Regulation of tumor stroma angiogenesis by fibroblast cell-derived tetrahydrobiopterin


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Liye Chen1, Jihui Wang2, Jiliang Li1, Russell Leek1, Helen Turley1, David Kerr1, Adrian Harris1, Shijie Cai1

1University of Oxford, UK, 2College of Biology & Food Engineering, Dalian Polytechnic University, China

Abstract

Cancer-associated stromal fibroblasts are the predominant component of cancer stroma. Previous study with gene microarray shows a significant up-regulation of GCH-1 gene expression in fibroblasts from patients with metastatic colorectal cancer in liver. This finding suggests GCH-1 expression of GTP cyclohydrolase (GTPCH), a rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis potentially provides a favourable microenvironment for tumour angiogenesis. In this regard, we hypothesise that: (a) fibroblast cell derived-BH4 activates endothelial nitric oxide synthase (eNOS) for synthesis of nitric oxide (NO), an important signaling for tumour angiogenesis; (b) the activated eNOS by BH4 is phosphoinositide-3 kinase (PI3K)/Akt dependent.

In this study, we constructed a murine fibroblast cell line (3T3tet-off) with Dox-regulated expression of HA-tagged GTPCH (HA-GTPCH) construct “GCHtet-off”. Relatively to 3T3tet-off cells transfected with empty vector alone, GTPCH expression significantly enhanced cell proliferation in vitro and increased 4-fold of Ki-67 (a cell proliferation marker) and CD34 (an angiogenesis marker) in tumour stroma when xenografts were carried out. We also found BH4 synthesis increased Akt and eNOS phosphorylation in COS cells transfected with human eNOS plasmid DNA, corresponding to increased human umbilical vein endothelial cells (HUVEC) migration, tube formation and NO production, which however were abrogated by Ly294002, a specific inhibitor of PI3K.

Our results suggest BH4 synthesis by GTPCH in fibroblast cells of tumor stroma plays an important role in the regulation of angiogenesis in tumour stroma. BH4 in activation of eNOS appears to be PI3-K/Akt signaling dependent.

Cancer-associated stromal fibroblasts are the predominant component of cancer stroma. Previous study with gene microarray shows a significant up-regulation of GCH-1 gene expression in fibroblasts from patients with metastatic colorectal cancer in liver. This finding suggests GCH-1 expression of GTP cyclohydrolase (GTPCH), a rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis potentially provides a favourable microenvironment for tumour angiogenesis. In this regard, we hypothesise that: (a) fibroblast cell derived-BH4 activates endothelial nitric oxide synthase (eNOS) for synthesis of nitric oxide (NO), an important signaling for tumour angiogenesis; (b) the activated eNOS by BH4 is phosphoinositide-3 kinase (PI3K)/Akt dependent.

In this study, we constructed a murine fibroblast cell line (3T3tet-off) with Dox-regulated expression of HA-tagged GTPCH (HA-GTPCH) construct “GCHtet-off”. Relatively to 3T3tet-off cells transfected with empty vector alone, GTPCH expression significantly enhanced cell proliferation in vitro and increased 4-fold of Ki-67 (a cell proliferation marker) and CD34 (an angiogenesis marker) in tumour stroma when xenografts were carried out. We also found BH4 synthesis increased Akt and eNOS phosphorylation in COS cells transfected with human eNOS plasmid DNA, corresponding to increased human umbilical vein endothelial cells (HUVEC) migration, tube formation and NO production, which however were abrogated by Ly294002, a specific inhibitor of PI3K.

Our results suggest BH4 synthesis by GTPCH in fibroblast cells of tumor stroma plays an important role in the regulation of angiogenesis in tumour stroma. BH4 in activation of eNOS appears to be PI3-K/Akt signaling dependent.

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