Blocking soluble Fas Ligand ameliorates pemphigus: PC111 efficacy in ex-vivo human pemphigus models.

Pemphigus is a life-threatening, chronic, autoimmune bullous disease affecting both the skin and the mucous membranes. Based on the mainstream concept that blister formation occurs upon binding of autoantibodies to their antigen proteins (desmoglein1, DSG1 and desmoglein3, DSG3), current therapies mostly aim to suppress the immune system. To avoid the severe side effects associated with the chronic use of immunosuppressive treatments, we have developed PC111, a fully human monoclonal antibody targeting human Fas ligand (FasL). We have provided a number of in vitro and in vivo evidences showing that soluble FasL induces keratinocyte apoptosis followed by acantholysis. An anti-murine FasL prevents blister formation in the pemphigus neonatal mouse model. To confirm the mechanism of action (MoA) and the efficacy of PC111 in a human pemphigus context, we used the keratinocyte dissociation assay and two independent Human Skin Organ Cultures (HSOC) pemphigus models. PC111 reduced acantholysis in vitro, as shown by the dose-dependent reduction of fragments in the monolayer cultures. In the first HSOC model, normal human skin was subcutaneously injected with a scFv antibody fragment directed against DSG1 and DSG3, resulting in a severe acantholysis (70-100%) after 24 hours. PC111 inhibited blister formation to around 50% of control. In the second model, normal human skin was injected with a mixture of pemphigus patients' autoantibodies resulting in a less severe acantholysis (20-30%). PC111 significantly suppressed blister formation to more than 75% up to 72 hours. These results confirm PC111 MoA and demonstrates the efficacy of the anti-FasL antibody also in a pemphigus setting.

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. METHODS: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. RESULTS: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. CONCLUSION: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development.

S2k guidelines on the management of paraneoplastic pemphigus/paraneoplastic autoimmune multiorgan syndrome initiated by the European Academy of Dermatology and Venereology (EADV).

BACKGROUND: Paraneoplastic pemphigus (PNP), also called paraneoplastic autoimmune multiorgan syndrome (PAMS), is a rare autoimmune disease with mucocutaneous and multi-organ involvement. PNP/PAMS is typically associated with lymphoproliferative or haematological malignancies, and less frequently with solid malignancies. The mortality rate of PNP/PAMS is elevated owing to the increased risk of severe infections and disease-associated complications, such as bronchiolitis obliterans. OBJECTIVES: These guidelines summarize evidence-based and expert-based recommendations (S2k level) for the clinical characterization, diagnosis and management of PNP/PAMS. They have been initiated by the Task Force Autoimmune Blistering Diseases of the European Academy of Dermatology and Venereology with the contribution of physicians from all relevant disciplines. The degree of consent among all task force members was included. RESULTS: Chronic severe mucositis and polymorphic skin lesions are clue clinical characteristics of PNP/PAMS. A complete assessment of the patient with suspected PNP/PAMS, requiring histopathological study and immunopathological investigations, including direct and indirect immunofluorescence, ELISA and, where available, immunoblotting/immunoprecipitation, is recommended to achieve a diagnosis of PNP/PAMS. Detection of anti-envoplakin antibodies and/or circulating antibodies binding to the rat bladder epithelium at indirect immunofluorescence is the most specific tool for the diagnosis of PNP/PAMS in a patient with compatible clinical and anamnestic features. Treatment of PNP/PAMS is highly challenging. Systemic steroids up to 1.5 mg/kg/day are recommended as first-line option. Rituximab is also recommended in patients with PNP/PAMS secondary to lymphoproliferative conditions but might also be considered in cases of PNP/PAMS associated with solid tumours. A multidisciplinary approach involving pneumologists, ophthalmologists and onco-haematologists is recommended for optimal management of the patients. CONCLUSIONS: These are the first European guidelines for the diagnosis and management of PNP/PAMS. Diagnostic criteria and therapeutic recommendations will require further validation by prospective studies.

In Vivo Melanoma Cell Morphology Reflects Molecular Signature and Tumor Aggressiveness.

