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Abstract
Background Epidermal growth factor (EGF) signaling has well-established roles in cellular proliferation in normal tissue homeostasis and tumorigenesis. EGF receptor inhibitor therapy is associated with the development of a papulopustular rash and other cutaneous inflammatory effects.1 2 These dose-dependent toxicities are linked to treatment response and survival, and may reflect the interplay between EGF and the immune response.3 4 However, the effects of EGF signaling on inflammation in the skin and elsewhere are not entirely understood.5 6 In this study, we aimed to elucidate the immunomodulatory role of EGF in human keratinocytes exposed to the proinflammatory cytokine interferon-γ (IFN-γ).
Methods Human keratinocyte cell lines (HaCaT) were exposed to IFN-γ, EGF, or both (48 hours). Differential gene expression analyses of RNA expression was performed using DESeq2.7 Fold change in gene expression on the log2 scale were calculated for each experimental treatment group relative to control. Web Gestalt was used to identify differentially expressed biologic pathways and gene networks, and further investigated in publicly available cutaneous squamous cell (cSCC) cell lines (GSE98767) and cSCC and basal cell carcinoma (BCC) tumor samples (GSE125285).8
Results As compared to untreated control cells, 2792 genes were differentially expressed following IFN-γ treatment, 938 following EGF treatment, and 1248 following the combination of IFN-γ and EGF (figure 1). To assess the impact of EGF on the cellular response to IFN-g, we identified IFN-g-induced genes whose expression was significantly altered by EGF (figure 2). We found that the induction of CXCL10 by IFN-g was among those significantly attenuated in the presence of EGF (padjusted= 0.01) and selected CXCL10 as a model to further define the impact of EGF on immune gene expression. We found that in cutaneous SCC (cSCC) cell lines as well as cSCC and basal cell carcinoma tumor samples, the correlation between IFN-γ and CXCL10 expression was abrogated in samples with higher EGF expression (figure 3).
EGF modulates IFN-γ-induced gene expression in human keratinocytes. A. Heatmap showing differentially expressed genes (Padjusted <0.01) induced by IFN-γ alone, EGF alone, or IFN-γ plus EGF (excluding genes that were not differentially expressed in any treatment group relative to control). B. Venn-diagram showing differentially expressed genes (Padjusted < 0.01) induced by IFN-γ and/or EGF. C. Fold change of the top 10 genes induced after treated with IFN-γ alone. The top 10 genes which were induced by IFN-γ include CXCL10, CD74, several HLA-D genes, IDO1, GBP5, C1S, and BST2
Pathway analysis of genes induced by IFN-γ then differentially regulated by EGF. A. Heatmap showing log2fold change in gene expression of top IFN-γ-regulated genes whose expression was significantly dampened or augmented by EGF (Pinteraction < 0.05). The EGF* IFN-γ interaction fold-change (far left) column indicates the excess fold change due to interaction between EGF and IFN-γ. Within this column, blue and red shading indicates dampening and augmentation of IFN-γ-induced gene expression by EGF, respectively. B. Sub-network graph from Network Topology Analysis (NTA) of IFN-γ-regulated genes of which expression was either 2-fold higher or lower when EGF was added; genes in the top enriched GO Biological Process category are highlighted in red (GO:0019886 [antigen processing and presentation of exogenous peptide antigen via MHC class II]; Padjusted = 2.65 x 10-7); blue shading of CXCL10 denoting it as the most strongly upregulated gene by IFN-γ in this gene set to be dampened by EGF treatment. C. IFN-γ-induced genes attenuated by EGF, clustered according to significantly enriched KEGG pathways. Differentially expressed genes are listed in order of their score within the gene set enrichment analyses. Bolded italics type indicates common genes in multiple enriched pathways
Correlation between IFN-γ and CXCL10 expression stratified by EGF expression. A. Cutaneous squamous cell carcinoma cell lines (GSE98767, n=44). B. Cutaneous squamous and basal cell carcinoma tumor samples (GSE125285, n=35)
Conclusions EGF has pleotropic roles in cancer including immunologic effects relevant to anti-tumor immunity. These studies demonstrate that EGF alters the transcriptional response to IFN-g including the induction of CXCL10 by IFN-g. Moreover, these studies suggest that in the setting of high EGF levels, there is a modulation of IFN-g-regulated chemokine expression. Further research is needed to clarify the role of EGF in modulating inflammation, and to understand this process in the pathogenesis of EGF receptor inhibitor-induced cutaneous toxicities and skin cancers.
Acknowledgements Emory Integrated Genomics Core
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