Abstract
Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma.
Highlights
Acid ceramidase (N-acylsphingosine deacylase, #EC 3.5.1.23; AC) is a lysosomal cysteine hydrolase encoded by the ASAH1 gene, which catalyzes the conversion of ceramide into fatty acid and sphingosine[1]
The present study provides the first detailed description of the impact exerted by complete depletion of the ASAH1 gene, encoding for the lipid amidase AC, in a human melanoma cell line
The results show that AC ablation perturbs ceramide metabolism and directs A375 cells toward either apoptosis or senescence
Summary
Acid ceramidase (N-acylsphingosine deacylase, #EC 3.5.1.23; AC) is a lysosomal cysteine hydrolase encoded by the ASAH1 gene, which catalyzes the conversion of ceramide into fatty acid and sphingosine[1]. SiRNA-guided silencing of the ASAH1 gene reduces hepatocellular carcinoma growth in vivo[18] and synergizes with silencing of Akt to enhance death in a variety of cancer cell lines in vitro[20, 21]. In addition to these roles in apoptosis and chemoresistance, ceramide is implicated in the regulation of cellular senescence. In the present study we used CrispR/ Cas9-mediated gene editing to remove the ASAH1 gene and its protein product from A375 melanoma cells, which are known for their high invasiveness and self-renewal capabilities[26]
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