Psoriasis is a chronic inflammatory skin disease affecting millions of people worldwide. Although growing evidence links chronic inflammation with increased cancer risk, the association between psoriasis and cutaneous squamous cell carcinoma (cSCC) is still elusive. Using cell transplantation and chemical-induced models of cSCC combined with inducible genetically engineered mouse models of psoriasis, we investigated how chronic skin and systemic inflammation affects squamous skin tumor initiation and progression. Here we show that in the context of severe psoriasis-like disease, neutrophil-dependent inflammation prevents squamous skin tumor development. Cellular and molecular analyses of psoriasis-like skin at the tumor initiation stage revealed a marked infiltration of CD54-expressing neutrophils, associated with the release of cytotoxic granules and neutrophil extracellular traps (NETs), as well as enhanced senescence and the expression of senescence-associated secretory phenotype in keratinocytes. Furthermore, single-cell RNA sequencing demonstrated that inflammatory N1-like neutrophils mediate reprogramming of the cell-cell communication networks, while keratinocytes displayed diminished responsiveness to mitogenic signals, including epidermal growth factor and Wnt/β-catenin. Importantly, neutrophil depletion ameliorated psoriasis-like inflammation, abolished the senescence-like phenotype in keratinocytes and restored tumor growth. We propose that the release of neutrophil granules and NETs in psoriasis-like skin eliminate tumor cells and/or mediate oxidative and inflammatory stress-induced senescence in keratinocytes, thereby preventing tumor growth. Taken together, we have defined an innate control of skin tumorigenesis in psoriasis-like disease, which will be relevant for developing cancer prevention strategies.

The serrated neoplasia pathway is an alternate route to colorectal cancer (CRC) development where BRAFV600E is the most common initiating genetic alteration. BRAFV600E-driven tumorigenesis requires gene expression changes mediated by activation of the ERK MAPK signaling pathway. However, the key effectors of this process are elusive. Here, we identify the ERK-regulated transcription factor Fosl1, one such effector. We show that Fosl1 is dispensable for the initiation of BRAFV600E-driven serrated neoplasia in mice but promotes progression of the disease by regulating the expression of genes involved in inflammation, immunity, cell cycle control, fetal-like programming, and gastric metaplasia. Notably, transgenic Fosl1 expression alone was sufficient to induce tumors with a BRAFV600E-like serrated morphology and transcriptional profile. These findings reveal a mechanism through which oncogenic BRAF-driven ERK signaling reprograms transcription to drive serrated neoplasia.

Distinct expression of the alarmins S100A8 (A8), S100A9 (A9) and their antimicrobial Calprotectin (CP) hetero-complex has been documented in Inflammatory Skin Diseases (ISDs). The cell-specific functions of A8 and A9 in local and extra-cutaneous manifestations of ISD were evaluated using genetically engineered mouse models (GEMMs). ISDs such as Atopic Dermatitis are accompanied by Osteopenia, a musculoskeletal complication, while skin infections can lead to osteomyelitis and septic arthritis. A9 or A8 were inactivated in epidermal cells or neutrophils in the JunB?ep genetic mouse model for skin inflammation. Inactivation of epidermal A9 promoted, while A8 inactivation ameliorated experimental ISD. Skin inflammation was also ameliorated when A9 was inactivated in neutrophils or in all cells. However, complete A9 knock-out was associated with worsened systemic effects, such as neutrophilic inflammation and bone loss. Staphylococcus aureus overgrowth, bone destruction and increased A8 protein expression were also observed in the distal phalanges of the digits. Epidermal A8 inactivation ameliorated bone loss, but promoted bone destruction in the digits, likely through A8-positive neutrophilic infiltrates. These data show that site- and cell-type-specific A8 and A9 expression modulates chronic skin and systemic inflammation. These findings pave the way for novel therapies targeting divergent functions of A8 and A9 to restore homeostasis and prevent systemic complications. Marie Sklodowska-Curie postdoctoral grant INTERACT-101111333, European Research Council. APART-MINT postdoctoral fellowship/11998, Austrian Academy of Sciences (ÖAW). European Research Council Advanced grant (ERC AdG 2016-741888-CSI-Fun). Mucosal and Regional Immunology (MUC)

