The NLRP3 inflammasome - interleukin 1β axis in uveal melanoma

Abstract

Uveal melanoma (UM) is the most common primary intraocular cancer in the adult population. Recent studies suggested that the NLRP3 inflammasome could be a therapeutic target for cutaneous melanoma (CM), but the role of NLRP3 in UM remains unknown. Here, we analyzed the NLRP3-IL-1β axis in 5 UM and 4 CM cell lines. Expression of NLRP3 mRNA in UM and CM was low, and expression in UM was lower than in CM (P < 0.001). NLRP3 protein levels were below detection limit for all cell lines. UM exhibited lower baseline IL-1β secretion than CM, especially when compared to the Hs294t cell line (P < 0.05). Bioinformatic analysis of human tumor samples showed that UM has significantly lower expression of NLRP3 and IL-1β compared with CM. In conclusion, our work shows evidence of extremely low NLRP3 expression and IL-1β secretion by melanoma cells and highlight differences between CM and UM.

Keywords: IL-1; NLRP3; inflammasome; melanoma; skin; uveal.

Fig. 1

Uveal melanoma has lower NLRP3 expression compared with cutaneous melanoma. (A) NLRP3 mRNA expression levels of UM and CM cell lines measured by qPCR. (B) Comparison of NLRP3 mRNA expression of melanoma cell lines with monocyte cell line THP‐1. (C, D) NLRP3 protein expression analysis by western blotting in melanoma cell lines and the positive control THP‐1 at 42 s of membrane exposure. Saturated pixels are highlighted in red. (E, F) Overexposure of the same membrane at 10 min. (G) Coomassie blue staining of the membrane. Statistical significance was analyzed with ANOVA followed by post hoc Tukey HSD test. All values are expressed as mean ± SEM (n = 3).

Fig. 2

Uveal melanoma has minimal baseline secretion of IL‐1β. (A) Baseline secretion levels of IL‐1β by melanoma cell lines. (B, C) IL‐1β precursor (pro‐IL‐1β) protein expression analysis by WB in melanoma cell lines and the positive control (THP‐1‐derived macrophage) at 204 s of membrane exposure. Saturated pixels are highlighted in red. (D, E) Overexposure of the same membrane at 10 min. A faint band on OCM3 is indicated by the blue arrow. (F) Coomassie blue staining of the membrane. Statistical significance was analyzed with ANOVA followed by post hoc Tukey HSD test. All values are expressed as mean ± SEM (n = 5).

Fig. 3

Bioinformatic analysis from human samples reveals the low expression of NLRP3 and IL‐1β on uveal melanoma samples and normal choroidal melanocytes. (A, B) Analysis of UM and CM gene expression levels from the Cancer Genome Atlas (TCGA) database. UM cases show significantly lower levels of NLRP3 and IL‐1β compared with CM. (C) Single‐cell clustering of normal human choroidal tissue showing the heatmap of NLRP3 (blue) and IL‐1β (red) co‐expression. Melanocytes (green circle) have low expression of both genes. (D, E) Reclustering of melanocytes reveals that most cells do not express NLRP3 or IL‐1β. Statistical significance was analyzed with Mann–Whitney U test. Outliers are marked with X.