Background:Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models.Methods:Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used.Results:In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg−1) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis.Conclusions:Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC.
We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under aerobic and anaerobic conditions, respectively, to investigate the sequential metabolism of chemicals by the liver and microflora in vitro. The culture device consisted of two chambers separated by a permeable polycarbonate membrane. In the aerobic compartment, hepatocytes were cultivated as a monolayer on the membrane and in the anaerobic compartment fecal microflora as a suspension. To characterize the metabolic capacity of the microflora and hepatocytes, various marker enzymes were studied. Azoreductase, nitroductase, beta-glucuronidase, beta-glucosidase and sulphatase were tested in the microflora of the feces from three volunteers who had had significantly different eating habits for years (daily meat, mixed diet, vegetarian). The microflora exhibited significant activities and the various enzymes differed only moderately in the samples from the three volunteers. For rat hepatocytes the activities of various cytochrome P450 forms and conjugating enzymes served as markers. The enzyme activities were tested in the coculture system during a 4-h culture period intended for the test protocol. Deethylation of ethoxycoumarin and 2alpha-, 6beta- and 16alpha-hydroxylation of testosterone decreased by about 30%, 25%, 40% and 20%, respectively, while there was no loss of glucuronidation and sulphonation of 3-OH-benzo(a)pyrene nor of glutathione conjugation of 1-chloro-2,4-dinitrobenzene during the 4-h culture period. The activities of the tested hepatic phase I and II enzymes were not changed after coculture of the hepatocytes with the microflora for 4 h. The applicability of the in vitro system for studying the metabolic interaction of liver and microflora was demonstrated using 7-ethoxycoumarin and the developmental drug EMD 57033, a thiadiazinon derivative from Merck KGaA, as model compounds. Both compounds were oxidized and conjugated by liver cells. In the coculture of hepatocytes and fecal microflora the resulting glucuronides and sulphoconjugates were split by hydrolytic enzymes of the intestinal microflora.
Activation of the fibroblast growth factor receptor (FGF/FGFR) system has been reported in a large number of human cancers including human hepatocellular carcinoma (HCC). Furthermore, expression of FGFR2 has been associated with aggressive tumor growth and poor prognosis in this tumor entity. Moreover, HCC is known to be a hypervascularized tumor and activation of the FGF/FGFR system is a crucial event in tumor angiogenesis. Hence, we addressed the issue of targeting the FGFR system in HCC (Huh-7, HepG2) and stromal cells (endothelial cells (EC), vascular smooth muscle cells (VSMC), hepatic stellate cells (HSC)). The FGFR inhibitor BGJ398 (Novartis Oncology, Basel, Switzerland) was used to inhibit FGFR activation. Effects of targeting FGFR on growth of cells were determined by MTT assays. Inhibition of constitutive and growth factor induced cell motility was investigated in modified Boyden Chamber assays. Activation of signaling pathways upon treatment with BGJ398 was assessed by Western Blot analyses whereas effects on mediators of tumor angiogenesis (VEGF-A, PDGF-B) where measured by RT-PCR and ELISA, respectively. Results show that inhibition of the FGF/FGFR system with BGJ398 led to dose-dependent inhibition of growth in tumor cells and ECs whereas only minor effects were observed on VSMC and HSCs. Moreover, motility of tumor and stromal cells (ECs, VSMCs, HSCs) was significantly impaired upon constitutive conditions (P<0.05 for all). In addition, FGFR inhibition led to decrease in HSC motility even when cell were incubated with conditioned media from cancer cells (P<0.05). With regards to activation of signaling intermediates, FGFR inhibition impaired constitutive and growth factor induced phosphorylation of Akt and ERK in HCC cell lines, ECs and HSCs whereas only minor effect was observed in VSMCs. Moreover, treatment with BGJ398 led to inhibition of c-Myc expression and significantly reduced VEGF-A production from cancer cells, as determined by Western blotting and ELISA, respectively. Evaluation of effectiveness of BGJ398 in animal models is currently ongoing. However, results so far indicate that targeting FGFR impairs angiogenic signaling and motility in HCC cell lines and stromal cells. Therefore, inhibition of FGFR warrants further evaluation and might be an interesting novel approach for anti-neoplastic treatment protocols in human HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4373. doi:1538-7445.AM2012-4373
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