Psoriasis (Ps) is a chronic inflammatory skin disease with systemic manifestations, such as psoriatic arthritis (PsA), cardiovascular and psychiatric complications, and subsequent negative effects on patients’ quality of life. Although biologics targeting specific disease mediators have become a mainstay in Ps treatment, exploration of new disease targets to improve treatment is still needed. Here we show that fatty-acid binding protein 5 (Fabp5) promotes skin inflammation through a therapeutically relevant modulation of the ferroptotic response. In epidermal-specific inducible c-Jun and JunB knockout (DKO*) mice, a preclinical model for Ps with PsA-like manifestations, dermal fat is reduced, serum free fatty acids (FFA) decreased, and β-hydroxybutyric acids (β-OHB) altered. Comparing RNA-seq and proteomic datasets from DKO* mice and Ps patients revealed shared alterations in fatty acid metabolism and ferroptosis signatures. Specifically, increased expression of Fabp5 and decreased expression of glutathione peroxidase 4 (Gpx4), a lipid-modifying enzyme and ferroptosis suppressor, are observed in the epidermis of DKO* mice and Ps patients. Treatment of DKO* mice with the Fabp inhibitor BMS increased Gpx4 expression, reduced lipid peroxidation products and neutrophil infiltration, ameliorated the skin phenotype, and alleviated keratinocyte hyperproliferation without affecting systemic IL-17a signaling and PsA-like manifestations. Importantly, dysregulated epidermal Fabp5 and Gpx4 expression was normalized after anti-IL17a or anti-TNFα antibody administration in DKO* mice, as well as in Ps patients treated with the corresponding Ps biologics. Furthermore, treatment with the ferroptosis inhibitor, liproxstatin-1, suppressed Ps-like skin thickening in DKO* mice, but did not affect the joint phenotype. These results support a functional and disease-relevant link between Fabp5, Gpx4 and ferroptosis in the skin that should be therapeutically exploited.

Genetically engineered mouse models (GEMMs) are instrumental for modelling local and systemic features of complex diseases, such as cancer. Non-invasive, longitudinal cell detection and monitoring in tumors, metastases and/or the micro-environment is paramount to achieve a better spatiotemporal understanding of cancer progression and to evaluate therapies in preclinical studies. Bioluminescent and fluorescent reporters marking tumor cells or their microenvironment are valuable for non-invasive cell detection and monitoring in vivo. Here, we report the generation of a dual reporter allele allowing simultaneous bioluminescence and fluorescence detection of cells that have undergone Cre-Lox recombination in mice. The single copy knock-in allele in the permissive collagen I locus was evaluated in the context of several cancer GEMMs, where Cre expression was achieved genetically or by ectopic virus-mediated delivery. The new reporter allele was also combined with gene-targeted alleles widely used in bone, prostate, brain and pancreas cancer research, as well as with alleles inserted into the commonly used Rosa26 and collagen I loci. This allele is, therefore, a useful addition to the portfolio of reporters to help advance preclinical research.

Regulatory T (Treg) cells are a critical immune component guarding against excessive inflammation. Treg cell dysfunction can lead to chronic inflammatory diseases with current therapies aimed at inhibiting effector T cells rather than rescuing Treg cell function. We utilized single-cell RNAsequencing data from patients with chronic inflammation to identify SAT1, the gene encoding spermidine/spermine N1-acetyltransferase (SSAT), as a driver of skin-resident Treg cell dysfunction. CRISPRa-driven SAT1 expression in human skin-derived Treg cells impaired their suppressive function and induced a pro-inflammatory phenotype. During cutaneous type-17 inflammation, keratinocyte 4-1BBL induces SAT1 on Treg cells. In a mouse model of psoriasis, pharmacological inhibition of SSAT rescued Treg cell number and function. Together, these data show that SAT1 expression has severe functional consequences on Treg cells and suggest a therapeutic target to treat chronic inflammatory disease.

Increased expression of the homodimeric S100A8 (A8) and S100A9 (A9) alarmins and their Calprotectin (CP) antimicrobial hetero-complex has been reported in Inflammatory Skin Diseases (ISDs) such as Atopic Dermatitis (AD), but the functional consequences of this increase are not known. We evaluated the cell- and tissue-specific functions of A8 and A9 in the local and the extra-cutaneous manifestations of ISD using genetically engineered mouse models. The genes encoding for the A9 or A8 proteins were inactivated in epidermal cells or neutrophils in the JunB∆ep genetic mouse model for AD. Overall, epidermal inactivation of A9 aggravated, while similar A8 inactivation ameliorated experimental ISD. Epidermal differentiation and skin inflammation was also ameliorated when A9 was inactivated in neutrophils or in all cells. However, complete A9 knock-out was associated with worsened systemic effects, such as neutrophilic inflammation and bone loss. In addition, the distal phalanges of the digits displayed increased A8 protein expression, SA overgrowth and bone destruction. Epidermal A8 inactivation ameliorated bone loss, but promoted bone destruction in the digits, likely through A8-positive neutrophilic infiltrates. These data show that site- and cell-type-specific A8 and A9 expression modulates chronic skin and systemic inflammation with distinct effects on the skin differentiation and on the musculoskeletal system. These findings pave the way for novel therapies targeting the divergent functions of A8 and A9 to restore epidermal homeostasis and prevent systemic complications.

