Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Alabama, 35233, USA.
Department of Pathology, Yale University School of Medicine, New Haven, CT, 06510, USA; Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, 08028, Spain.
Mol Metab. 2021 Jun;48:101217. doi: 10.1016/j.molmet.2021.101217. Epub 2021 Mar 23.
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.
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.
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.
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.
代谢失调是癌细胞的一个关键特征，已被证明可促进癌症生长和转移。然而，并非所有黑色素瘤的代谢驱动因素都已被发现。基于我们发现鞘氨醇酰基水解酶 1（ASAH1）在黑色素瘤中过度表达，本研究旨在确定其在黑色素瘤肿瘤生长和转移中的作用，了解其作用机制，并评估 ASAH1 靶向治疗黑色素瘤的疗效。
我们使用了公开的黑色素瘤数据集和黑色素瘤患者来源的样本以及正常皮肤组织，并使用免疫组织化学方法分析了这些样本中 ASAH1 mRNA 和 ASAH1 蛋白的表达。我们使用短发夹 RNA（shRNA）在多种黑色素瘤细胞系中敲低 ASAH1，并用一系列细胞培养实验和小鼠黑色素瘤异种移植实验来监测 ASAH1 敲低对黑色素瘤肿瘤生长和转移的影响。进行了一项无偏代谢组学分析，以确定 ASAH1 的作用机制。基于代谢组学研究结果，探索了过氧化物酶体介导的活性氧（ROS）产生在介导 ASAH1 作用中的作用。单独使用 ASAH1 抑制剂或与 BRAFV600E 抑制剂联合使用，评估 ASAH1 靶向治疗黑色素瘤的治疗价值。
我们确定 ASAH1 在很大比例的黑色素瘤细胞中过度表达，并通过丝裂原活化蛋白激酶（MAPK）途径依赖性转录因子 E2F1 进行调节。ASAH1 的表达对于维持黑色素瘤细胞培养物和小鼠黑色素瘤肿瘤生长和转移模型中的肿瘤生长和转移属性是必需的。为了确定 ASAH1 促进黑色素瘤肿瘤生长和转移的机制，我们对表达 ASAH1 shRNA 的黑色素瘤细胞进行了大规模、无偏的代谢组学分析。我们发现，ASAH1 抑制通过神经酰胺介导的过氧化物酶体增殖物激活受体γ（PPARγ）激活增加过氧化物酶体生物发生。ASAH1 缺失增加了神经酰胺和过氧化物酶体衍生的 ROS，从而抑制了黑色素瘤的生长。ASAH1 的药理学抑制也减弱了黑色素瘤的生长，并增强了 BRAF 激酶抑制剂在细胞培养物和小鼠中的疗效。
总之，这些结果表明 ASAH1 是一种可成药的黑色素瘤肿瘤生长和转移驱动因子，通过抑制过氧化物酶体生物发生，从而抑制过氧化物酶体衍生的 ROS 产生来发挥作用。这些研究还强调了 ASAH1 抑制剂在黑色素瘤治疗中的治疗应用。
