Mus musculus papillomavirus 1 is a key driver of skin cancer development upon immunosuppression.

Epidemiological and experimental data implicate cutaneous human papillomavirus infection as co-factor in the development of cutaneous squamous cell carcinomas (cSCCs), particularly in immunocompromised organ transplant recipients (OTRs). Herein, we established and characterized a skin cancer model, in which Mus musculus papillomavirus 1 (MmuPV1) infection caused cSCCs in cyclosporine A (CsA)-treated mice, even in the absence of UV light. Development of cSCCs and their precursors were observed in 70% of MmuPV1-infected, CsA-treated mice on back as well as on tail skin. Immunosuppression by systemic CsA, but not UV-B irradiation, was a prerequisite, as immunocompetent or UV-B-irradiated mice did not develop skin malignancies after infection. In the virus-driven cSCCs the MmuPV1-E6/E7 oncogenes were abundantly expressed, and transcriptional activity and productive infection demonstrated. MmuPV1 infection induced the expression of phosphorylated H2AX, but not degradation of proapoptotic BAK in the cSCCs. Transfer of primary cells, established from a MmuPV1-induced cSCC from back skin, into athymic nude mice gave rise to secondary cSCCs, which lacked viral DNA, demonstrating that maintenance of the malignant phenotype was virus independent. This papillomavirus-induced skin cancer model opens future investigations into viral involvement, pathogenesis, and cancer surveillance, aiming at understanding and controlling the high incidence of skin cancer in OTRs.

Inhibition of p38/MK2 Signaling Prevents Vascular Invasion of Melanoma.

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination in vivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.

CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma.

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

Dermal Blimp1 Acts Downstream of Epidermal TGFß and Wnt/ß-Catenin to Regulate Hair Follicle Formation and Growth.

B-lymphocyte-induced maturation protein 1 (Blimp1) is a transcriptional repressor that regulates cell growth and differentiation in multiple tissues, including skin. Although in the epidermis Blimp1 is important for keratinocyte and sebocyte differentiation, its role in dermal fibroblasts is unclear. Here we show that Blimp1 is dynamically regulated in dermal papilla cells during hair follicle (HF) morphogenesis and the postnatal hair cycle, preceding dermal Wnt/ß-catenin activation. Blimp1 ablation in E12.5 mouse dermal fibroblasts delayed HF morphogenesis and growth and prevented new HF formation after wounding. By combining targeted quantitative PCR screens with bioinformatic analysis and experimental validation we demonstrated that Blimp1 is both a target and a mediator of key dermal papilla inductive signaling pathways including transforming growth factor-ß and Wnt/ß-catenin. Epidermal overexpression of stabilized ß-catenin was able to override the HF defects in Blimp1 mutant mice, underlining the close reciprocal relationship between the dermal papilla and adjacent HF epithelial cells. Overall, our study reveals the functional role of Blimp1 in promoting the dermal papilla inductive signaling cascade that initiates HF growth.

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells.

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2.

Epidermal ß-catenin activation remodels the dermis via paracrine signalling to distinct fibroblast lineages.

Sustained epidermal Wnt/ß-catenin signalling expands the stem cell compartment and induces ectopic hair follicles (EFs). This is accompanied by extensive fibroblast proliferation and extracellular matrix (ECM) remodelling in the underlying dermis. Here we show that epidermal Hedgehog (Hh) and Transforming growth factor-beta (TGF-ß) signalling mediate the dermal changes. Pharmacological inhibition or genetic deletion of these pathways prevents ß-catenin-induced dermal reprogramming and EF formation. Epidermal Shh stimulates proliferation of the papillary fibroblast lineage, whereas TGF-ß2 controls proliferation, differentiation and ECM production by reticular fibroblasts. Hh inhibitors do not affect TGF-ß target gene expression in reticular fibroblasts, and TGF-ß inhibition does not prevent Hh target gene induction in papillary fibroblasts. However, when Hh signalling is inhibited the reticular dermis does not respond to epidermal ß-catenin activation. We conclude that the dermal response to epidermal Wnt/ß-catenin signalling depends on distinct fibroblast lineages responding to different paracrine signals.

Fate of Prominin-1 Expressing Dermal Papilla Cells during Homeostasis, Wound Healing and Wnt Activation.

