Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Promotes Angiogenesis and Ischemia-Induced Neovascularization Via NADPH Oxidase 4 (NOX4) and Nitric Oxide-Dependent Mechanisms.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the ability to inhibit angiogenesis by inducing endothelial cell death, as well as being able to promote pro-angiogenic activity in vitro. These seemingly opposite effects make its role in ischemic disease unclear. Using Trail(-/-) and wildtype mice, we sought to determine the role of TRAIL in angiogenesis and neovascularization following hindlimb ischemia. METHODS AND RESULTS: Reduced vascularization assessed by real-time 3-dimensional Vevo ultrasound imaging and CD31 staining was evident in Trail(-/-) mice after ischemia, and associated with reduced capillary formation and increased apoptosis. Notably, adenoviral TRAIL administration significantly improved limb perfusion, capillary density, and vascular smooth-muscle cell content in both Trail(-/-) and wildtype mice. Fibroblast growth factor-2, a potent angiogenic factor, increased TRAIL expression in human microvascular endothelial cell-1, with fibroblast growth factor-2-mediated proliferation, migration, and tubule formation inhibited with TRAIL siRNA. Both fibroblast growth factor-2 and TRAIL significantly increased NADPH oxidase 4 (NOX4) expression. TRAIL-inducible angiogenic activity in vitro was inhibited with siRNAs targeting NOX4, and consistent with this, NOX4 mRNA was reduced in 3-day ischemic hindlimbs of Trail(-/-) mice. Furthermore, TRAIL-induced proliferation, migration, and tubule formation was blocked by scavenging H2O2, or by inhibiting nitric oxide synthase activity. Importantly, TRAIL-inducible endothelial nitric oxide synthase phosphorylation at Ser-1177 and intracellular human microvascular endothelial cell-1 cell nitric oxide levels were NOX4 dependent. CONCLUSIONS: This is the first report demonstrating that TRAIL can promote angiogenesis following hindlimb ischemia in vivo. The angiogenic effect of TRAIL on human microvascular endothelial cell-1 cells is downstream of fibroblast growth factor-2, involving NOX4 and nitric oxide signaling. These data have significant therapeutic implications, such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with cardiovascular disease and diabetes.