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.

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.

Speckle-tracking echocardiography: state of art and its applications.

Echocardiographic evaluation of left ventricular ejection fraction (LVEF) provides important information regarding both myocardial function and prognosis. This parameter presents various limitations and does not allow early detection of myocardial dysfunction. LVEF may be related to hemodynamic load, geometric assumptions, to image quality, and it does not reflect myocardial contractility. It has been hypothesized that speckle tracking echocardiography (STE) may allow overcoming such limits. STE through the measurement of strain and strain rate, which detect myocardial deformation, allows earlier identification of myocardial dysfunction in different settings both in presence of systolic and diastolic dysfunction, helps to predict left ventricular remodeling after acute myocardial infarction (AMI), and helps to decide the timing of surgery in asymptomatic severe valvular heart disease which is still a problematic issue. Increasingly interest regards the role of STE for the assessment of cardiomyopathies, myocarditis, and pulmonary hypertension. STE may be applied to the evaluation of systolic and diastolic dysfunction. STE is useful in all conditions in which cardiac dysfunction is not still overt, but a subclinical involvement is undoubtedly present such as in presence of cardiovascular risk factors and in cardio-oncology at earlier stages. It has been confirmed its role in predicting left ventricular remodeling after AMI which represents an important prognostic datum and in deciding the timing of surgery in asymptomatic valvular diseases. STE is an important tool to detect myocardial impairment even at earlier stages. 3DSTE and layer-specific strain represent promising fields of clinical application of STE.

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.

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.

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.

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.

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.

CD271 mediates stem cells to early progeny transition in human epidermis.

CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of ß1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.

Isolation and characterization of squamous cell carcinoma-derived stem-like cells: role in tumor formation.

In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of ß1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2-4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.

Apoptotic pathways in the pathogenesis of pemphigus: targets for new therapies.

Pemphigus is a group of rare autoimmune blistering diseases of the skin in which autoantibodies to desmosome cadherins, desmogleins, induce loss of cell-cell adhesion (acantholysis). In addition to steric hindrance and activation of intracellular phosphorylation cascade signaling pathways, apoptosis has been suggested to contribute to the mechanism by which pathogenic IgG induces acantholysis. We review the literature examining the role of apoptosis in pemphigus. Current data recognize a central role of apoptosis in the mechanisms of blister induction. In particular, here we stress the key role of FasL in pemphigus, as it is able to first induce apoptosis, then acantholysis. Being pro-apoptotic molecules important in blister formation, they could represent new specific targets for pemphigus treatment.

A previously unreported function of ß(1)B integrin isoform in caspase-8-dependent integrin-mediated keratinocyte death.

Integrins regulate adhesive cell-matrix interactions and mediate survival signals. On the other hand, unligated or free cytoplasmic fragments of integrins induce apoptosis in many cell types (integrin-mediated death). We have previously shown that ß(1) integrin expression protects keratinocyte stem cells from anoikis, whereas the role of the ß(1)B integrin isoform has not been clarified. In this study we report that suspended keratinocytes undergo apoptosis through the activation of caspase-8, independently of the Fas/Fas ligand system. Indeed, anti-ß(1) integrin-neutralizing antibodies induced apoptosis in short hairpin RNA Fas-associated death domain-treated cells. Moreover, before and during suspension, caspase-8 directly associated with ß(1) integrin, which in turn internalized and progressively degraded, shedding the cytoplasmic domain. ß(1)B was expressed only in the cytoplasm in a perinuclear manner and remained unaltered during suspension. At 24 hours, as ß(1)A was located close to the nucleus, ß(1)B colocalized with ß(1)A and coimmunoprecipitated with caspase-8. Caspase-8 was activated earlier in ß(1)B integrin-transfected keratinocytes, and these cells underwent a higher rate of apoptosis than mock cells. In contrast, caspase-8 was not activated in small interfering RNA (siRNA) ß(1)B-transfected cells. These results indicate that when ß(1)A is unligated, ß(1)B is responsible for "integrin-mediated death" in human keratinocytes.