Background/Objectives: Radiolabeled fibroblast activation protein inhibitors (FAPIs) are emerging as promising imaging agents assessing fibrotic diseases. This study evaluates [68Ga]Ga-DATA5m.SA.FAPi for imaging pulmonary fibrosis in two mouse models, bleomycin-induced (BLM) and a transgenic (fra-2tg) model, both displaying characteristics of human pulmonary fibrotic diseases. Methods: In the BLM model, C57BL/6 mice were treated with bleomycin or isotonic sodium chloride (controls) for 4, 5, and 6 weeks, followed by [68Ga]Ga-DATA5m.SA.FAPi PET/CT scans. Fra-2tg mice and wildtype (WT) littermates underwent at 7, 11, and 18/19 weeks of age a PET/CT scan. The selected timepoints correspond to early, middle, and late disease stages for each model. Imaging was complemented by ex vivo quantification, histological, and immunohistochemical (IHC) analyses. Results: In BLM mice, pulmonary [68Ga]Ga-DATA5m.SA.FAPi uptake showed a trend toward increase as early as 5 weeks of treatment compared with the controls, which was confirmed by ex vivo analysis (BLM: 3.31 ± 0.29%ID/g, n = 5; control: 1.61 ± 0.29%ID/g, n = 4; p = 0.0035). In fra-2tg mice, no significant differences could be detected. IHC revealed elevated pulmonary FAP expression specifically at early (BLM) and mild (fra-2tg) disease stages, whereas for BLM, tracer uptake was more pronounced at later stages. Conclusions: Our findings complement and extend observations from previous studies and support the potential of FAPI tracers as molecular imaging agents for pulmonary fibrosis.

Background & Aims Quantification of the human S100A8/S100A9 tetrameric protein complex in stool, referred to as fecal calprotectin, is an extensively validated biomarker supporting the diagnosis and management of gastrointestinal diseases. Here, we studied the quaternary protein structures (termed configuration) of S100A8 and S100A9 and their biological function in inflammatory bowel diseases (IBD). Methods We dissected fecal S100A8 and S100A9 configurations in patients with IBD by size-exclusion chromatography coupled with tandem mass spectrometry and systematically defined human S100A8 and S100A9 homodimer functions compared with the calprotectin heterotetramer (CP) in the intestine of mice and in human epithelium and T cells. Moreover, we report a protein interaction network of fecal S100A8 and S100A9 in IBD. Results Stool from patients with active IBD contained abundant S100A8 and S100A9 dimers besides CP. Fecal S100A9 detection associated with clinical and endoscopic disease activity in IBD patients with low CP concentration. Oral exposure to human recombinant S100A8 and S100A9 homodimers, but not to CP, worsened intestinal inflammation in toxic and genetic mouse models. Functional profiling revealed that human S100A8 and S100A9 homodimers enhanced activation of cluster of differentiation 4+ and 8+ T cells, which promoted experimental colitis. In turn, genetic inactivation of S100a9 protected against experimental enteritis and colitis, and pharmacologic inhibition of S100A9 ameliorated chronic colitis. Conclusions Collectively, this study links the detection of fecal S100A9 dimers with clinical and endoscopic disease activity in IBD and identifies inflammatory actions of S100A8 and S100A9 homodimers in the intestine. Our findings pave the way for novel diagnostic and therapeutic approaches in patients with inflammatory diseases of the intestine.

Psoriasis is a chronic inflammatory skin disease involving a complex cross-talk between immune and epidermal cells. Psoriasis is difficult to treat and often complicated by systemic manifestations such as psoriatic arthritis. SQSTM1/p62 is a multifunctional adaptor protein controlling autophagy, cell differentiation, and inflammation that was found elevated in human psoriatic skin. We functionally evaluated the role of p62 in the cutaneous and systemic psoriasis-like phenotypes of a mouse model with inducible epidermal inactivation of c-Jun and JunB (ie, DKO∗). A male-specific aggravation of skin and joint disease was observed in DKO∗ mice when crossed with p62-/- mice (DKO∗ p62-/-). Thickened epidermis, disturbed keratinocyte differentiation, enhanced immune cell infiltration, and increased CXCL1 expression were exclusively observed in the skin of male DKO∗ p62-/- mice. Increased androgen receptor protein expression and activation of androgen receptor signaling as well as upregulated inflammasome and KEAP1/NRF2 activities were apparent in the skin of male DKO∗ p62-/- mice and were likely responsible for disease worsening. Our results describe a sex-specific anti-inflammatory role for p62 in psoriasis-like disease that could be relevant in the clinical setting.

