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

CD271 downregulation promotes melanoma progression and invasion in 3-dimensional models and in zebrafish

CD271 is a neurotrophin receptor variably expressed in melanoma. While contradictory data are reported on its role as a marker of tumor initiating cells, little is known on 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, while 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, as compared to 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 absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis

Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models

Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients' care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research

A previously unreported function of beta1B 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 b1 integrins expression protects keratinocyte stem cells from anoikis, while the role of the b1B integrin isoform has never been clarified. Here we report that suspended keratinocytes undergo apoptosis via the activation of caspase-8, independently of Fas/Fas Ligand system. Indeed, anti-b1 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 b1 integrin, that in turn internalized and progressively degraded, shedding the cytoplasmic domain. b1B was expressed only in the cytoplasm in a perinuclear fashion and remained unaltered during suspension. At 24 hrs, as b1A located close to the nucleus, b1B co-localized with b1A and co-immunoprecipitated with caspase-8. Caspase-8 was activated earlier in b1B integrin transfected keratinocytes, and these cells underwent a higher rate of apoptosis than mock cells. By contrast, caspase-8 was not activated in siRNA b1B transfected cells. These results indicate that when b1A is unligated, b1B is responsible for “integrin-mediated death” in human keratinocytes

A novel DLX3–PKC integrated signaling network drives keratinocyte differentiation

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3- dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3–PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis

E-FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro.

Epidermal fatty acid-binding protein (E-FABP) is a lipid carrier, originally discovered in human epidermis. We show that E-FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E-FABP induces overexpression of K10 and involucrin. On the other hand, E-FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF-κB and JNK signalling pathways. E-FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E-FABP as compared to the same population in normal epidermis. E-FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high-calcium conditions, E-FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E-FABP plays an important role in keratinocyte differentiation. Moreover, E-FABP modulates differentiation in psoriatic keratinocytes

Characterization of Skin Interfollicular Stem Cells and Early Transit Amplifying Cells during the Transition from Infants to Young Children

In the interfollicular epidermis, keratinocyte stem cells (KSC) generate a short-lived population of transit amplifying (TA) cells that undergo terminal differentiation after several cell divisions. Recently, we isolated and characterized a highly proliferative keratinocyte cell population, named “early” TA (ETA) cell, representing the first KSC progenitor with exclusive features. This work aims to evaluate epidermis, with a focus on KSC and ETA cells, during transition from infancy to childhood. Reconstructed human epidermis (RHE) generated from infant keratinocytes is more damaged by UV irradiation, as compared to RHE from young children. Moreover, the expression of several differentiation and barrier genes increases with age, while the expression of genes related to stemness is reduced from infancy to childhood. The proliferation rate of KSC and ETA cells is higher in cells derived from infants’ skin samples than of those derived from young children, as well as the capacity of forming colonies is more pronounced in KSC derived from infants than from young children’s skin samples. Finally, infants-KSC show the greatest regenerative capacity in skin equivalents, while young children ETA cells express higher levels of differentiation markers, as compared to infants-ETA. KSC and ETA cells undergo substantial changes during transition from infancy to childhood. The study presents a novel insight into pediatric skin, and sheds light on the correlation between age and structural maturation of the skin

Role of neurotrophins on dermal fibroblast survival and differentiation

Neurotrophins (NTs) belong to a family of growth factors that play a critical role in the control of skin homeostasis. NTs act through the low-affinity receptor p75NTR and the high-affinity receptors TrkA, TrkB and TrkC. Here we show that dermal fibroblasts (DF) and myofibroblasts (DM) synthesize and secrete all NTs and express NT receptors. NTs induce differentiation of DF into DM, as shown by the expression of \u3b1-SMA protein. The Trk inhibitor K252a, TrkA/Fc, TrkB/Fc or TrkC/Fc chimera prevents DF and DM proliferation. In addition, p75NTR siRNA inhibits DF proliferation, indicating that both NT receptors mediate DF proliferation induced by endogenous NTs. Autocrine NTs also induce DF migration through p75NTR and Trk, as either silencing of p75NTR or Trk/Fc chimeras prevent this effect, in absence of exogenous NTs. Finally, NGF or BDNF statistically increase the tensile strength in a dose dependent manner, as measured in a collagen gel through the GlaSbox device. Taken together, these results indicate that NTs exert a critical role on fibroblast and could be involved in tissue remodelling and wound healin