– Wim Declercq, VIB Center for Inflammation Research, UGent – PhD project 9, 10
A primary determinant of SCCs is the accumulation of ultraviolet light (UV)-induced somatic mutations. However, these mutations are also found in normal sun-exposed epidermis, and SCC frequently develop in the context of skin infection or chronic inflammation. Loss of epidermal barrier integrity facilitates an inflammatory microenvironment and microbial spreading. Transcriptional control of pro-inflammatory cytokines is likely to play a role in promoting cancer development. RIPK4 is a kinase involved in epidermal differentiation and homeostasis and is in the top 10 of frequently mutated genes in aggressive cutaneous SCC with UV signature. By genetic and biochemical studies, we will assess to what extent RIPK4 mutations can promote cancer formation (ESR9). Inflammatory signaling can also be ignited by endogenous nucleic acid sensors, and in this way play a role in cancer progression. We will test this hypothesis in the context of the Z-DNA-sensing protein ZBP1/DAI/DLM1, which is frequently upregulated in stromal tumor cells, such as macrophages (ESR10).
Project 9 – Investigating the relationship between skin barrier loss, skin inflammation and tumor susceptibility
Hypothesis: Loss of epidermal barrier integrity facilitates an inflammatory microenvironment promoting skin tumor formation.
Background and Significance: The primary cause of squamous cell carcinoma is the accumulation of ultraviolet light (UV)-induced somatic mutations. However, squamous cell cancer develops often within the context of skin infection or chronic inflammation. In mice where the epidermal barrier is weakened by wounding, infection of flagellated bacteria drive wound-induced skin SCC development. Also in humans there is a clear association between chronic wounds and skin cancer. RIPK4 is a kinase involved in epidermal differentiation and homeostasis and is in the top 10 of frequently mutated genes in a set of aggressive cutaneous SCC with UV signature. Tamoxifen-inducible deletion of RIPK4 (RIPK4iEKO) in adult epidermis causes keratinocyte-intrinsic defects in the transition from proliferation to differentiation and aberrant STAT signaling, resulting in hyperplasia, transepidermal water loss and signs of ‘subclinical’ skin inflammation. Crossing RIPK4fl/fl mice with K14-CreER; p53fl/fl mice results in SCC development within 5-8 months with 100% penetrance while K14-CreER;p53fl/fl;RIPK4+/+ mice remain tumor-free during that period, thereby defining RIPK4 as a tumor suppressor. We will investigate the contribution of commensal skin micro-organisms to mild skin inflammation and tumor development and how the inflammatory microenvironment changes during tumor development and contributes to it, comparing K14-CreER;p53fl/fl;RIPK4+/+ and K14-CreER;p53fl/fl;RIPK4-/- mice. Understanding the role of skin microbiota and skin inflammation in skin cancer is important to discover new therapeutic strategies, e.g. selective antibiotics or (genetically engineered) probiotic bacteria that compete for pathogenic tumor-promoting bacteria or inflammation-suppressing strategies. Furthermore, we will evaluate the use of inflammatory inhibitors, such as STAT3 inhibitors.
Objectives: (1) We will characterize the inflammatory changes over time in non-tumor skin and tumor-bearing skin in the K14-CreER;p53fl/fl;RIPK4fl/fl model; (2) Complementary, we will study the changes in the transcriptome of epidermal keratinocytes from non-tumor skin and tumors to identify the deregulated inflammatory pathways; (3) Preliminary data suggest epidermal infiltration of CD8+ cells. We will test whether these cells could be a source of IL-17 production thereby promoting the inflammatory status of the skin, potentially contributing to skin cancer; (4) We will functionally study the contribution of the cutaneous microbiome to immune activation and tumor promotion, by housing the mice in a germ-free environment; 5) We will test the potential of STAT3 inhibitors to reduce tumor growth in K14-CreER;p53fl/fl;RIPK4fl/fl mice.
Project 10 – Investigating the role of Z-DNA binding protein 1 (ZBP1) signaling in skin cancer
Hypothesis: ZBP1 signaling is involved in skin tumor growth modulation.
Background and significance: The Z-DNA-sensing protein ZBP1/DAI/DLM1, not to be confused with β-actin zip code-binding protein (also abbreviated as ZBP1), has been identified as a gene upregulated in stromal tumor cells (such as macrophages), however, it can also be upregulated in cancer cells, suggesting a role in tumor growth modulation. Tumor-associated macrophages can have tumor-suppressing or tumor-promoting activities. Interestingly, ZBP1 was suggested to promote macrophage M2 polarization, which could be associated with tumor development and progression. The exact role of ZBP1 in tumor initiation and progression has not been defined. ZBP1 has been reported to be activated by binding to Z-DNA, thereby inducing cell death, NF-κB and IRF-3 activation. Recently, this idea has been challenged and it has been shown that ZBP1 can act as a sensor of viral-derived and endogenous RNA molecules. Preliminary data generated in our lab, making use of knockin mice expressing an unresponsive nucleic acid-sensing dead ZBP1 mutant (ZBP1 sensing-dead knockin mice), indicate that ZBP1 can act as a sensor of endogenous nuclear acids in contributing to skin inflammation. Given the interconnection between chronic inflammation and cancer, we will assess the contribution of ZBP1 to tumor formation making use of genetically engineered mouse models. Such studies can indicate whether ZBP1 expression and activity can be beneficial or detrimental during skin carcinogenesis and its expression may have prognostic value for cancer progression.
Objectives: (1) We will analyze the skin tumor susceptibility of ZBP1 deficient and ZBP1 sensing-dead knockin mice compared to wild-type mice using the two stage DMBA/TPA skin carcinogenesis model. Tumors will be analysed for tumor progression (growth rate, transformation to SCC, cytokine expression, immune cell infiltration, etc); (2) Study the contribution of ZBP1 expression in macrophages, cancer associated fibroblasts or keratinocytes, making use of tissue-specific knock-out lines, in DMBA/TPA-induced skin carcinogenesis. Tumors will be analyzed for tumor progression (growth rate, transformation to SCC, cytokine expression, immune cell infiltration, etc); (3) Analyze the expression of human ZBP1 in skin and breast tumor tissues using IHC and study the association with clinical outcome.
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