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Background & Aims Recently, overexpression of the fibroblast growth factor receptor 3 (FGFR3) splice variants FGFR3‐IIIb and FGFR3‐IIIc was found in ~50% of hepatocellular carcinoma (HCC). Here, we aim to identify FGFR3‐IIIb/IIIc ligands, which drive the progression of HCC. Methods FACS, MTT assay and/or growth curves served to identify the FGFR3‐IIIb/IIIc ligand being most effective to induce growth of hepatoma/hepatocarcinoma cell lines, established from human HCC. The most potent FGF was characterized regarding the expression levels in epithelial and stromal cells of liver and HCC and impact on neoangiogenesis, clonogenicity and invasive growth of hepatoma/hepatocarcinoma cells. Results Among all FGFR3‐IIIb/IIIc ligands tested, FGF9 was the most potent growth factor for hepatoma/hepatocarcinoma cells. Replication and/or sprouting of blood/lymphendothelial cells was stimulated as well. FGF9 occurred mainly in stromal cells of unaltered liver but in epithelial cells of HCC. Every fifth HCC exhibited overexpressed FGF9 and frequent co‐upregulation of FGFR3‐IIIb/IIIc. In hepatoma/hepatocarcinoma cells FGF9 enhanced the capability for clonogenicity and disintegration of the blood and lymphatic endothelium, being most pronounced in cells overexpressing FGFR3‐IIIb or FGFR3‐IIIc, respectively. Any of the FGF9 effects in hepatoma/hepatocarcinoma cells was blocked completely by applying the FGFR1‐3‐specific tyrosine kinase inhibitor BGJ398 or siFGFR3, while siFGFR1/2/4 were mostly ineffective. Conclusions FGF9 acts via FGFR3‐IIIb/IIIc to enhance growth and aggressiveness of HCC cells. Accordingly, blockade of the FGF9‐FGFR3‐IIIb/IIIc axis may be an efficient therapeutic option for HCC patients.

This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46. KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics. Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme. Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy. Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization. Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L. Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway.

Fibroblast growth factor receptors (FGFRs) are frequently up-regulated in subsets of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight that FGFR3 splice variants IIIb and IIIc impact considerably on the malignant phenotype of HCC cells. The occurrence of FGFR3 variants was analyzed in human HCC samples. In hepatoma/hepatocarcinoma cell lines, FGFR3 isoforms were overexpressed by lentiviral constructs or down-modulated by small interfering RNA (siRNA; affecting FGFR3-IIIb and -IIIc) or an adenoviral kinase-dead FGFR3-IIIc construct (kdFGFR3). Elevated levels of FGFR3-IIIb and/or -IIIc were found in 53% of HCC cases. FGFR3-IIIb overexpression occurred significantly more often in primary tumors of large (pT2-4) than of small size (pT1). Furthermore, one or both isoforms were enhanced mostly in cases with early tumor infiltration and/or recurrence at the time of surgery or follow-up examinations. In hepatoma/hepatocarcinoma cells, up-regulated FGFR3-IIIb conferred an enhanced capability for proliferation. Both FGFR3-IIIb and FGFR3-IIIc suppressed apoptotic activity, enhanced clonogenic growth, and induced disintegration of the blood/lymph endothelium. The tumorigenicity of cells in severe combined immunodeficiency mice was augmented to a larger degree by variant IIIb than by IIIc. Conversely, siRNA targeting FGFR3 and kdFGFR3 reduced clonogenicity, anchorage-independent growth, and disintegration of the blood/lymph endothelium in vitro. Furthermore, kdFGFR3 strongly attenuated tumor formation in vivo. Conclusions: Deregulated FGFR3 variants exhibit specific effects in the malignant progression of HCC cells. Accordingly, blockade of FGFR3-mediated signaling may be a promising therapeutic approach to antagonize growth and malignant behavior of HCC cells.(Hepatology 2015;62:1767–1778)

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.

