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IntroductionMycosis fungoides (MF), the most common type of Cutaneous T cell Lymphoma (CTCL), is characterized by an inflamed skin intermixed with proliferating malignant mature skin-homing CD4+ T cells. Detailed genomic analyses of MF skin biopsies revealed several candidate genes possibly involved in genesis of these tumors and/or potential targets for therapy. These studies showed, in addition to common loss of cell cycle regulator CDKN2A, activation of several oncogenic pathways, most prominently and consistently involving JAK/STAT signaling. SOCS1, an endogenous inhibitor of the JAK/STAT signaling pathway, was identified as a recurrently deleted gene in MF, already occurring in the earliest stages of the disease.MethodsTo explore the mechanisms of MF, we create in vivo mouse models of autochthonous CTCLs and these genetically engineered mouse models (GEMMS) can also serve as valid experimental models for targeted therapy. We describe the impact of allelic deletion of Socs1 in CD4 T cells of the skin. To achieve this, we crossed inducible Cre-transgenic mice in the CD4 lineage with transgenic mice carrying floxed genes of Socs1. We first determined optimal conditions for Socs1 ablation with limited effects on circulating CD4 T-cells in blood. Next, we started time-course experiments mimicking sustained inflammation, typical in CTCL. FACS analysis of the blood was done every week. Skin biopsies were analyzed by immunocytochemical staining at the end of the experiment.ResultsWe found that the Socs1 knockout transgenic group had thicker epidermis of treated skin compared with the control group and had more CD3 and CD4 in the skin of the transgenic group compared to the control group. We also noted more activation of Stat3 by staining for P-Stat3 in Socs1 knockout compared to wt CD4+T cells in the skin. The results also indicated that single copy loss of Socs1 in combination with sustained inflammation is insufficient to start a phenotype resembling early stage mycosis fungoides within eight weeks in these mice.ConclusionIn sum, we developed and optimized an autochthonous murine model permitting selective knockout of Socs1 in skin infiltrating CD4 T-cells. This paves the way for more elaborate experiments to gain insight in the oncogenesis of CTCL.

Primary cutaneous CD8(+) aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) is a rare variant of cutaneous T-cell lymphoma with an aggressive clinical course and a very poor prognosis. Until now, neither a systematic characterization of genetic alterations driving pcAECyTCL has been performed, nor effective therapeutic regimes for patients have been defined. Here, we present the first high-resolution genetic characterization of pcAECyTCL by using whole-genome and RNA sequencing. Our study provides a comprehensive description of genetic alterations (i.e., genomic rearrangements, copy number alterations and small-scale mutations) with pathogenic relevance in this lymphoma, including events that recurrently impact genes with important roles in the cell cycle, chromatin regulation and the JAK-STAT pathway. In particular, we show that mutually exclusive structural alterations involving JAK2 and SH2B3 predominantly underlie pcAECyTCL. In line with the genomic data, transcriptome analysis uncovered upregulation of the cell cycle, JAK2 signaling, NF-kappa B signaling and a high inflammatory response in this cancer. Functional studies confirmed oncogenicity of JAK2 fusions identified in pcAECyTCL and their sensitivity to JAK inhibitor treatment. Our findings strongly suggest that overactive JAK2 signaling is a central driver of pcAECyTCL, and consequently, patients with this neoplasm would likely benefit from therapy with JAK2 inhibitors such as Food and Drug Adminstration-approved ruxolitinib.

UV radiation in sunlight has long been recognized as the main exogenous cause of skin carcinomas. We present a brief historical perspective on the progress in understanding the pathogenesis of skin carcinomas, and recent advances. Sun-exposed skin carries numerous UV-related mutations, and skin carcinomas rank among the tumors with the highest mutational loads. In this multitude of mutations only a few are crucial in driving the tumor. Some are known from hereditary (skin) cancer syndromes and other recurrent ones have been validated in transgenic mice. Considering the continuous renewal of the epidermis, the question arises whether the lifelong residing stem cells are the main targets in skin carcinogenesis, a multistep process that would require ample time to evolve. Therefore, classic quiescent stem cells have been studied as potential tumor-initiating cells, as well as more recently discovered actively dividing stem cells (either Lgr5+ or Lgr6+). Interesting differences have emerged between experimental UV and two-stage chemical carcinogenesis, e.g., the latter appears to originate from follicular stem cells, in contrast to the former.

