Christoffer Gebhardt

RAGE signaling sustains inflammation and promotes tumor development

A broad range of experimental and clinical evidence has highlighted the central role of chronic inflammation in promoting tumor development. However, the molecular mechanisms converting a transient inflammatory tissue reaction into a tumor-promoting microenvironment remain largely elusive. We show that mice deficient for the receptor for advanced glycation end-products (RAGE) are resistant to DMBA/TPA-induced skin carcinogenesis and exhibit a severe defect in sustaining inflammation during the promotion phase. Accordingly, RAGE is required for TPA-induced up-regulation of proinflammatory mediators, maintenance of immune cell infiltration, and epidermal hyperplasia. RAGE-dependent up-regulation of its potential ligands S100a8 and S100a9 supports the existence of an S100/RAGE-driven feed-forward loop in chronic inflammation and tumor promotion. Finally, bone marrow chimera experiments revealed that RAGE expression on immune cells, but not keratinocytes or endothelial cells, is essential for TPA-induced dermal infiltration and epidermal hyperplasia. We show that RAGE signaling drives the strength and maintenance of an inflammatory reaction during tumor promotion and provide direct genetic evidence for a novel role for RAGE in linking chronic inflammation and cancer.

Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Calgranulins S100A8 and S100A9 are negatively regulated by glucocorticoids in a c-Fos-dependent manner and overexpressed throughout skin carcinogenesis.

The two calgranulins S100A8 and S100A9 were found to be differentially expressed at sites of acute and chronic inflammation. Here we have employed the phorbol ester-induced multistage skin carcinogenesis protocol in mice to determine the expression of both genes in inflamed skin and in skin tumors. We show that expression is coordinately induced by the phorbol ester TPA in epithelial cells as well as infiltrating leukocytes. By comparing S100A8 and S100A9 mRNA levels in wild type and c-Fos deficient mice (c-fos−/−) we found that expression is negatively regulated by c-Fos/AP-1. Glucocorticoids, which exhibit potent anti-inflammatory and anti-tumor promoting activities repressed TPA-mediated S100A8 and S100A9 induction in wild type, but not in c-fos−/− mice, thus identifying both genes as the first examples of AP-1 target genes whose repression of TPA-induced transcription by glucocorticoids depends on c-Fos. Finally, we show that enhanced expression is not restricted to the initial TPA-induced inflammatory response but is observed at all stages of skin carcinogenesis. These data identify S100A8 and S100A9 as novel, tumor-associated genes and may point to an as yet unrecognized function of both genes in the development of epithelial skin tumors.

Baseline Biomarkers for Outcome of Melanoma Patients Treated with Pembrolizumab

Purpose: Biomarkers for outcome after immune-checkpoint blockade are strongly needed as these may influence individual treatment selection or sequence. We aimed to identify baseline factors associated with overall survival (OS) after pembrolizumab treatment in melanoma patients. Experimental Design: Serum lactate dehydrogenase (LDH), routine blood count parameters, and clinical characteristics were investigated in 616 patients. Endpoints were OS and best overall response following pembrolizumab treatment. Kaplan–Meier analysis and Cox regression were applied for survival analysis. Results: Relative eosinophil count (REC) ≥1.5%, relative lymphocyte count (RLC) ≥17.5%, ≤2.5-fold elevation of LDH, and the absence of metastasis other than soft-tissue/lung were associated with favorable OS in the discovery (n = 177) and the confirmation (n = 182) cohort and had independent positive impact (all P < 0.001). Their independent role was subsequently confirmed in the validation cohort (n = 257; all P < 0.01). The number of favorable factors was strongly associated with prognosis. One-year OS probabilities of 83.9% versus 14.7% and response rates of 58.3% versus 3.3% were observed in patients with four of four compared to those with none of four favorable baseline factors present, respectively. Conclusions: High REC and RLC, low LDH, and absence of metastasis other than soft-tissue/lung are independent baseline characteristics associated with favorable OS of patients with melanoma treated with pembrolizumab. Presence of four favorable factors in combination identifies a subgroup with excellent prognosis. In contrast, patients with no favorable factors present have a poor prognosis, despite pembrolizumab, and additional treatment advances are still needed. A potential predictive impact needs to be further investigated. Clin Cancer Res; 22(22); 5487–96. ©2016 AACR.

S100A8 and S100A9 are novel nuclear factor kappa B target genes during malignant progression of murine and human liver carcinogenesis

The nuclear factor‐kappaB (NF‐κB) signaling pathway has been recently shown to participate in inflammation‐induced cancer progression. Here, we describe a detailed analysis of the NF‐κB–dependent gene regulatory network in the well‐established Mdr2 knockout mouse model of inflammation‐associated liver carcinogenesis. Expression profiling of NF‐κB–deficient and NF‐κB–proficient hepatocellular carcinoma (HCC) revealed a comprehensive list of known and novel putative NF‐κB target genes, including S100a8 and S100a9. We detected increased co‐expression of S100A8 and S100A9 proteins in mouse HCC cells, in human HCC tissue, and in the HCC cell line Hep3B on 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. Conclusion: We identified S100A8 and S100A9 as novel NF‐κB target genes in HCC cells during inflammation‐associated liver carcinogenesis and provide experimental evidence that increased co‐expression of both proteins supports malignant progression by activation of ROS‐dependent signaling pathways and protection from cell death. (HEPATOLOGY 2009.)

