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