Purpose of study. Chemical carcinogens are categorized as either genotoxic or non-genotoxic. While most genotoxic carcinogens are known to interfere with DNA, the mode of action of non-genotoxic carcinogens (NGC) is far from being understood. Many steroid hormones act as NGC and account for cancer of the breast, prostate, or other organs. As ligands of nuclear receptors hormones induce growth in target tissues and support the outgrowth of mutated cells to malignancy. An almost identical principle is underlying the action of NGC producing liver tumors in long-term rodent bioassays. This group of compounds comprises drugs prescribed to hundred-thousands of humans, like hypolipidemic and antidiabetic drugs, or synthetic steroid hormones. A thorough knowledge of the mode of action of NGC is of utmost importance in order to estimate the risk of exposed humans. Conventional notion regards the action of NGC an autonomous process largely confined to parenchymal cells. Here, we aim to elucidate the role of the hepatic mesenchyme for the action of two prototypical NGC, phenobarbital (PB), an anti-epileptic drug, and cyproterone acetate (CPA), a gestagen used in contraceptive pills. Experimental procedures. Mesenchymal liver cells (MC) and hepatocytes (HC) were isolated from control and PB/CPA-treated rat livers by collagenase perfusion and subsequent low-speed centrifugation. Transcriptomics was performed by oligo-array analyses (Affymetrix). Cytokine levels were determined by ELISA. Nuclear translocation of NFkB was shown by immunoblotts of nuclear extracts and by reporter gene assay. Results: Analyses by oligo-arrays revealed that PB and CPA altered the transcriptome profiles of HC and MC. In PB-treated MC, there were deregulations of pro-inflammatory cytokines and chemokines of the TNF-, CCL- and CXCL-family. PB treatment in vivo or in vitro elevated the production and release of tumor necrosis factor alpha (TNFalpha)from MC. The secretome of PB-treated MC induced in HC a pro-inflammatory reaction and nuclear translocation of nuclear factor-kB (NFkB), an effect also observed with recombinant TNFalpha. Both, TNFalpha and the secretome of PB-treated MC, protected HC from pro-apoptotic stimuli. Transcriptome analyses of CPA-treated MC revealed enhanced expression not only of pro-inflammatory cytokines but also of several potent growth factors. Accordingly, the supernatant of CPA-treated MC enhanced considerably the DNA replication of preneoplastic hepatocytes. Conclusion: PB and CPA appear to alter considerably the function of the hepatic mesenchyme. The resulting release of cytokines induces growth and/or activates anti-apoptotic pathways in HC which may contribute to the tumor promoting activity of these compounds. Thus, epithelial-mesenchymal interactions appear crucial in NGC-driven hepatocarcinogenesis. Project MARCAR, funded by IMI JU under grant agreement Nr 115001. Citation Format: Bettina Grasl-Kraupp, Teresa Riegler, Marzieh Nejabat, Jakob Paur, Johannes Eichner, Michael Roemer, Andreas Zell, Rolf Schulte-Hermann, Wolfgang Huber. Non-genotoxic hepatocarcinogens induce growth and anti-apoptotic pathways in hepatocytes via mesenchymal cytokines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2015-810

Purpose of the study. To provide mechanistic evidence that fibroblast growth factor receptor 3 (FGFR3) and its two splice variants, FGFR3-IIIb and -IIIc, impact considerably on the malignant phenotype of hepatocellular carcinoma (HCC). Experimental procedures. The occurrence of FGFR3 and its two isoforms was analyzed in human HCC samples by qRT-PCR and immunostaining. In hepatoma/hepatocarcinoma cell lines FGFR3-isoforms were overexpressed by lentiviral constructs or were down-modulated by siRNA or an adenoviral kinase-dead FGFR3 (kdFGFR3) construct. Cell lines were tested for anchorage-independent growth, proliferation at low-densities and their cell-cycle distribution, as well as their ability to form tumors after subcutaneous injection into the flanks of SCID-mice. Results. Upregulated FGFR3 was found in almost 50% of HCC patients, which was often due to the enhanced expression of FGFR3-IIIb. Furthermore, patients with FGFR3-IIIb overexpression showed early tumor recurrence indicating that this isoform may be a major player in tumor progression. In hepatoma/hepatocarcinoma cells, upregulated FGFR3-IIIb or FGFR3-IIIc conferred an enhanced capability for proliferation and tumor growth. Both isoforms enhanced downstream signaling and increased the cells’ ability to grow at low densities. The tumorigenicity of cells in SCID-mice was augmented more by variant IIIb than by IIIc. This stronger impact of FGFR-IIIb on cell proliferation could also be seen in analysis of the cell cycle distribution, which was shifted to the S-phase. Conversely, siFGFR3 and kdFGFR3 affected both receptor-variants and strongly reduced clonogenic growth at low density and anchorage-independent growth. Furthermore, kdFGFR3 arrested the cells in G2/M and strongly attenuated tumor formation in vivo. Conclusions: Upregulated FGFR3 is associated with development and progression of hepatocellular carcinoma. Accordingly, blockade of FGFR3-mediated signaling may be a promising therapeutic approach to antagonize growth and malignant behavior of HCC cells. Citation Format: Jakob Paur, Lisa Nika, Christiane Maier, Julia Kostka, Petra Heffeter, Sonja Kappel, Daniela Kandioler, Klaus Holzmann, Brigitte Marian, Walter Berger, Michael Grusch, Bettina Grasl-Kraupp. Fibroblast growth factor receptor 3 enhances progression of hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1671. doi:10.1158/1538-7445.AM2015-1671