C. Hall1, L. Ehrlich2, T. Shepperd2, A. O’Brien2, G. Alpini2, S. Glaser2, T. C. Lairmore1 1Scott & White Healthcare,Temple, Texas, USA 2Texas A & M Health Science Center College Of Medicine,Temple, TX, USA
Introduction:
Cholangiocarcinoma (CCA) is a malignancy of the intrahepatic and extrahepatic biliary epithelium that is associated with low five-year survival despite multidisciplinary treatment strategies. Tumor angiogenesis correlates with CCA progression, metastases, and patient survival. The apelin receptor (APLNR), which is activated by the apelin peptide, is a G-protein coupled receptor that has been implicated in the growth and angiogenesis of other malignancies, such as colon, breast, prostate and hepatocellular carcinoma, but has not been studied in CCA. The purpose of this study is to quantify APLNR expression in CCA, characterize the proliferative and angiogenic effects of receptor activation, and determine if inhibition of the APLNR axis can inhibit tumor growth in a murine xenograft model.
Methods:
In vitro, CCA cell lines (CCLP, HuH-28, HuCCT-1, SG231, TFK-1 and Mz-ChA-1) and benign cholangiocytes (H69) were used to measure the expression of apelin and the APLNR via flow cytometry, ELISA and immunofluorescence. Immunohistochemistry (IHC) and qPCR was used to measure APLNR expression in human CCA tissues. Mz-ChA-1 cells were treated with increasing concentrations of apelin and ML221, an APLNR antagonist. Expression of proliferative (Ki-67 and PCNA) and angiogenic (VEGF-A, ANG1, ANG2) genes were measured via qPCR. Phosphorylation of the ERK1/2 pathway, a known pathway for cholangiocyte proliferation, was measured using flow cytometry and immunoblots. In vivo, Mz-ChA-1 cells were injected into the flanks of NU/NU immunocompromised mice, which were treated with ML221 (150 ?g/kg) via tail vein injection for 4 weeks.
Results:
APLNR expression and apelin secretion was upregulated in human CCA cells and tissues compared to benign controls. In vitro, treatment of Mz-ChA-1 cells with apelin increased proliferation and angiogenesis via activation of the ERK1/2 pathway in a dose-dependent response, whereas, ML221 inhibited these affects. Treatment of Mz-ChA-1 cells with apelin also increased expression of the apelin gene, suggesting an autocrine/paracrine mechanism of receptor activation. Treatment of CCA tumors in NU/NU mice with ML221 significantly decreased tumor growth in the xenograft model (Figure 1).
Conclusion:
APLNR is increased in CCA tissues and the autocrine/paracrine effects of APLNR receptor signaling regulate tumor growth and angiogenesis, both in vitro and in vivo. Inhibition of the APLNR axis decreases tumor growth in our xenograft CCA model. Targeting APLNR signaling has the potential to serve as a novel, tumor directed therapy for CCA by inhibiting cell proliferation and angiogenesis.
