Molecular mechanisms for leptin regulation of VEGF in breast cancer cells.

Abstract

Abstract Abstract #3071 Background: We have previously reported that in vitro and in vivo leptin signaling mediates proliferation of mouse 4T1 mammary tumor (MT) cells and levels of VEGF and VEGFR2. Therefore, we hypothesize that leptin can contribute to MT growth by augmenting angiogenesis through the regulation of VEGF expression. To test this we assessed whether leptin regulates VEGF promoter activity in mouse MT cells and if leptin-induced factors (HIF-1α, NFκB, AP1, SP1) regulate the VEGF promoter. Materials and Methods: Mouse MT cells (4T1, EM6 and MMT) were transiently transfected with molecular engineered luciferase-reporters (pGL3) for mouse VEGF promoter (full-length) and transcription factor-binding deletions for hypoxia responsive element (HRE), AP1, SP1 and NFκB. Leptin dose-response effects on reporter activity and VEGF levels (protein and mRNA) were investigated using ELISA, Western blot and real-time PCR, respectively. To further assess leptin regulation of VEGF expression in MT cells the effects of hypoxia (CoCl2, hypoxia mimetic agent) alone and combined with leptin on VEGF promoter activity were investigated. To assess which leptin-induced signaling pathways/transcription factors are linked to VEGF regulation in MT cells inhibitors for: (1) JAK2/STAT3, MEK-1/MAPK and PI-3K/Akt-1 pathways and; (2) target molecules NFκB; SP1; AP1 and HIF-1α were used. The effects of leptin/CoCl2 and inhibitors on luciferase activity of transfected cells were compared using pRL-TK renilla luciferase as control and the Dual-Luciferase Reporter Assay System. Results: Our data suggest that leptin signaling can regulate the transcriptional activity of VEGF gene in breast cancer cells by activating gene transcription at several sites of the VEGF promoter. Leptin-induced HIF-1α has a relevant role in the regulation of VEGF expression in MT cells. Leptin can also increase VEGF-levels (protein and mRNA) through the specific activation of AP1, SP1, and NFκB. PI-3K activity was mainly linked to leptin regulatory effects on VEGF. However, diverse mechanisms for leptin regulation of VEGF expression were found in the different MT cells. Discussion: Our results provide novel information on the molecular mechanisms for leptin-induced angiogenic effects in breast cancer. Our data further suggest that disruption of leptin signaling could impact angiogenesis and MT growth by inhibiting VEGF expression. Thus targeted disruption of leptin-signaling could serve as method of prevention/treatment of breast cancer. This work was supported in part by the Susan G. Komen Foundation for the Cure (to R.R.G.; BC 504370); UAB Breast SPORE Award (to R.R.G) and Contraceptive Research and Development Program (CONRAD; CIG-07-114 to R.R.G). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3071.