Profile of gene expression induced by the tumour promotor TPA in murine epithelial cells

Malignant transformation of mouse skin by chemical carcinogens and tumour promoters, such as the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), is a multistage process that leads to squamous cell carcinoma (SCC) formation. In an effort to identify tumour‐associated genes, we studied the influence of short‐term TPA‐treatment on the gene expression profile of murine skin. A comprehensive microarray with some 5,000 murine gene specific cDNA fragments was established and hybridised with pooled RNA derived from control and TPA‐treated dorsal skin samples. Of these genes, 54 were up‐ and 35 were down‐regulated upon TPA application. Additionally, we performed suppression subtractive hybridisation (SSH) with respective RNA pools to generate and analyse a cDNA library enriched for TPA‐inducible genes. Expression data of selected genes were confirmed by quantitative real‐time PCR and Northern blot analysis. Comparison of microarray and SSH data revealed that 26% of up‐regulated genes identified by expression profiling matched with those present in the SSH library. Besides numerous known genes, we identified a large set of unknown cDNAs that represent previously unrecognised TPA‐regulated genes in murine skin with potential function in tumour promotion. Additionally, some TPA‐induced genes, such as Sprr1A, Saa3, JunB, Il4rα, Gp38, RalGDS and Slpi exhibit high basal level in advanced stages of skin carcinogenesis, suggesting that at least a subgroup of the identified TPA‐regulated genes may contribute to tumour progression and metastasis.

Elevated chronic inflammatory factors and myeloid‐derived suppressor cells indicate poor prognosis in advanced melanoma patients

Chronic inflammation is considered to be one of the hallmarks for tumor initiation and progression. Moreover, a long‐term production and accumulation of inflammatory factors lead to a local and systemic immunosuppression associated with cancer progression. However, the correlation between inflammatory mediators, immunosuppressive cells and the clinical outcome of malignant melanoma patients was poorly investigated. In this study, we performed a complex analysis of various inflammatory factors, myeloid‐derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the peripheral blood of patients suffering from malignant melanoma of different stages. We demonstrated that levels of serum IL‐1β, IFN‐γ and CXCL10 were significantly increased in advanced melanoma patients. In addition, these factors were found to be associated with an increased frequency of MDSCs and Tregs as compared to age‐ and gender‐matched healthy donors. Importantly, advanced melanoma patients with signs of progression displayed markedly elevated concentrations of IL‐1β and CXCL10 as compared to patients with stable disease. Moreover, an enrichment of circulating monocytic (Mo)‐MDSCs significantly correlated with a decreased progression free survival of these patients. Our data highlight a complex association between circulating inflammatory mediators, Mo‐MDSCs and the clinical outcome as well as suggest that their levels in patients with advanced melanoma are of important prognostic value allowing the identification of those with high risk of disease progression.

The Role of Myeloid-Derived Suppressor Cells (MDSC) in Cancer Progression

The immunosuppressive tumor microenvironment represents not only one of the key factors stimulating tumor progression but also a strong obstacle for efficient tumor immunotherapy. Immunosuppression was found to be associated with chronic inflammatory mediators including cytokines, chemokines and growth factors produced by cancer and stroma cells. Long-term intensive production of these factors induces the formation of myeloid-derived suppressor cells (MDSCs) representing one of the most important players mediating immunosuppression. Moreover, MDSCs could not only inhibit anti-tumor immune reactions but also directly stimulate tumor growth and metastasis. Therefore, understanding the mechanisms of their generation, expansion, recruitment and activation is required for the development of novel strategies for tumor immunotherapy.

c-Fos-Dependent Induction of the Small Ras-Related GTPase Rab11a in Skin Carcinogenesis

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.

Myeloid-derived suppressor cells in malignant melanoma

Melanoma is known for its rapid progression, metastasis to distant organs and therapeutic resistance. Despite high melanoma immunogenicity, the results of immunotherapeutic clinical studies are mostly unsatisfactory. One explanation is the development of strong immunosuppression mediated by highly immunosuppressive regulatory leukocytes, in particular, myeloid‐derived suppressor cells (MDSCs). These cells were found to be enriched and activated in the melanoma microenvironment, inducing a profound impairment of anti‐tumor immune responses and leading to the tumor progression. Therefore, understanding the mechanisms of MDSC generation, migration to the tumor site and activation as well as their targeting is important for the development of novel strategies for effective melanoma immunotherapy. We suggest that such therapeutic approaches should involve the inhibition of MDSC‐mediated immunosuppressive melanoma microenvironment combined with other immunologic treatments.