c-Myc promotes lymphatic metastasis of pancreatic neuroendocrine tumor through VEGFC upregulation

Abstract

Pancreatic neuroendocrine tumor (pNET) is a pancreatic neoplasm with neuroendocrine differentiation. pNET in early stage can be treated with surgical resection with long-term survival, whereas the prognosis of pNET with locoregional or distant metastasis is relatively poor. Lymphangiogenesis is essential for tumor metastasis via the lymphatic system and may overhead distant metastasis. c-Myc overexpression is involved in tumorigenesis. The role of c-Myc in lymphangiogenesis is unclear. In this study, we evaluated the mechanism and effect of c-Myc on lymphangiogenesis of pNET via interaction of lymphatic endothelial cells (LECs) and pNET cells. Lymph node metastasis was evaluated in pNET xenograft mice. Potential target agents to inhibit lymph node metastasis were evaluated in an animal model. We found that vascular endothelial growth factor C (VEGFC) expression and secretion was increased in pNET cell lines with c-Myc overexpression. c-Myc transcriptionally upregulates VEGFC expression and the secretion of pNET cells by directly binding to the E-box of the VEGFC promoter and enhances VEGF receptor 3 phosphorylation and the tube formation of LECs. c-Myc overexpression is associated with lymph node metastasis in pNET xenograft mice. Combinational treatment with an mTOR inhibitor and c-Myc inhibitor or VEGFC-neutralizing chimera protein reduced lymph node metastasis in the mice with c-Myc overexpression. The mTOR inhibitor acts on lymphangiogenesis by reducing VEGFC expression in pNET cells and inhibiting the tube formation of LECs. In conclusion, mTOR and c-Myc are important for lymphangiogenesis of pNET and are potential therapeutic targets for prevention and treatment of lymph node metastasis in pNET.

Keywords: c-Myc; lymphangiogenesis; mTOR; pancreatic neuroendocrine tumor; vascular endothelial growth factor C.

FIGURE 1

c‐Myc positively correlates with vascular endothelial growth factor C (VEGFC) expression in pancreatic neuroendocrine tumor (pNET) cells. A, VEGFC expression in QGP‐1 and NIT‐1 cells with and without knockdown of c‐Myc. B, VEGFC expression in QGP‐1 and NIT‐1 cells with and without overexpression of c‐Myc. C, Secretory amount of VEGFC in QGP‐1 and NIT‐1 cells with and without overexpression of c‐Myc. VC, vector control

FIGURE 2

c‐Myc transcriptionally upregulates vascular endothelial growth factor C (VEGFC) expression. A, RNA expression of QGP‐1 and NIT‐1 cells with and without c‐Myc overexpression or knockdown by RT‐PCR. VC, vector control. B, The luciferase activity of the VEGFC promoter in 293T cells transfected with three VEGFC promoter plasmids (−1046/+38, −439/+38, −185/+38) with stimulation of condition medium from QGP‐1 cells with and without c‐Myc overexpression. *P < .01. C, Chromatin immunoprecipitation (ChIP) assay for VEGFC and c‐Myc in QGP‐1 cells with and without c‐Myc overexpression. D, Luciferase activity of the VEGFC promoter in 293T cells transfected with wild‐type or E‐box–mutant VEGFC promoter (−1046/+38) and stimulation of condition medium from QGP‐1 cells with or without c‐Myc overexpression. **P < .01

FIGURE 3

c‐Myc enhanced vascular endothelial growth factor receptor 3 (VEGFR3) phosphorylation and tube formation of lymphoepithelial cell (LECs). A, Expression of phosphorylated VEGFR3 and Prox1 in LECs with the addition of condition medium derived from QGP‐1 cells with and without c‐Myc overexpression and the addition of VEGFR3/Fc in the LEC culture in condition medium from QGP‐1 cells with c‐Myc overexpression. B, Tube formation of LECs treated with condition medium in the three conditions of (A). Relative tube formation of LECs treated with condition medium derived from QGP‐1 cells without c‐Myc overexpression (vector control [VC]) was defined as 1. *P < .05

FIGURE 4

The effect of mTOR inhibitor, c‐Myc inhibitor, and vascular endothelial growth factor C (VEGFC)‐neutralizing recombinant protein on tumor growth and lymph node metastasis in a pancreatic neuroendocrine tumor (pNET) mouse model. A, Tumor growth curves of c‐Myc–overexpressing QGP‐1 xenograft mice treated with RAD001, 10058‐F4, VEGFR3/Fc, or a combination of RAD001 and 10058‐F4 or VEGFR3/Fc (vector control [VC] vs c‐Myc overexpression group,P = .003; c‐Myc overexpression vs c‐Myc overexpression + RAD001,P = .005; c‐Myc overexpression vs c‐Myc overexpression + 10058‐F4,P = .009; c‐Myc overexpression vs c‐Myc overexpression + RAD001 + 10058‐F4,P = .01; c‐Myc overexpression vs c‐Myc overexpression + RAD001 + VEGFR3/Fc,P = .02, Wilcoxon’s rank‐sum test). B, H&amp;E staining of the lymph node of mice in each group (upper) and the weight (mean ± standard error) and number of mice with lymph node metastasis in each group

FIGURE 5

mTOR inhibitor inhibits the phosphorylation of vascular endothelial growth factor receptor 3 (VEGFR3) and tube formation of human and murine lymphoepithelial cells (LECs). A, Phosphorylated VEGFR3 expression (upper) and tube formation (lower) in human LECs treated with and without RAD001. **P < .001. B, Phosphorylated VEGFR3 expression (upper) and tube formation (lower) in murine LECs (SVEC4‐10) treated with and without RAD001. *P = .03

FIGURE 6

The putative model of lymphatic metastasis in pancreatic neuroendocrine tumor (pNET). c‐Myc can be regulated by PTEN and LKB1 via the AKT/mTOR axis, and it can backregulate PTEN to activate the mTOR pathway. c‐Myc can upregulate VEGFC expression and the secretion of QGP‐1 cells (pNET) and promotes lymphangiogenesis and lymph node metastasis. mTOR is constitutively activated in pNET and can be negatively regulated by PTEN and/or LKB1. mTOR promotes lymphangiogenesis in pNET via increasing vascular endothelial growth factor C (VEGFC) expression of pNET cells and inhibiting tube formation of lymphoepithelial cells (LECs) in pNET. Combined treatment with an mTOR inhibitor, RAD001, and a c‐Myc inhibitor (10058‐F4) or a VEGFC‐neutralizing chimeric protein (VEGFR/Fc) reduces the lymphangiogenesis and lymph node metastasis of pNET in a mouse model