Abstract A34: VEGFR-2 expression in carcinoid cancer cells and its role in tumor growth and metastasis
Authors: Silva Scott R. , Bowen Kanika A. , Rychahou Piotr G. , Jackson Lindsey N. , Lee Eun Y. , Townsend Courtney M. , Evers B. Mark 
Published: May 2012

Abstract
Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that have been increasing in incidence since the 1970s. Carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR-2); however, its role is not completely understood. The aims of this study were 1) to assess the expression and regulation of VEGFR-2 in carcinoid tumors and cells and 2) to evaluate the effect of VEGFR-2 on carcinoid growth and metastasis.
Methods: 1) The expression of VEGFR-2 in carcinoid tumor sections and in the novel carcinoid cell line BON, which was established and characterized in our laboratory, was assessed by immunohistochemistry andWestern blot, respectively. As VEGFR-2 signals through the PI3K/Akt pathway, signaling through Akt was adjusted chemically and through RNAi to delineate its effect on VEGFR-2 expression. 2) BON cell proliferation was measured following treatment with the VEGFR-2 inhibitor sunitinib malate to assess the effect of VEGF signaling on cell growth. A BON cell line with stably decreased VEGFR-2 expression, designated as BON shVEGFR-2, was generated by transfecting cells with shRNA to VEGFR-2. Boyden chamber and scratch assays were performed on BON shVEGFR-2 cells to assess the effect of VEGFR-2 on invasion and migration.
Results: 1) Immunhistochemical analysis confirmed positive expression of VEGFR-2 in the endothelial and epithelial components of carcinoid tumor sections. Consistent with this finding, BON cells also expressed VEGFR-2. Interestingly, we noted that the regulation of VEGFR-2 expression is inversely related to PI3K/Akt signaling. Inhibition of PI3K/Akt signaling with wortmannin upregulated VEGFR-2 expression, as noted with western blot and mRNA analysis. shRNA against PTEN increased phospho-Akt, resulting in decreased expression of VEGFR-2 in BON cells. BON shPTEN cells injected into the pancreas of nude mice formed increased liver metastases, compared to BON shControl cells, and the liver metastatic cells retained decreased VEGFR-2 expression. 2) Treatment with sunitinib malate had only a modest effect on BON cell inhibition. Consistent with our in vivo studies, BON shVEGFR-2 cells have decreased expression of E-cadherin compared to BON shControl cells. Additionally, the migratory and invasive potential of BON shVEGFR-2 cells is approximately four and 1.75 times that of control BON cells, respectively, at 48 h.
Conclusions: We demonstrate the expression and activity of VEGFR-2 in BON cells. The expression of VEGFR-2 on the epithelial component of carcinoid tumors and in the BON cell line, suggests an alternate role for VEGFR-2, in addition to its well-defined role in angiogenesis. The modest inhibition of BON cell growth with sunitinib malate suggests that VEGFR-2 on carcinoid tumor cells has a limited role in cell proliferation. Interestingly, we show that the level of VEGFR-2 expression in BON cells is inversely proportional to the degree of total Akt signaling, suggesting a negative feedback loop in the regulation of VEGFR-2. The finding that decreased VEGFR-2 expression results in increased invasion and migration suggests that VEGFR-2 may inhibit carcinoid metastasis. Thus, while VEGFR-2 inhibition prevents angiogenesis, it may also increase the metastatic potential of surviving carcinoid cells.
Citation Information: Cancer Res 2009;69(23 Suppl):A34.