; red dashed lines indicate inhibitory relationships; and continuous gray lines indicate neither stimulatory nor inhibitory relationship, according to Chilibot categories. (a) None of the 3 selected autophagy-related genes has any direct known interactive relationship with melanoma; rather, the relationships are all mediated by autophagy. This indicates that the proposed role of BAG1, PEX3, and WIPI1 in melanoma is novel. (b) Strong interactive relationships of the 3 genes occur with intracellular vesicles, and, through these, they may relate to melanoma. (c) Strong interactive relationships of all 222 ARGs taken from Supplementary Table 1 with melanoma were investigated. Neither BAG1, nor PEX3 nor WIPI1, has a direct strong interactive relationship with melanoma. BAG1 may have indirect strong interactive relationships with melanoma, mediated by BIRC5, BID, PARP1, FAS, BAX, DNAJB, and many others. WIPI1 interaction with melanoma is mediated by PRKAB1, ATG12, ATG5, ATG7, BECN1, and MAP1LC36. Interestingly, ARSA is the only autophagy-related gene having known strong interactive relationships with both BAG1, PEX3, and WIPI1 and with melanoma.

Figure 5

STRING analysis of WIPI1, BAG1, and PEX3 network. BCL2, PEX14, and ATG9A physically/functionally connect BAG1, PEX3, and WIPI1 (respectively) via HDAC1.