Signalling and transcription factor networks in cancer and in inflammation associated carcinogenesis
Cancer is a multistage disorder in which genetic and epigenetic changes result in characteristic alterations affecting intrinsic cellular programs. A broad range of experimental and clinical evidence highlight a key function of the inflammatory microenvironment during cancer development by the release of potent soluble mediators that regulate tumour cell proliferation and survival, metabolism, angiogenesis and genomic integrity. Additionally, infiltrating immune cells activate proteolytic cascades that are key determinants of matrix remodelling and thereby critically contribute to tumour cell invasion and malignant progression. During the last years, our group used a well-established mouse model of skin carcinogenesis and state-of-the-art genome-wide technologies to elucidate relevant alterations in the genetic program during tumour promotion and malignant progression (Figure 1). In addition to well-established AP-1-target genes, such as MMP-13 (which we have analysed by loss-of-function-approaches in mice for its role during tissue homeostasis and remodeling inbone and skin) this study revealed a comprehensive list of differentially expressed genes some of which represent novel Fos/AP-1 target genes. Detailed functional analysis of distinct candidate genes using genetically modified cell culture and mouse model systems revealed novel insight into the molecular nature of tumour cell behaviour.
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Figure 1: Schematic representation of the two-stage skin carcinogenesis model. Single application of the mutagen DMBA followed by repeated treatment with the phorbol ester TPA results in the induction of an inflammatory response and hyperplasia, which is a pre-requisite for the subsequent tumour formation. For experimental details see Gebhardt et al., 2005; Hummerich et al., 2006.
The secreted serine proteinase KLK6 in malignant progression of epithelial tumour cells
One candidate gene with enhanced expression in TPA-treated mouse back skin and advanced tumour stages of the two-step skin tumour model is the brain and skin serine proteinase (Bssp), the mouse ortholog of human kallikrein-related peptidase 6 (KLK6). Expression of Bssp was greatly diminished in mice lacking Fos or the MAP kinase Erk-1, two critical components of the Ras-Erk-AP-1 pathway. More recent data demonstrated KLK6 up-regulation also in human patients with squamous skin tumours and other epithelial malignancies. Detailed functional analysis of Bssp/KLK6 using genetically modified cell culture and mouse model systems unravelled a crucial role for this proteinase in cell cycle regulation, cell-cell adhesion, cell migration and invasion that is most likely due to enhanced E-cadherin shedding and activation of b-catenin/TCF-dependent transcription. Cell culture experiments further revealed an essential role for ADAM proteases in KLK6-induced ectodomain shedding, suggesting the existence of a novel KLK-ADAM cascade in tumour cell proliferation and invasion.
S100-RAGE signalling in inflammation and cancer
S100a8 and S100a9 are two other proteins that exhibit strong up-regulation in our tumour model system and show enhanced expression in human epithelial malignancies. Both belong to a large family of small Ca2+-binding EF-hand proteins. Their expression is closely linked to processes of acute and chronic inflammation accompanied by the development of degenerative disorders and cancer. Several membrane receptors have been discussed to interact with the secreted S100a8/S100a9 complex of which the receptor for advanced glycation end products (Rage) is the most attractive one, since it is also implicated in innate immune response, chronic inflammation and cancer. Accordingly, we could show that Rage-deficient mice are resistant to chemically induced skin carcinogenesis due to a defect in the conversion of an acute pro-inflammatory stimulus into sustained tissue activation.
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Figure 2: RAGE function in inflammation-associated carcinogenesis.
RAGE is expressed in all cell types implicated in tumour formation, including tumour cells, endothelial cells, myeloid cells, MDSCs, and lymphocytes. Signalling pathways downstream of RAGE that are activated by the accumulation of its ligands (AGE, HMGB1, S100 proteins) regulate cellular interactions during neoplastic transformation and malignant progression (Riehl et al., 2009)
Besides the importance of S100-Rage signalling in inflammation-associated skin carcinogenesis, we identified S100a8 and S100a9 as novel NF-kB target genes driving malignant progression in inflammation-associated liver carcinogenesis. Detailed analysis of the NF-kB-dependent gene regulatory network in the well-established Mdr2 knockout mouse model of inflammation-associated liver carcinogenesis revealed a comprehensive list of known and novel putative NF-kB target genes, including S100a8 and S100a9. We detected increased co-expression of S100a8 and S100a9 proteins in mouse hepatocellular carcinoma cells, in human HCC tissue and in the HCC cell line Hep3B upon ectopic RelA expression. Finally, we found a synergistic function for S100a8 and S100a9 in Hep3B cells resulting in a significant induction of reactive oxygen species (ROS), accompanied by enhanced cell survival.
These data provide experimental evidence that S100a8 and S100a9 represent important key players in linking inflammation and cancer as well as in malignant progression.
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Figure 3: Model of NF-kB dependent gene interaction network in hepatocellular carcinoma development.
Expression profiling of NF-kB-deficient and -proficient hepatocellular carcinoma (HCC) revealed a NF-kB-dependent gene regulatory network including several known and novel NF-kB target genes, such as S100a8 and S100a9. For experimental details see Németh et al., 2009.