The physiologic conditions and signaling mechanisms

The physiologic conditions and signaling mechanisms that regulate apelin secretion and APLNR expression in malignant tissues appear to be multifactorial. Previous studies have shown that hyperplastic and neoplastic cholangiocytes secrete a variety of hormones, peptides, and growth factors that help regulate cell proliferation [36]. Hypoxia appears to be a major factor in tumor angiogenesis by increasing expression of VEGF and hypoxia-inducible factor (HIF) [5], [37]. Similarly, Heo et al. demonstrated that apelin expression was significantly up regulated under hypoxic conditions in oral squamous cell carcinoma, which correlated with increased cell proliferation and migration [38]. Additionally, high levels of serum apelin in gastric and esophageal cancer patients correlates with high levels of C-reactive protein, indicating that apelin may be involved in the systemic inflammatory response of certain malignancies [39]. Studies in chronic liver diseases have also shown that hypoxia and inflammatory conditions are capable of inducing apelin expression, which creates an angiogenic and fibroproliferative response [40]. Additionally, Wan et al. demonstrated that apelin is a target gene for microRNA-224 (miR-224) and that low miR-224 levels correlates with elevated apelin levels in prostate cancer tissues, which is associated with increased cancer progression, advanced stage, and decreased disease-free survival [41]. Benign and malignant cholangiocytes proliferate in response to numerous peptides, hormones, and growth factors during normal physiologic conditions and in response to biliary injury [2], [36]. Activation of the PKA/Src/MEK/ERK1/2 phosphorylation cascade is a common pathway that promotes cholangiocyte proliferation [42]. ERK1/2 signaling has also been implicated in the proliferation and autophagy of lung adenocarcinoma 71 5 australia when stimulated with apelin [43]. Additionally, apelin-mediated ERK signaling has also been shown to regulate cardiomyocyte hypertrophy and to activate the expression of inflammatory cytokines in microglial cells [44], [45]. Our results show that ML221 treatment decreased Mz-ChA-1 tumor expression of p-ERK and t-ERK, indicating that this pathway may become less active with ML221 treatment. Previous studies have demonstrated that apelin's involvement in cell proliferation is not limited to the ERK1/2 phosphorylation cascade. Masri et al. demonstrated that apelin induces a time-dependent phosphorylation of p70S6K, which is associated with transduction of PI3K and ERK phosphorylation cascades [46]. More recent studies have shown that apelin-mediated activation of PI3K/Akt is associated with proliferative and anti-apoptotic properties [47]. Zeng et al. demonstrated that apelin is neuroprotective by inhibiting apoptosis in cortical neurons via phosphorylation of Akt and ERK1/2 [48]. Additionally, apelin has been shown to stimulate proliferation and inhibit apoptosis in mouse osteoblasts via activation of JNK and PI3K/AKT signaling pathways [49]. APLNR signaling has also been shown to induce nitric oxide synthase in endothelial cells and decrease intracellular reactive oxygen species, however, it is unclear if these signaling properties also help regulate cell proliferation [47]. Our data shows that ML221 treatment decreased expression of angiogenic factors in a dose-dependent response. Angiogenesis has been considered critical to for the development and progression of CCA [50]. In recent studies, tumor-associated angiogenesis and lymphangiogenesis has been implicated in intrahepatic and hilar CCA disease progression, lymph node metastases, and overall prognosis [51], [52], [53]. Furthermore, microvascular density has been shown to significantly decrease 5-year survival rates [51]. Additionally, angiogenesis was linked to a poor prognosis in patients with node-negative intrahepatic cholangiocarcinoma [54]. These studies emphasize why targeting the mechanisms of angiogenesis and neovascularization in CCA, such as the apelin/APLNR axis, may help improve long-term survival.