N-acylsphingosine amidohydrolase 1 promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production

Objective: Metabolic deregulation is a key hallmark of cancer cells and has been shown to drive cancer growth and metastasis. However, not all metabolic drivers of melanoma are known. Based on our finding that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in melanoma, the objective of these studies was to establish its role in melanoma tumor growth and metastasis, understand its mechanism of action, and evaluate ASAH1 targeting for melanoma therapy. Methods: We used publicly available melanoma datasets and patient-derived samples of melanoma and normal skin tissue and analyzed them for ASAH1 mRNA expression and ASAH1 protein expression using immunohistochemistry. ASAH1 was knocked down using short-hairpin RNAs in multiple melanoma cell lines that were tested in a series of cell culture-based assays and mouse-based melanoma xenograft assays to monitor the effect of ASAH1 knockdown on melanoma tumor growth and metastasis. An unbiased metabolomics analysis was performed to identify the mechanism of ASAH1 action. Based on the metabolomics findings, the role of peroxisome-mediated reactive oxygen species (ROS) production was explored in regard to mediating the effect of ASAH1. The ASAH1 inhibitor was used alone or in combination with a BRAFV600E inhibitor to evaluate the therapeutic value of ASAH1 targeting for melanoma therapy. Results: We determined that ASAH1 was overexpressed in a large percentage of melanoma cells and regulated by transcription factor E2F1 in a mitogen-activated protein (MAP) kinase pathway-dependent manner. ASAH1 expression was necessary to maintain melanoma tumor growth and metastatic attributes in cell cultures and mouse models of melanoma tumor growth and metastasis. To identify the mechanism by which ASAH1 facilitates melanoma tumor growth and metastasis, we performed a large-scale and unbiased metabolomics analysis of melanoma cells expressing ASAH1 short-hairpin RNAs (shRNAs). We found that ASAH1 inhibition increased peroxisome biogenesis through ceramide-mediated PPARγ activation. ASAH1 loss increased ceramide and peroxisome-derived ROS, which in turn inhibited melanoma growth. Pharmacological inhibition of ASAH1 also attenuated melanoma growth and enhanced the effectiveness of BRAF kinase inhibitor in the cell cultures and mice. Conclusions: Collectively, these results demonstrate that ASAH1 is a druggable driver of melanoma tumor growth and metastasis that functions by suppressing peroxisome biogenesis, thereby inhibiting peroxisome-derived ROS production. These studies also highlight the therapeutic utility of ASAH1 inhibitors for melanoma therapy.