HL156 compounds, novel metformin derivatives, inhibit angiogenesis through AMPK mediated downregulation FOXM1 in gastric cancer cells

Abstract

Metformin, which is widely used as an anti-diabetic drug, has recently gained significant attention as a potential anti-cancer agent. Although a couple of potential mechanisms explaining anti-tumor effects have been suggested, its detailed molecular mechanisms remain elusive. HL156 compounds are novel biguanide derivatives based on metformin formula with more potent anti-tumor effects than metformin. In cell based assays, HL156 activates AMP-activated kinase (AMPK) more potently than metformin in various cancer cell lines.Our preliminary analysis of Reverse phase protein array (RPPA) data from gastric cancer patients indicated that proteins related to regulating angiogenesis were increased in tumors of patients with poor prognosis. In addition, a pilot in vivo efficacy study showed that HL156 significantly suppressed metastasis in breast cancer xenograft models.Motivated by these, we hypothesized that HL156 would inhibit angiogenesis that is one of the critical steps of metastatic process. Further, the anti-angiogenic mechanism of HL156 would be mediated through AMPK activation which downregulated oncogenic transcription factor FOXM1. Culturing HUVEC cells in the conditioned media treated with HL156 compounds to gastric cancer cells downregulated in vitro endothelial cell network formation. In the same conditioned media, the expression of VEGFA, a major angiogenic factor, was prominently suppressed. Also, the expression of FOXM1, a pleiotropic oncogenic transcription factor, was inhibited by HL156, which is correlated with activation of AMPK. To further interrogate the molecular mechanisms by which FOXM1 was down-regulated, we assessed CDK4/6 expression in gastric cancer cells after HL 156 treatment. CDK4/6, which phosphorylates and activates FOXM1, was downregulated leading to the decrease of phospho-FOXM1 (Ser35) level. Electrophoretic mobility shift assay showed that HL156 treatment repressed the FOXM1 binding to the VEGFA promoter.Taken together, these results suggest that HL156 alleviates tumor angiogenesis by VEGFA inhibition through AMPK mediated CDK4/6 - FOXM1 downregulation in gastric cancer cells.