Supplementary Figure 2 from Sphingosine-1-Phosphate Produced by Sphingosine Kinase 1 Promotes Breast Cancer Progression by Stimulating Angiogenesis and Lymphangiogenesis
PDF file - 3.7MB, Effect of knockdown of SphK1 on S1P secretion from 4T1-luc2 cells and on in vitro hemangiogenesis and lymphangiogenesis. (A-C) 4T1-luc2 cells were stably transfected with control-shRNA or SphK1-shRNA. (A) Expression of SphK1 was determined by qPCR and normalized to GAPDH mRNA. *, P < 0.001. (B) SphK1 proteins were determined by western blot. (C) S1P in the culture medium from wild type (WT) 4T1-luc2 cells, 4T1-luc2 cells transfected with control-shRNA and SphK1-shRNA were determined by LC-ESI-MS/MS. *, P <0.01. (D, E) HUVECs and HLECs tagged with GFP were cultured on reduced growth factor basement membrane matrix-coated 48 well plates and incubated for 6 hours with culture media from the indicated 4T1-luc2 cells. (D) Representative images. (E) Two random fields per well were photographed. Total tube length was determined and means � SD are shown. *, P <0.05.
History
ARTICLE ABSTRACT
Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I–treated animals. Taken together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer–induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer. Cancer Res; 72(3); 726–35. ©2012 AACR.Usage metrics