Endogenous survivin modulates survival and proliferation in UVB-treated human keratinocytes.

Survivin is a bi-functional member of inhibitor of apoptosis protein family, as it is able to both inhibit apoptosis and to regulate cell cycle. We investigated the role of survivin in human keratinocytes under normal conditions and during UVB irradiation. Survivin siRNA decreases proliferation and induces apoptosis in human keratinocytes, in a mode consistent with the mitotic catastrophe. Low doses UVB increase survivin expression at earlier times, while high doses down-regulate survivin level. Low doses UVB induce cell cycle arrest in G2/M, while high doses UVB cause apoptosis. Moreover, overexpression of survivin protects keratinocytes from UVB-induced apoptosis, and silencing of survivin renders keratinocytes more susceptible to UVB-induced cell death. Finally, survivin siRNA increases UVB-induced reduction of cell proliferation. Taken together, these results indicate that survivin plays a critical role in epidermal homeostasis in normal conditions and during UVB exposure, with possible implication in skin carcinogenesis.

Neurotrophins and their receptors stimulate melanoma cell proliferation and migration.

Melanoma is a highly aggressive skin tumor that originates in the epidermis from melanocytes. As melanocytes share with the nervous system a common neuroectodermal origin and express all neurotrophins (NTs), we evaluated the expression and function of NTs and their receptors in melanoma. We report that primary and metastatic melanoma cell lines synthesize and secrete all NTs. Moreover, melanoma cells express the low-affinity (p75NTR) and the high-affinity tyrosine kinase NT receptors (Trk). The inhibition of Trk receptors by either K252a or Trk/Fc chimeras prevents proliferation, indicating that autocrine NTs are responsible for this effect. NT-3, NT-4, and nerve growth factor (NGF) induce cell migration, with a stronger effect on metastatic cell lines. Transfection with p75NTR small interfering RNA (p75NTRsiRNA) or treatment with K252a inhibits NT-induced melanoma cell migration, indicating that both the low- and high-affinity NT receptors mediate this effect. All melanoma cell lines express the p75NTR coreceptor sortilin by which proNGF stimulates migration in melanoma cells, but not in cells transfected with p75NTRsiRNA. These results indicate that NTs, through their receptors, play a critical role in the progression of melanoma.

Survivin identifies keratinocyte stem cells and is downregulated by anti-beta1 integrin during anoikis.

Survivin belongs to the family of inhibitor of apoptosis proteins and is involved in regulation of cell death as well as cell division. Here, we show that wild-type (WT) survivin is expressed in a subpopulation of basal keratinocytes in normal human skin at the cytoplasmic level. WT survivin is highly expressed in keratinocyte stem cells (KSCs), whereas its mRNA level decreases in transit amplifying (TA) cells and disappears in postmitotic (PM) cells. Likewise, WT survivin protein is expressed in KSCs, almost undetectable in TA cells, and absent in PM cells. Real time polymerase chain reaction demonstrates that the putative antiapoptotic isoforms survivin-2B and survivin-DeltaEx3 are expressed at the highest levels in KSCs, whereas they tend to decrease in TA cells and disappear in PM cells. On the contrary, the putative proapoptotic variants of survivin, survivin-3B, and survivin-2alpha tend to be high in PM and TA cells and are almost absent in KSCs. By confocal microscopy, survivin is predominantly expressed at the nuclear level in KSCs, which proliferate significantly better than TA cells, which, in turn, express mostly cytosolic WT survivin. Blocking beta1 integrin signal downregulates WT survivin mRNA and protein expression and induces apoptosis (anoikis) in KSCs. On the other hand, inhibition of beta1 integrin upregulates mRNA expression of survivin-2alpha. Taken together, these results indicate that survivin identifies human KSCs. Expression of nuclear survivin could reflect the different behavior between KSCs in vitro and in vivo, in terms of proliferation. Finally, survivin could be part of the "niche" protection by preventing anoikis in KSCs.