Background: There is currently no therapy targeting cancer-related cardiomyopathy and treatment of heart failure in the oncological setting is nonspecific. Moreover, cancer is often assocaited with cachexia and the pathophysiology of cancer-cachexia induced cardiac (dys)functions are not fully known. Methods: Colon-26 adenocarcinoma (C26; n=30) or shIL-6 (C26 shIL-6; n=30) cells were inoculated subcutaneously into the flank of syngeneic adult male BALB/cmice, meanwhile control mice were injected with PBS (n=25). Twenty days after the cells injection, cardiac function was assessed using transthoracic echocardiography, ex vivo isolated working heart methods. In addition, intracellular Ca 2+ transient and force-calcium relationships were assessed in isolated single ventricular cardiomyocytes (CMs). Cardiac inflammation, metabolism and fibrosis were also assessed. Results: Despite that tumor size was comparable between the cancer groups, C26 group showed a loss of subcutaneous fat and skeletal muscle confirming a cachectic phenotype in association with an elevation of serum IL-6 levels. Tumor-bearing mice groups show a tendecy towards to both LV systolic and diastolic dysfunction. Sarcomere dysfunction, including significantly reduced maximum calcium-activated tension (Tmax) and increased calcium sensitivity (decreased EC 50 ) was found in skinned cardiomyocyte preparation from both tumor-bearing mice in compared to controls (p<0.05, respectively). Intracellular Ca 2+ transient was exclusively increased in CM isolated from cachectic mice, suggesting the SERCA2 upregulation. Infiltration of macrophage or T-cells, nor interstital fibrosis were difference. β-myosin heavy chain expression is upregulated in a cell autonomous fashion in C26 mice. Comprehensive energetic and metabolims analysis showed shift to a profound glucose metabolsim and reduction in fatty acid oxidation in LV samples. This was futher proven in h9c2 cells were incubated with C26 or shIL6 cell culture medium. Discussion: Our results suggest that LV dysfunction in cancer mice is associated with sarcomere dysfunction while additional abnormal intracellular Ca 2+ handling is solely present in mice with cachectic phenotype. These functional alterations are independent from changes in myocardial fibrosis and inflammation but may rely on the cardiac metabolism alterations. These data provide new insights into how cancer and cancer-cachexia impacts the cardiac performance even prior to cancer therapy treatments.

Hepatocellular carcinoma (HCC), a leading cause of cancer-related death with limited therapies, is a complex disease developing in a background of Hepatitis Virus infection or systemic conditions, such as the metabolic syndrome. Investigating HCC pathogenesis in model organisms is therefore crucial for developing novel diagnostic and therapeutic tools. Genetically engineered mouse models (GEMMs) have been instrumental in recapitulating the local and systemic features of HCC. Early studies using GEMMs and patient material implicated members of the dimeric Activator Protein-1 (AP-1) transcription factor family, such as c-Jun and c-Fos, in HCC formation. In a recent report, we described how switchable, hepatocyte-restricted expression of a single-chain c-Jun~Fra-2 protein, functionally mimicking the c-Jun/Fra-2 AP-1 dimer, results in spontaneous and largely reversible liver tumors in GEMMs. Dysregulated cell cycle, inflammation, and dyslipidemia are observed at early stages and tumors display molecular HCC signatures. We demonstrate that increased c-Myc expression is an essential molecular determinant of tumor formation that can be therapeutically targeted using the BET inhibitor JQ1. Here, we discuss these findings with additional results illustrating how AP-1 GEMMs can foster preclinical research on liver diseases with novel perspectives offered by the constantly increasing wealth of HCC-related datasets.