Prominin-1/CD133 (Prom1) is expressed by fibroblasts in the dermal papilla (DP) of the hair follicle (HF). By examining endogenous Prom1 expression and expression of LacZ in the skin of Prom1CreERLacZ (Prom1C-L) mice, in which a CreERT2-IRES-nuclear LacZ cassette is knocked into the first ATG codon of Prom1, we confirmed that Prom1 is expressed in the DP of all developing HFs and also by postnatal anagen follicles. To analyze the fate of Prom1+ DP cells, we crossed Prom1C-L mice with Rosa26-CAG flox/stop/flox tdTomato reporter mice and applied 4-hydroxytamoxifen (4OHT) to back skin at postnatal day (P) 1 and P2. We detected tdTomato+ cells in ~50% of DPs. The proportion of labeled cells per DP increased between P5 and P63, while the total number of cells per DP declined. Following full thickness wounding, there was no migration of tdTomato-labeled cells out of the DP. When ß-catenin was activated in Prom1+ DP cells there was an increase in the size of anagen and telogen DP, but the proportion of tdTomato-labeled cells did not increase. We conclude that Prom1+ DP cells do not contribute to dermal repair but are nevertheless capable of regulating DP size via ß-catenin-mediated intercellular communication.

Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis.

The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage-dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co-repressor complex function, increased levels of c-Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis.

Epidermal EGFR controls cutaneous host defense and prevents inflammation.

The epidermal growth factor receptor (EGFR) plays an important role in tissue homeostasis and tumor progression. However, cancer patients treated with EGFR inhibitors (EGFRIs) frequently develop acneiform skin toxicities, which are a strong predictor of a patient's treatment response. We show that the early inflammatory infiltrate of the skin rash induced by EGFRI is dominated by dendritic cells, macrophages, granulocytes, mast cells, and T cells. EGFRIs induce the expression of chemokines (CCL2, CCL5, CCL27, and CXCL14) in epidermal keratinocytes and impair the production of antimicrobial peptides and skin barrier proteins. Correspondingly, EGFRI-treated keratinocytes facilitate lymphocyte recruitment but show a considerably reduced cytotoxic activity against Staphylococcus aureus. Mice lacking epidermal EGFR (EGFR(Δep)) show a similar phenotype, which is accompanied by chemokine-driven skin inflammation, hair follicle degeneration, decreased host defense, and deficient skin barrier function, as well as early lethality. Skin toxicities were not ameliorated in a Rag2-, MyD88-, and CCL2-deficient background or in mice lacking epidermal Langerhans cells. The skin phenotype was also not rescued in a hairless (hr/hr) background, demonstrating that skin inflammation is not induced by hair follicle degeneration. Treatment with mast cell inhibitors reduced the immigration of T cells, suggesting that mast cells play a role in the EGFRI-mediated skin pathology. Our findings demonstrate that EGFR signaling in keratinocytes regulates key factors involved in skin inflammation, barrier function, and innate host defense, providing insights into the mechanisms underlying EGFRI-induced skin pathologies.

Autocrine VEGF signaling synergizes with EGFR in tumor cells to promote epithelial cancer development.

It is established that tumor cell-derived VEGF acts on endothelial cells to promote angiogenesis and tumor growth. Here, we demonstrate that in K5-SOS-dependent mouse skin tumors, autocrine VEGF is required for tumor cell proliferation in a cell-autonomous and angiogenesis-independent manner. VEGF is upregulated in SOS-expressing tumors, and its deletion in epidermal cells delays tumorigenesis by suppressing angiogenesis and tumor cell proliferation. Epidermis-specific Flt1 deletion also impairs tumorigenesis and proliferation. Surprisingly, complete tumor inhibition occurs in the absence of VEGF in EGFR mutant mice, demonstrating that VEGFR and EGFR synergize in neoplastic cells to promote tumor growth. Mechanistically, K5-SOS upregulates VEGF, Flt1, and Neuropilin-1 in an Erk-dependent manner, thereby activating an autocrine proliferation loop, whereas EGFR prevents tumor cells from apoptosis. Moreover, Flt1 is upregulated in human SCC, and its inhibition in SCC cells impairs proliferation. Thus, in addition to regulating angiogenesis, VEGF has to be considered as a potent growth factor for epidermal tumors.

The epidermal growth factor receptor: from development to tumorigenesis.

The epidermal growth factor receptor (EGFR) is activated by many ligands and belongs to a family of tyrosine kinase receptors, including ErbB2, ErbB3, and ErbB4. These receptors are de-regulated in many human tumors, and EGFR amplification, overexpression, and mutations are detected at a high frequency in carcinomas and glioblastomas, which are tumors of epithelial and glial origin, respectively. From the analysis of EGFR-deficient mice, it seems that the cell types mostly affected by the absence of EGFR are epithelial and glial cells, the same cell types where the EGFR is found to be overexpressed in human tumors. Therefore, it is important to define molecularly the function of EGFR signaling in the development of these cell types, because this knowledge will be of fundamental importance to understand how aberrant EGFR signaling can lead to tumor formation and progression. A molecular understanding of the pathways that control the development of a given tissue or cell type will also provide the basis for developing better combination therapies targeting different key components of the EGFR signaling network in the respective cancerous cells. Here, we will review the current knowledge, mostly derived from the analysis of genetically modified mice and cells, about the function of the EGFR in specific organs and tissues and in sites where the EGFR is found to be overexpressed in human tumors.