Glypican 6 is a putative biomarker for metastatic progression of cutaneous melanoma

Abstract

Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize. By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases. Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma. Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors. Our results suggest that GPC6 may play a role in tumor metastatic progression. In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines. We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed. This result suggested that GPC6 was a putative target of miR-509-3p in melanoma. Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p.

Fig 1. GPC6 expression in normal and melanoma cell lines and TCGA melanoma tumors.

Data from the two platforms were not normalized for direct comparison. (A) GPC6 expression in TCGA melanoma samples from four tissue sites (RNA-seq from TCGA). (B) Boxplots of the expression of GPC6 in normal melanocytes (four replicates) and melanoma cell line (mel-2183, two replicates) measured by Affymetrix arrays (GSE15805).

Fig 2. miR-509-1 expression and correlation with GPC6.

(A) Boxplots of miR-509-1 expression levels in TCGA melanoma samples for the four tissue sites. (B) Scatter plot of miR-509-1 and GPC6 expression levels in TCGA melanoma samples (N = 389). The red line indicates the regression line.

Fig 3. GPC6 is a putative target of miR-509-3p and overexpression of miR-509-3p downregulates GPC6 mRNA levels.

(A) Predicted binding sites of miR-509-3p on the 3’-UTR of human GPC6 gene (NM005708). (B-C) A375 cells were transfected with 4nM negative control mimic (mimic ctrl) or miR-509-3p mimic for 48 hours. RNA was collected and RNA levels of miR-509-3p (B) and GPC6 (C) were measured using Taqman assays relative to Rplp0 from n = 3 experiments. Means ± S.D. **, p < 0.001; ***, p < 0.0001 for miR-509-3p mimic versus mimic control.

Fig 4. Correlation between GPC6 and ZEB1 expression.

(A) GPC6 expression is correlated with ZEB1 in TCGA cutaneous skin melanoma samples. (B) Boxplots of RNA-seq expression levels of ZEB1 in TCGA cutaneous melanoma samples from primary site (skin), and metastases to cutaneous/subcutaneous tissue, lymph node, and distant sites.