DSE inhibits melanoma progression by regulating tumor immune cell infiltration and VCAN

Abstract

Dermatan sulfate epimerase (DSE) is a C5 epiminase that plays a key role in converting chondroitin sulfate into dermal sulfate. DSE is often upregulated during carcinogenesis of some types of cancer and can regulate growth factor signaling in cancer cells. However, the expression and function of DSE in human melanoma have not been reported. In this study, we investigated the influence of tumor-derived DSE in melanoma progression and the potential mechanism of their action. First, proteomic analysis of collected melanoma tissues revealed that DSE was significantly down-regulated in melanoma tissues. DSE silenced or overexpressed melanoma cells were constructed to detect the effect of DSE on melanoma cells, and it was found that the up-regulation of DSE significantly inhibited the proliferation, migration and invasion of melanoma cells. Data analysis and flow cytometry were used to evaluate the immune subpopulations in tumors, and it was found that the high expression of DSE was closely related to the invasion of killer immune cells. Mechanistically, DSE promoted the expression of VCAN, which inhibited the biological activity of melanoma cells. Together, these results suggest that DSE is downregulated in melanoma tissues, and that high expression of DSE can promote melanoma progression by inducing immune cell infiltration and VCAN expression.

Fig. 1. DSE is specifically low expressed in melanoma patients.

A Proteomics was used to analyze human melanoma and paracancerous tissues, and heat maps showed proteins specifically expressed in human melanoma tissues; B Volcanic maps showed differentially up-expressed (291) and down-expressed (301) proteins; C Combined analysis of specific expression in human melanoma and differential genes in human melanoma in TCGA; D qRT PCR analysis of mRNA levels of DSE in human melanoma and normal tissues (mean ± s.e.m. ***P < 0.001); E Western blot analysis of DSE levels in human melanoma and normal tissues; F The expression of DSE in human melanoma and normal tissues was analyzed by fluorescence staining (mean ± s.e.m. ***P < 0.001); G Expression of DSE protein in human melanoma cell line; H TCGA data were used to analyze the effect of DSE expression level on the prognosis of patients with human melanoma. Statistical significance was assessed by two-tailed unpaired Student’s t-test (D–G). Data are representative of three (D–G) independent experiments.

Fig. 2. DSE inhibits proliferation, invasion and migration of melanoma cells.

A A2508 cells were infected with lentivirus (PLVE) and DSE, respectively, and the expression levels of DSE protein in cells were analyzed by western blotting; B CCK8 assay to analysis cell viability of A2508 cells stably overexpressing DSE (mean ± s.e.m. *P < 0.05); C Colony formation assay to analyze cell proliferation of A2508 cells stably overexpressing DSE; (mean ± s.e.m. ***P < 0.001); D Transwell assay to analyze cell invasion and migration of A25O8 cells stably overexpressing DSE (mean ± s.e.m. **P < 0.01); E Knockdown of DSE in A875 cells and Western blot analysis of DSE protein expression levels in those cells; F CCK8 assay was used to analyze the cell viability of DSE knockout A875 cells; (mean ± s.e.m. **P < 0.01); G Colony formation assay to analyze cell proliferation of DSE knockout A875 cells (mean ± s.e.m. ***P < 0.001); H Transwell assay to analyze cell invasion and migration of DSE knockout A875 cells (mean ± s.e.m. **P < 0.01). Data are representative of three (A–H) independent experiments.

Fig. 3. DSE inhibits melanoma growth and metastasis in vivo.

A Analysis of the effect of DSE on tumor growth using cells overexpressing or knocking out DSE injected subcutaneously into nude mice (n = 4 mice, mean ± s.e.m. **P < 0.01); B Weight of tumor tissue in A (n = 4 mice, mean ± s.e.m. *P < 0.05, **P < 0.01); C, D Expression levels of Ki67 in tumor tissue in A (n = 4 mice, mean ± s.e.m. ***P < 0.001); E Tail vein injection of cells overexpressing or knocking out DSE into nude mice to analyze lung metastasis (n = 4 mice, mean ± s.e.m. **P < 0.01).

Fig. 4. DSE enhances the antitumor immune microenvironment.

A Analysis of immune infiltration scores in TCGA human melanoma patients; B Correlation analysis of immune infiltration scores with DSE expression levels; C Subcutaneous inoculation of B16 cells into C57/B6 mice and collection of lymphocytes infiltrating within the tumor for flow analysis of CD4⁺ and CD8⁺ T cell ratios (n = 5 mice, mean ± s.e.m. **P < 0.01); D Flow analysis of GZMB in CD8 + T cells, TNF and IFNG expression levels in CD8⁺ T cells (n = 5 mice, mean ± s.e.m. **P < 0.01); E Flow analysis of PD-1, TIM-3 and LAG-3 expression levels in CD8⁺ T cells (n = 5 mice, mean ± s.e.m. **P < 0.01).

Fig. 5. DSE in melanoma patient tissues is proportional to antitumor immune effector molecules.

A Immunofluorescence staining of DSE, IFNG, GZMB and TNF in melanoma patient tissue; B Statistical analysis of the correlation between DSE and IFNG, GZMB and TNF expression levels; C Immunofluorescence staining of DSE, PD-1 in melanoma patient tissue, TIM3 and LAG3; D Statistical analysis of the correlation between DSE and PD-1, TIM3 and LAG3 expression levels.

Fig. 6. DSE regulates the expression of VCAN.

A Transcriptome analysis of DSE-knockdown A875 cells; B Volcano plot showing differentially expressed genes in DSE-knockdown A875 cells; C–E VCAN protein and mRNA expression in DSE-overexpressing or knockdown cells level (mean ± s.e.m. **P < 0.01); F The correlation between DSE and VCAN expression in melanoma tissues was analyzed by immunofluorescence. Data are representative of three (C–E) independent experiments.

Fig. 7. DSE regulates melanoma proliferation, invasion and migration through VCAN.

A CCK8 assay to analyze the cell viability of A2508 cells overexpressing DSE and knocking out VCAN (mean ± s.e.m. **P < 0.01); B A2508 cells overexpressing DSE and knocking out VCAN were injected into nude mice to observe tumor growth; (n = 4 mice, mean ± s.e.m. **P < 0.01 and ***P < 0.001); C tumor weight in B (n = 4 mice, mean ± s.e.m. **P < 0.01); D Transwell assay analyzing DSE overexpressing and VCAN knockout Invasion and migration of A2508 cells (mean ± s.e.m. **P < 0.01); E Tail vein injection of cells overexpressing or knocking out DSE into nude mice to analyze lung metastasis (n = 4 mice, mean ± s.e.m. **P < 0.01 and ***P < 0.001). Data are representative of three (A–D) independent experiments.