Melanoma is the deadliest type of skin cancer characterized by high cellular heterogeneity, which contributes to therapy resistance and unpredictable disease outcome. Recently, by correlating reflectance confocal microscopy morphology with histopathological type, we identified four distinct melanoma subtypes: dendritic cell, round cell, dermal nest, and combined-type melanomas. In this study, each reflectance confocal microscopy melanoma subtype expressed a specific biomolecular profile and biological behavior in vitro. Markers of tumor aggressiveness, including Ki-67, MERTK, nestin, and stemness markers were highest in the most invasive combined-type and dermal nest melanomas than in dendritic cell and round cell melanomas. This was also confirmed in multicellular tumor spheroids. Transcriptomic analysis showed modulation of cancer progression-associated genes from dendritic cell to combined-type melanomas. The switch from E- to N-cadherin expression proved the epithelial-to-mesenchymal transition from dendritic cell to combined-type subtypes. The dermal nest melanoma was predominantly located in the dermis, as also shown in skin reconstructs. It displayed a unique behavior and a molecular profile associated with a high degree of aggressiveness. Altogether, our results show that each reflectance confocal microscopy melanoma subtype has a distinct biological and gene expression profile related to tumor aggressiveness, confirming that reflectance confocal microscopy can be a dependable tool for in vivo detection of different types of melanoma and for early diagnostic screening.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

Dermocosmetics: beneficial adjuncts in the treatment of acne vulgaris.

Introduction: Dermocosmetics are increasingly being recognized as an integral part of acne management. Dermocosmetics may minimize the side effects of acne medications, provide synergistic effects by improving the efficacy of other treatments, and limit exposure to environmental factors such as ultraviolet radiation. We aimed to provide an overview of the active ingredients and different types of preparations used in dermocosmetics for acne, and highlight supporting evidence for their use in clinical practice.Methods: A literature search for selected key words was performed using PubMed. Additional papers were identified based on author expertize.Results and discussion: The different types of active ingredients in dermocosmetics for acne can be classified as: sebum-controlling, antimicrobial, anti-inflammatory, anti-oxidant and/or keratolytic. Such agents may modulate the pathogenic pathways in acne. Dermocosmetics can be formulated as emulsions/creams, cleansers or camouflaging make-up. Dermocosmetics are useful treatment adjuncts for acne and have been shown to improve the clinical signs of acne, reduce transepidermal water loss and modify sebum production. Dermocosmetics have also been associated with reducing side effects of pharmacological treatments, high levels of patient satisfaction and increased adherence to treatment regimens. Together this evidence supports the use of dermocosmetics in clinical practice.

Wnt/CTNNB1 Signal Transduction Pathway Inhibits the Expression of ZFP36 in Squamous Cell Carcinoma, by Inducing Transcriptional Repressors SNAI1, SLUG and TWIST.

The Wnt/CTNNB1 pathway is often deregulated in epithelial tumors. The ZFP36 gene, encoding the mRNA binding protein Tristetraprolin (TTP), is downregulated in several cancers, where it has been described to behave as a tumor suppressor. By this report, we show that Wnt/CTNNB1 pathway is constitutively activated, and ZFP36 expression is downregulated in Squamous Cell Carcinoma (SCC) cell lines compared to normal keratinocytes. Moreover, we suggest that the decrease of ZFP36 expression might depend on the activity of transcriptional repressors SNAI1, SLUG and TWIST, whose expression is induced by Wnt/CTNNB1, highlighting a potential regulatory mechanism underlying ZFP36 downregulation in epithelial cancers.

Molecular Approach to Cutaneous Squamous Cell Carcinoma: From Pathways to Therapy.

Cutaneous squamous cell carcinoma (cSCC) represents the second most frequent skin cancer,recently showing a rapid increase in incidence worldwide, with around >1 million cases/year in theUnited States and 2500 deaths [1] [...].

Development of a Desmocollin-3 Active Mouse Model Recapitulating Human Atypical Pemphigus.

Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. It is often associated with autoantibodies to the desmosomal adhesion proteins Desmoglein 3 (DSG3) and Desmoglein 1 (DSG1). Recently, auto-antigens, such as desmocollins and others have been described in PV and in atypical pemphigus forms such as Pemphigus Herpetiformis (PH), Pemphigus Vegetans (PVeg), and Paraneoplastic Pemphigus (PP). Desmocollins belong to a cadherin subfamily that provides structure to the desmosomes and play an important role in cell-to-cell adhesion. In order to verify the pathogenic activity of anti-Desmocollin 3 (DSC3) antibodies, we developed an active disease model of pemphigus expressing anti-DSC3 autoantibodies or anti-DSC3 and anti-DSG3 antibodies. This approach included the adoptive transfer of DSC3 and/or DSG3 lymphocytes to Rag2-/- immunodeficient mice that express DSC3 and DSG3. Our results show that the presence of anti-DSC3 auto-antibodies is sufficient to determine the appearance of a pathological phenotype relatable to pemphigus, but with features not completely super-imposable to those observed in the DSG3 active model, suggesting that the DSC3 active model might mimic the atypical pemphigus. Moreover, the presence of both anti-DSC3 and anti-DSG3 antibodies determines a more severe phenotype and a slower response to prednisolone. In conclusion, we have developed an adult DSC3 pemphigus mouse model that differs from the DSG3 model and supports the concept that antigens other than desmogleins may be responsible for different phenotypes in human pemphigus.

Do DLX3 and CD271 Protect Human Keratinocytes from Squamous Tumor Development?

Well-regulated epidermal homeostasis depends on the function of different classes of factors, such as transcription regulators and receptors. Alterations in this homeostatic balance may lead to the development of cutaneous squamous tumorigenesis. The homeobox transcription factor DLX3 is determinant for a p53-dependent regulation of epidermal differentiation and modulates skin carcinogenesis. The maintenance of skin homeostasis also involves the action of neurotrophins (NTs) and their receptors, Trk and CD271. While Trk receptor overexpression is a hallmark of cancer, there are conflicting data on CD271 expression and function in cutaneous SCC (cSCC). Previous studies have reported NT receptors expression in head and neck SSC (HNSCC). We show that CD271 is expressed at low levels in primary cSCC cells and the number of CD271+ cells correlates with cell cohesion in SCC spheroids. In normal epidermis, CD271 is expressed in proliferative progenitor cells and DLX3 in terminally differentiated keratinocytes. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) increase DLX3 expression. In the absence of a functional BDNF receptor TrkB in keratinocytes, we hypothesize that the BDNF-dependent DLX3 response could be mediated via CD271. Altogether, our results support a putative CD271-DLX3 connection in keratinocytes, which might be crucial to preventing squamous skin cancer.

Mechanisms Underlying the Clinical Effects of Apremilast for Psoriasis.

Psoriasis is a chronic, systemic, inflammatory disease with manifestations resulting from a dysregulated immune response. In psoriatic skin, expression of all phosphodiesterase 4 (PDE4) isoforms (A-D), part of a family of enzymes known to regulate cyclic adenosine monophosphate levels and immune homeostasis, is elevated compared with healthy controls. Agents that inhibit the enzymatic activity of PDE4, the predominant PDE in most immune cells, exert well-recognized anti-inflammatory effects. Apremilast is a selective PDE4 inhibitor approved for the treatment of adults with moderate to severe plaque psoriasis and/or psoriatic arthritis. In vitro and in vivo investigations have demonstrated the beneficial impact of apremilast treatment on PDE4 activity, inflammatory signal expression, and dermal psoriasiform signs. In patients with moderate to severe psoriasis, treatment with apremilast is associated with significant reductions in plasma levels of interleukin (IL)-17F, IL-17A, IL-22, and tumor necrosis factor-α compared with placebo as early as week 4; decreases in cytokine levels were sustained with continued treatment. Multivariate analyses demonstrated that while changes in IL-17F are the most important predictor of improvement in Psoriasis Area and Severity Index scores, apremilast exerts synergistic attenuating effects among a key group of cytokines involved in the pathology of psoriasis, and these effects correlate with improved skin symptoms. These in vitro and clinical data demonstrate that the beneficial effects of apremilast on known inflammatory mediators are associated with its clinical efficacy. J Drugs Dermatol. 2018;17(8):835-840.

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Progress in melanoma modelling in vitro.