The observation that mutations in the phospholipase C gamma 1 (PLCG1) gene (among which p.S345F was shown to be activating) are frequent (20%) in tumoral cutaneous T-cell lymphoma (CTCL) samples raised the possibility of targeting therapies against the PLCG1 signaling pathway. However, new data by Caumont et al. in this issue of JID show that PLCG1 mutations are far less prevalent than expected in CTCLs, which tempers the initial enthusiasm. This new study finds that only 3-5% of the CTCL tumor genomes (mycosis fungoides and Sézary syndrome) harbor PLCG1 mutations.

Cutaneous squamous cell carcinoma (SCC) is the second most common type of skin cancer in the Caucasian population worldwide, having a propensity for invasion, local recurrence and metastasis. Stromal cancer-associated fibroblasts (CAFs) are suspected to play an important role in SCC carcinogenesis. In this study, we characterized CAFs isolated from primary cutaneous SCCs and compared them to normal fibroblasts (NFs) isolated from healthy dermis. Human skin CAFs in monolayers displayed different morphology, increased proliferation and migration compared to NFs. CAB caused strong contraction of collagen matrices in which they were seeded and released high levels of the extracellular matrix component pro-collagen I. CAFs decreased proliferation and differentiation in the epidermis of human skin equivalents (FISEs) seeded with SCC cell lines, without affecting basement membrane composition. Finally, CAFs significantly increased invasion and dermal-epidermal detachment of SCC cell lines SCC-12B2 and SCC-13, respectively, when cultured in HSEs. These distinct features of CAFs point out a specific role in cutaneous SCC development.

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma. © 2010 John Wiley & Sons A/S.

Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub-sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (= 4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (>2 mm) from the Rapa-fed group showed a reduction in UV-signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf-positive in the Rapa-fed groups, but a correspondingly reduced expression of Hif1 alpha target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV-induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients.

Primary cutaneous anaplastic large cell lymphoma (C-ALCL) has an indolent clinical course and favorable prognosis. On the contrary, primary cutaneous peripheral T-cell lymphoma not otherwise specified (PTL-NOS) shows aggressive clinical behavior. To identify genomic events relevant in the pathogenesis of these cutaneous T-cell lymphomas (CTCLs), we carried out array-based comparative genomic hybridization (CGH) analysis. Simultaneously, gene expression profiling was conducted to gain insight into gene expression programs associated with the different clinical behavior of these CTCLs. C-ALCL was characterized by gains on chromosome 7q and 17q and losses on 6q and 13q. PTL-NOS similarly showed gains on 7q and 17q, but was distinguished by gains on chromosome 8 and loss of a focal overlapping region on 9p21. We identified minimal common regions harboring candidate oncogenes and tumor suppressor genes in C-ALCL and PTL-NOS. Genes with a role in lymphocyte chemotaxis, apoptosis, and proliferation were overrepresented among genes differentially expressed between these lymphomas. C-ALCL showed higher expression of the skin-homing chemokine receptor genes CCR10 and CCR8, which may explain the lower tendency to disseminate to extracutaneous sites. Furthermore, C-ALCL and PTL-NOS showed aberrant expression of distinct genes implicated in apoptosis and proliferation, such as IRF4/MUM1 and PRKCQ, which may account for differences in clinical aggressiveness.

Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total spectrum of chemokine variants. We have recently shown that the native chemokine CXCL10 is processed at the C terminus, thereby shedding the last four amino acids. The present study was performed to elucidate the mechanism in vivo and in vitro and to study the biological activity of this novel isoform of CXCL10. Using a combination of protein purification and mass spectrometric techniques, we show that the production of C-terminally truncated CXCL10 by primary keratinocytes is inhibited in vivo by a specific inhibitor of pro-protein convertases (e.g. furin) but not by inhibition of matrix metalloproteinases. Moreover, CXCL10 is processed by furin in vitro, which is abrogated by a mutation in the furin recognition site. Using GTRγS binding, Ca

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-γ stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-γ- inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-γ-inducible protein-9, revealed that interferon-γ-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-γ-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as β-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-γ-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.

Contains fulltext : mmubn000001_128869259.pdf (Publisher’s version ) (Open Access) Promotores : J. Denucé en F. van Herp 159 p.