Hepatocellular carcinoma (HCC) is a highly aggressive cancer with poor prognosis due to its extensive vascularization. Antiangiogenic therapy shows promise but faces limitations like adverse events and drug resistance. Tumor angiogenesis is a complex process involving various molecules in the tumor microenvironment (TME), including cytokines and inflammatory factors, which influence endothelial cell (EC) proliferation, migration, and tube formation. Cytokines can transform normal ECs into HCC-associated ECs (HAECs), promoting angiogenesis. Metabolic reprogramming of ECs is also implicated in tumor angiogenesis, with alterations in fatty acid oxidation, glycolysis, and glutamine metabolism impacting tumor development. However, the role of sphingolipid metabolism reprogramming in vascular ECs on HCC angiogenesis remains unclear. Sphingolipid metabolism, with ceramide as a central molecule, significantly contributes to carcinogenesis. Ceramide metabolism produces bioactive molecules like sphingosine-1-phosphate (S1P) and sphingosine (SPH). S1P, via its receptors S1PR1-5, affects vascular growth and maturation, and S1PR1, in particular, is implicated in tumor angiogenesis. The role of S1PR1 in HCC angiogenesis and its underlying mechanisms were the focus of this study. Ceramide synthases (CerS1-6) synthesize ceramides of varying fatty acyl chain lengths, each with different roles in physiological and pathological processes. This study investigated the role of S1PR1 in HCC vascular ECs, hypothesizing that S1PR1 upregulation in these cells promotes HCC angiogenesis and progression.

The literature extensively discusses the role of angiogenesis in HCC progression and the limitations of current antiangiogenic therapies. Studies have highlighted the involvement of cytokines and inflammatory factors in tumor angiogenesis, with a focus on the transformation of normal ECs into HAECs. Metabolic reprogramming of ECs, affecting various metabolic pathways, is also recognized as a driver of angiogenesis. Sphingolipid metabolism, particularly ceramide and its metabolites S1P and SPH, are implicated in cancer development. S1P, through its receptors, notably S1PR1, impacts vascular growth and has been associated with tumor angiogenesis. Previous research has highlighted the importance of ceramide synthases (CerS) in regulating ceramide levels, with different CerS isoforms producing ceramides of varying chain lengths, each with distinct biological effects. Although some studies have suggested links between ceramide and angiogenesis, the specific role and mechanisms of action of endogenous ceramide in mammalian angiogenesis, especially concerning HCC, remained poorly understood before this study.

This study employed a combination of in vitro and in vivo experiments to investigate the role of S1PR1 in HCC angiogenesis. In vitro studies utilized tube-formation assays, luciferase reporter assays, and cell migration and proliferation assays. Tube formation assays assessed the ability of ECs to form capillary-like networks in Matrigel. Luciferase reporter assays quantified the activity of CerS3 and CerS6 promoters in response to S1PR1 manipulation. Cell migration and proliferation assays evaluated the effects of S1PR1 on EC behavior. In vivo studies used subcutaneous xenograft models in nude mice. HCC cells were injected subcutaneously, alone or in combination with ECs, either with or without S1PR1 knockdown. Tumor growth was monitored over time. Immunohistochemistry (IHC) and immunofluorescence (IF) were used to assess protein expression and localization in tissue samples. Quantitative PCR (qPCR) was performed to measure gene expression levels. Western blotting (WB) was used to detect protein levels. Statistical analysis involved Student's t-tests, one-way ANOVA, and other appropriate statistical methods. Specific assays included CCK-8 for cell proliferation, transwell assays for cell migration and invasion, and ELISA to measure S1P levels. Pharmacological inhibitors such as FB1 (CerS inhibitor) and stattic (STAT3 inhibitor), and W146 (S1PR1 antagonist) were used to investigate specific mechanisms and pathways. A PCR array was used to investigate the sphingolipid metabolic pathway. Proximity ligation assay (PLA) was utilized to examine the interaction between ceramide and PTEN.

This study revealed that S1PR1 is selectively and highly expressed in the blood vessels of HCC tissues compared to peritumoral tissues. High S1PR1 expression in ECs significantly promoted HCC angiogenesis and progression both in vitro and in vivo. HCC-derived proangiogenic factors (S1P, IL-6, and VEGFA) induced S1PR1 upregulation in ECs through STAT3 phosphorylation at Y705. Crucially, S1PR1 promoted angiogenesis by decreasing ceramide levels via CerS3 downregulation. S1PR1 downregulated CerS3 by inducing CerS6 translocation into the nucleus, inhibiting CerS3 at the transcriptional level. A high concentration of Lenvatinib significantly downregulated S1PR1 expression, enhancing S1PR1 knockdown-mediated angiogenesis inhibition. In vitro experiments showed that S1PR1 knockdown decreased the proliferation, migration, and invasion of HCC cells when co-cultured with ECs. In vivo, the tumor volume of xenografts generated by the Huh7 + EC-NC cell mixture was significantly larger than the Huh7 group and the Huh7 + EC-shS1PR1 group. The reduction in ceramide levels activated AKT/ERK signaling by inhibiting PTEN expression. The study also found an interaction between ceramide and PTEN, further supporting the mechanism of action. Treatment with FB1, a CerS inhibitor, promoted tumor growth, which was rescued by S1PR1 knockdown, indicating that S1PR1-regulated ceramide levels are important in HCC angiogenesis. W146, a specific S1PR1 antagonist, inhibited S1PR1 expression and reduced EC migration and tube formation induced by HCC cells. Lenvatinib, at higher concentrations, significantly decreased S1PR1 protein expression, and the combination of Lenvatinib and S1PR1 knockdown showed a synergistic antiangiogenic effect.

This study provides novel insights into the role of S1PR1 in HCC angiogenesis. The findings demonstrate that S1PR1 is not only highly expressed in HCC blood vessels but also plays a critical role in promoting tumor growth and metastasis by regulating ceramide metabolism in ECs. The identification of the S1PR1-CerS3-CerS6-ceramide axis offers a potential new therapeutic target. The study's finding that Lenvatinib downregulates S1PR1 suggests a potential mechanism for its antiangiogenic effects. The synergistic effect of combining Lenvatinib with S1PR1 knockdown further supports this concept. The observed interaction between ceramide and PTEN adds another layer of complexity to the understanding of ceramide's role in angiogenesis. However, the study should be extended to other cancer types or investigate further the underlying molecular mechanisms of the interaction between ceramide and PTEN. The contradictory findings of previous research on S1PR1's role in angiogenesis might stem from the different cell types and model systems used. Further investigations are warranted to clarify the precise role of S1PR1 and its isoforms in different cancer contexts.

This research identifies S1PR1 as a crucial regulator of angiogenesis in HCC, acting through a novel mechanism involving ceramide metabolic reprogramming. The findings highlight S1PR1 as a potential therapeutic target for HCC, and the synergistic effect observed with Lenvatinib suggests a promising combination therapy approach. Future research could focus on developing specific S1PR1 inhibitors or exploring the therapeutic potential of targeting the S1PR1-CerS3-CerS6 axis. Further investigation of the ceramide-PTEN interaction and its precise role in AKT/ERK signaling is also warranted.

The study primarily focused on in vitro and in vivo models, thus limiting the direct translation of findings to human patients. While the study suggests a potential role for S1PR1 as a target for Lenvatinib, the exact mechanism and the clinical implications remain to be determined through further research. The sample size in some of the in vivo experiments could be considered relatively small. Although the study suggests that the ceramide-PTEN interaction is crucial in this mechanism, further research is needed to validate and fully elucidate the exact nature of this interaction.