Melanoma is one of the most studied neoplasia, although laboratory techniques used for investigating this tumor are not fully reliable. Animal models may not predict the human response due to differences in skin physiology and immunity. In addition, international guidelines recommend to develop processes that contribute to the reduction, refinement and replacement of animals for experiments (3Rs). Adherent cell culture has been widely used for the study of melanoma to obtain important information regarding melanoma biology. Nonetheless, these cells grow in adhesion on the culture substrate which differs considerably from the situation in vivo. Melanoma grows in a 3D spatial conformation where cells are subjected to a heterogeneous exposure to oxygen and nutrient. In addition, cell-cell and cell-matrix interaction play a crucial role in the pathobiology of the tumor as well as in the response to therapeutic agents. To better study, melanoma new techniques, including spherical models, tumorospheres and melanoma skin equivalents, have been developed. These 3D models allow to study tumors in a microenvironment that is more close to the in vivo situation and are less expensive and time-consuming than animal studies. This review will also describe the new technologies applied to skin reconstructs such as organ-on-a-chip that allows skin perfusion through microfluidic platforms. 3D in vitro models, based on the new technologies, are becoming more sophisticated, representing at a great extent the in vivo situation, the "perfect" model that will allow less involvement of animals up to their complete replacement, is still far from being achieved.

Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus.

Pemphigus is a blistering disease characterized by pemphigus autoantibodies (PVIgG) directed mostly against desmogleins (Dsgs), resulting in the loss of keratinocyte adhesion (acantholysis). Yet, the mechanisms underlying blister formation remain to be clarified. We have shown previously that anti-Fas ligand (FasL) antibody (Ab) prevents PVIgG-induced caspase-8 activation and Dsg cleavage in human keratinocytes, and that sera from pemphigus patients contain abnormally increased levels of FasL. Here, we demonstrate that recombinant FasL induces the activation of caspases prior to Dsg degradation, and anti-FasL Ab prevents acantholysis in cultured keratinocytes. Moreover, the silencing of FasL reduces PVIgG-induced caspase-8 activation and Dsg3 cleavage. Following injection of PVIgG into mice, FasL is upregulated at 1-3 h and is followed by caspase-8-mediated keratinocyte apoptosis, before blister formation. The administration of anti-FasL Ab after PVIgG injection blocks blister formation in mice. Furthermore, we injected PVIgG into two different gene-targeted mutant mice that selectively lack either secreted soluble FasL (sFasL), FasLΔs/Δs mice, or the membrane-bound form of FasL (mFasL), FasLΔm/Δm mice. After PVIgG treatment, blisters are only visible in FasLΔm/Δm animals, lacking mFasL, but still producing sFasL, similar to wild-type (C57BL/6) animals. By contrast, a significant decrease in the relative acantholytic area is observed in the FasLΔs/Δs animals. These results demonstrate that soluble FasL plays a crucial role in the mechanisms of blister formation, and blockade of FasL could be an effective therapeutic approach for pemphigus.

Zebrafish as a Model Organism for the Development of Drugs for Skin Cancer.

Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future. This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation and apoptosis of skin cells. The development of animal models has allowed a better understanding of these pathomechanisms, with the possibility of carrying out toxicological screening and drug development. In particular, the zebrafish (Danio rerio) has been established as one of the most important model organisms for cancer research. This model is particularly suitable for live cell imaging and high-throughput drug screening in a large-scale fashion. Thanks to the recent advances in genome editing, such as the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) methodologies, the mechanisms associated with cancer development and progression, as well as drug resistance can be investigated and comprehended. With these unique tools, the zebrafish represents a powerful platform for skin cancer research in the development of target therapies. Here, we will review the advantages of using the zebrafish model for drug discovery and toxicological and phenotypical screening. We will focus in detail on the most recent progress in the field of zebrafish model generation for the study of melanoma and squamous cell carcinoma (SCC), including cancer cell injection and transgenic animal development. Moreover, we will report the latest compounds and small molecules under investigation in melanoma zebrafish models.

p75 Neurotrophin Receptor in the Skin: Beyond Its Neurotrophic Function.

p75 neurotrophin receptor (p75NTR), also known as CD271, is the low-affinity receptor that, together with the tyrosine kinase receptor tropomyosin-receptor kinase (Trk), mediate neurotrophin (NT) functions. Beside their classic role in skin innervation, NT and their receptors constitute a complex cutaneous network associated with a number of autocrine and paracrine activities. In this context, the role of p75NTR is becoming more and more important. This review will focus on the intriguing functions of p75NTR in healthy and diseased skin. First, p75NTR counterbalances the proliferative and survival activities of its cognate receptor Trk by inducing keratinocyte apoptosis. In addition, p75NTR identifies an early transit-amplifying (TA) keratinocyte population and plays a critical role in keratinocyte stem cell transition to its progeny as well as in epidermal differentiation. p75NTR is absent in psoriatic TA cells, thus rendering these cells resistant to apoptosis. On the other hand, p75NTR infection restores NT-induced apoptosis in psoriatic keratinocytes. Taken together, these results provide evidence for a critical role of p75NTR in epidermal homeostasis, while its lack may account for the TA defect in psoriasis. While the issue of p75NTR as a marker of melanoma initiating cells is still to be solved, there is strong evidence that downregulation of this receptor is a precondition to melanoma invasion and metastasis in vitro and in vivo. All in all, this review points to p75NTR as a major actor in both physiologic and pathologic conditions at the skin level.