ObjectivesThe activator protein-1 (AP-1) transcription factor component c-Fos regulates chondrocyte proliferation and differentiation, but its involvement in osteoarthritis (OA) has not been functionally assessed.Methodsc-Fos expression was evaluated by immunohistochemistry on articular cartilage sections from patients with OA and mice subjected to the destabilisation of the medial meniscus (DMM) model of OA. Cartilage-specific c-Fos knockout (c-FosΔCh) mice were generated by crossingc-fosfl/fltoCol2a1-CreERTmice. Articular cartilage was evaluated by histology, immunohistochemistry, RNA sequencing (RNA-seq), quantitative reverse transcription PCR (qRT-PCR) andin situmetabolic enzyme assays. The effect of dichloroacetic acid (DCA), an inhibitor of pyruvate dehydrogenase kinase (Pdk), was assessed in c-FosΔChmice subjected to DMM.ResultsFOS-positive chondrocytes were increased in human and murine OA cartilage during disease progression. Compared with c-FosWTmice, c-FosΔChmice exhibited exacerbated DMM-induced cartilage destruction. Chondrocytes lacking c-Fos proliferate less, have shorter collagen fibres and reduced cartilage matrix. Comparative RNA-seq revealed a prominent anaerobic glycolysis gene expression signature. Consistently decreased pyruvate dehydrogenase (Pdh) and elevated lactate dehydrogenase (Ldh) enzymatic activities were measuredin situ, which are likely due to higher expression of hypoxia-inducible factor-1α,Ldha, and Pdk1 in chondrocytes.In vivotreatment of c-FosΔChmice with DCA restored Pdh/Ldh activity, chondrocyte proliferation, collagen biosynthesis and decreased cartilage damage after DMM, thereby reverting the deleterious effects of c-Fos inactivation.Conclusionsc-Fos modulates cellular bioenergetics in chondrocytes by balancing pyruvate flux between anaerobic glycolysis and the tricarboxylic acid cycle in response to OA signals. We identify a novel metabolic adaptation of chondrocytes controlled by c-Fos-containing AP-1 dimers that could be therapeutically relevant.

Targeted therapies are available for lung adenocarcinoma (LUAD); however, more than 50% of patients either cannot benefit from them or become resistant, highlighting the need for alternative strategies. Our project focuses on FRA-2 (encoded by FOSL2 gene), a member of the AP-1 family that can act downstream of KRAS and EGFR pathways, two of the predominant genetic alterations observed in LUAD. Computational analyses of public databases have revealed a correlation between high FOSL2 expression and poor survival in KRAS-mutant LUAD. Consistently, tumour cell expression of Fra-2 has been found to correlate with shorter survival and higher tumour burden in a genetically engineered mouse model of LUAD driven by KRasG12V mutation and Tp53 loss. Cell viability was assessed in primary tumour cells with Fra-2 gain- and loss-of-function, cultured from KRasG12V/Tp53-knockout mouse tumours. We prospectively collected fresh samples of resected LUAD tumours from patients with complete clinical and molecular annotations (N=27; 9 KRASmut), which were subjected to characterization by flow cytometry and IHC. In vitro analyses of primary tumour cells confirmed that overexpression of Fra-2 increases tumour cell viability. However, the absence of Fra-2 did not affect cell growth, suggesting that in vivo observations in Fra-2-deficient tumours could depend on anti-tumoral mechanisms that are extrinsic to tumour cells. In LUAD patients, the percentage of tumour cells expressing FRA-2 correlates with FRA-2 expression in immune cells as well as in cancer-associated fibroblasts (αSMA). FRA-2 also tends to show a positive correlation with proliferation (KI-67) and a negative correlation with T cells. A more detailed analysis by flow cytometry revealed that FRA-2 expression was related to an increased tumour density of regulatory T cells and decreased PD-1 expression, particularly in KRAS mutant tumours. Our results suggest that FRA-2 could play a significant role as a mediator in the tumorigenic effects driven by KRAS mutations in LUAD, by promoting both tumour cell proliferation and immune evasion. These findings provide a new therapeutic opportunity in a clinically relevant setting.

Supplementary Tables 2-3 from Podoplanin Is a Novel Fos Target Gene in Skin Carcinogenesis

Cancer-associated cachexia (CAC) is a hypermetabolic syndrome characterized by unintended weight loss due to the atrophy of adipose tissue and skeletal muscle. A phenotypic switch from white to beige adipocytes, a phenomenon called browning, accelerates CAC by increasing the dissipation of energy as heat. Addressing the mechanisms of white adipose tissue (WAT) browning in CAC, we now show that cachexigenic tumors activate type 2 immunity in cachectic WAT, generating a neuroprotective environment that increases peripheral sympathetic activity. Increased sympathetic activation, in turn, results in increased neuronal catecholamine synthesis and secretion, β-adrenergic activation of adipocytes, and induction of WAT browning. Two genetic mouse models validated this progression of events. 1) Interleukin-4 receptor deficiency impeded the alternative activation of macrophages, reduced sympathetic activity, and restrained WAT browning, and 2) reduced catecholamine synthesis in peripheral dopamine β-hydroxylase (DBH)–deficient mice prevented cancer-induced WAT browning and adipose atrophy. Targeting the intraadipose macrophage-sympathetic neuron cross-talk represents a promising therapeutic approach to ameliorate cachexia in cancer patients.