Lupeol and Paclitaxel cooperate in hindering hypoxia induced vasculogenic mimicry via suppression of HIF-1α-EphA2-Laminin-5γ2 network in human oral cancer

Vasculogenic mimicry (VM), defined as an endothelial cell independent alternative mechanism of blood and nutrient supply by dysregulated tumor cells, is associated with poor prognosis in oral squamous cell carcinoma (OSCC). Here we aim to investigate the underlying molecular mechanism of the synergistic effect of phytochemical Lupeol and standard microtubule inhibitor Paclitaxel in reversing the hypoxia induced VM formation in OSCC. The results demonstrated that the hypoxia induced upregulation of HIF-1 alpha led to augmentation of signaling cascade associated with extracellular matrix remodeling and EMT phenotypes that are mechanistically linked to VM. Induction of HIF-1 alpha altered the expression of EMT/CSC markers (E-Cadherin, Vimentin, Snail, Twist and CD133) and enhanced the ability of cell migration/invasion and spheroid formation. Subsequently, the targeted knockdown of HIF-1 alpha by siRNA led to the perturbation of matrigel mediated tube formation as well as of Laminin-5 gamma 2 expression with the down-regulation of VE-Cadherin, total and phosphorylated (S-897) EphA2, pERK1/2 and MMP2. We also observed that Lupeol in association with Paclitaxel resulted to apoptosis and the disruption of VM associated phenotypes in vitro. We further validated the impact of this novel interventional approach in a patient derived tumor explant culture model of oral malignancy. The ex vivo tumor model mimicked the in vitro anti-VM potential of Lupeol-Paclitaxel combination through down-regulating HIF-1 alpha/EphA2/Laminin-5 gamma 2 cascade. Together, our findings elucidated mechanistic underpinning of hypoxia induced Laminin-5 gamma 2 driven VM formation highlighting that Lupeol-Paclitaxel combination may serve as novel therapeutic intervention in perturbation of VM in human OSCC.

Automated summary

In this study, we found that the upregulation of HIF-1α induced by hypoxia led to enhanced extracellular matrix remodeling and EMT phenotypes, resulting in increased VM formation. Knockdown of HIF-1α disrupted the signaling cascade and inhibited VM formation. Additionally, the combination of Lupeol and Paclitaxel induced apoptosis and disrupted VM-associated phenotypes.