Electrochemotherapy induces apoptotic death in melanoma metastases: a histologic and immunohistochemical investigation.

BACKGROUND: Electrochemotherapy (ECT) is increasingly used in the treatment of primary and secondary skin tumors, but little is known about the pathologic mechanism responsible for tumor cell destruction in humans. Knowledge of detailed mechanism of host response after ECT may improve the treatment efficacy related to patient selection and technique refinements. AIM: The aim of the study was to investigate the histopathology and mechanism of cell death after ECT in cutaneous melanoma metastases. METHODS: Skin biopsy specimens were sequentially obtained after ECT of cutaneous melanoma metastases, during a follow-up period of 2 months. Results from histologic evaluation and immunohistochemical characterization of the inflammatory infiltrate (CD3, CD4, CD8, CD56, Granzyme-B) were compared with a panel of apoptosis-related markers. MAIN OUTCOME MEASURES: Evidence of the mechanism of tumor cell damage, identification of histological and immunohistochemical signs of apoptosis and/or necrosis underlining a possible time course of tumor destruction and inflammatory reaction after ECT. RESULTS: Early signs of epidermal degeneration, an increase of the inflammatory infiltrate, and initial tumor cell morphological changes were already detected 10 min after ECT. The cell damage progression, as demonstrated by histological and immunohistochemical evidence using apoptotic markers (TUNEL and caspase-3 staining), reached a climax 3 days after treatment, to continue until 10 days after. Scarring fibrosis and complete absence of tumor cells were observed in the late biopsy specimens. A rich inflammatory infiltrate with a prevalence of T-cytotoxic CD3/CD8-positive cells was detected 3 h after ECT and was still appreciable 3 months later. CONCLUSION: This study attempts to define the time course and characteristics of tumor response to ECT. The observations suggest both a direct necrotic cell damage and a rapid activation of apoptotic mechanisms that occur in the early phases of the cutaneous reaction to ECT. A persistent immune response of T-cytotoxic lymphocytes could possibly explain the long-term local tumor control.

CD271 Down-Regulation Promotes Melanoma Progression and Invasion in Three-Dimensional Models and in Zebrafish.

CD271 is a neurotrophin receptor variably expressed in melanoma. Although contradictory data are reported on its role as a marker of tumor-initiating cells, little is known about its function in tumor progression. CD271 expression was higher in spheroids derived from freshly isolated cells of primary melanomas and in primary WM115 and WM793-B cell lines, and it decreased during progression to advanced stages in cells isolated from metastatic melanomas and in metastatic WM266-4 and 1205Lu cell lines. Moreover, CD271 was scarcely detected in the highly invasive spheroids (SKMEL28 and 1205Lu). CD271, originally expressed in the epidermis of skin reconstructs, disappeared when melanoma started to invade the dermis. SKMEL8 CD271(-) cells showed greater proliferation and invasiveness in vitro and were associated with a higher number of metastases in zebrafish compared with CD271(+) cells. CD271 silencing in WM115 induced a more aggressive phenotype in vitro and in vivo. On the contrary, CD271 overexpression in SKMEL28 cells reduced invasion in vitro, and CD271 overexpressing 1205Lu cells was associated with a lower percentage of metastases in zebrafish. A reduced cell-cell adhesion was also observed in the absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis.

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo.

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, ß1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in Ras(G12V)-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development.

Phosphodiesterase 4 in inflammatory diseases: Effects of apremilast in psoriatic blood and in dermal myofibroblasts through the PDE4/CD271 complex.

Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared with normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis as compared with normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by ß-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF- and transforming growth factor-ß1 (TGF-ß1)-induced fibroblast migration, and inhibits DF differentiation into DM mediated by NGF or TGF-ß1. Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with ß-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be important in modulating fibroblast functions.