Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function.

Variably reduced expression of the basement membrane component laminin-332 (α3aß3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a ß3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.

The role of oncogenic Ras in human skin tumorigenesis depends on the clonogenic potential of the founding keratinocytes.

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis.

The Role of p16INK4a Pathway in Human Epidermal Stem Cell Self-Renewal, Aging and Cancer.

The epidermis is a self-renewing tissue. The balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of the stem cell (SC) population in the epidermis during life. Aging and cancer may be considered related endpoints of accumulating damages within epidermal self-renewing compartment. p16INK4a is a potent inhibitor of the G1/S-phase transition of the cell cycle. p16INK4a governs the processes of SC self-renewal in several tissues and its deregulation may result in aging or tumor development. Keratinocytes are equipped with several epigenetic enzymes and transcription factors that shape the gene expression signatures of different epidermal layers and allow dynamic and coordinated expression changes to finely balance keratinocyte self-renewal and differentiation. These factors converge their activity in the basal layer to repress p16INK4a expression, protecting cells from senescence, and preserving epidermal homeostasis and regeneration. Several stress stimuli may activate p16INK4a expression that orchestrates cell cycle exit and senescence response. In the present review, we discuss the role of p16INK4a regulators in human epidermal SC self-renewal, aging and cancer.

The Secretome of Aged Fibroblasts Promotes EMT-Like Phenotype in Primary Keratinocytes from Elderly Donors through BDNF-TrkB Axis.

Age-related changes in the dermis can play a primary role in tumor initiation promoting the unrestrained proliferation of precancerous keratinocytes (KCs) through cytokines and GF secretion. We found a high percentage of epithelial-to-mesenchymal transition-like colonies raising in primary human KC cultures from old subjects after treatment with aged fibroblast supernatants (SPNs). Continuous extracellular signals were required for maintaining these changes. Conversely, the secretome did not induce epithelial-to-mesenchymal transition-like colonies in KCs from young subjects. SPN-treated aged KCs displayed the activation of pathways involved in the disjunction of cell‒cell adhesion, extracellular matrix remodeling, manifestation of a mesenchymal phenotype, and dedifferentiation programs. Moreover, they recovered proliferation and clonogenic ability and showed enhanced migration. We identified an age-related increase of the BDNF secretion from fibroblasts as well as of the expression of its receptor TrkB in KCs. BDNF treatment of aged KCs induced TrkB phosphorylation and recapitulated the modifications promoted by aged fibroblast SPN. Furthermore, the treatment with a specific antibody against BDNF or a TrkB antagonist inhibited the paracrine signaling preventing SPN-mediated morphological and molecular changes. Finally, BDNF induced signs of matrix invasion in a three-dimensional organotypic model. Therefore, we demonstrate that aged fibroblast SPN promotes phenotypic plasticity in KCs from the elderly through BDNF-TrkB axis.

Cockayne Syndrome Type A Protein Protects Primary Human Keratinocytes from Senescence.

Defects in Cockayne syndrome type A (CSA), a gene involved in nucleotide excision repair, cause an autosomal recessive syndrome characterized by growth failure, progressive neurological dysfunction, premature aging, and skin photosensitivity and atrophy. Beyond its role in DNA repair, the CSA protein has additional functions in transcription and oxidative stress response, which are not yet fully elucidated. Here, we investigated the role of CSA protein in primary human keratinocyte senescence. Primary keratinocytes from three patients with CS-A displayed premature aging features, namely premature clonal conversion, high steady-state levels of reactive oxygen species and 8-OH-hydroxyguanine, and senescence-associated secretory phenotype. Stable transduction of CS-A keratinocytes with the wild-type CSA gene restored the normal cellular sensitivity to UV irradiation and normal 8-OH-hydroxyguanine levels. Gene correction was also characterized by proper restoration of keratinocyte clonogenic capacity and expression of clonal conversion key regulators (p16 and p63), decreased NF-κB activity and, in turn, the expression of its targets (NOX1 and MnSOD), and the secretion of senescence-associated secretory phenotype mediators. Overall, the CSA protein plays an important role in protecting cells from senescence by facilitating DNA damage processing, maintaining physiological redox status and keratinocyte clonogenic ability, and reducing the senescence-associated secretory phenotype-mediated inflammatory phenotype.

miR-200a Modulates the Expression of the DNA Repair Protein OGG1 Playing a Role in Aging of Primary Human Keratinocytes.

Oxidative DNA damage accumulation may induce cellular senescence. Notably, senescent cells accumulate in aged tissues and are present at the sites of age-related pathologies. Although the signaling of DNA strand breaks has been extensively studied, the role of oxidative base lesions has not fully investigated in primary human keratinocyte aging. In this study, we show that primary human keratinocytes from elderly donors are characterized by a significant accumulation of the oxidative base lesion 8-OH-dG, impairment of oxidative DNA repair, and increase of miR-200a levels. Notably, OGG1-2a, a critical enzyme for 8-OH-dG repair, is a direct target of miR-200a and its expression levels significantly decrease in aged keratinocytes. The 8-OH-dG accumulation displays a significant linear relationship with the aging biomarker p16 expression during keratinocyte senescence. Interestingly, we found that miR-200a overexpression down-modulates its putative target Bmi-1, a well-known p16 repressor, and up-regulates p16 itself. miR-200a overexpression also up-regulates the NLRP3 inflammasome and IL-1ß expression. Of note, primary keratinocytes from elderly donors are characterized by NRPL3 activation and IL-1ß secretion. These findings point to miR-200a as key player in primary human keratinocyte aging since it is able to reduce oxidative DNA repair activity and may induce several senescence features through p16 and IL-1ß up-regulation.

Oxidative Stress Induces HSP90 Upregulation on the Surface of Primary Human Endothelial Cells: Role of the Antioxidant 7,8-Dihydroxy-4-methylcoumarin in Preventing HSP90 Exposure to the Immune System.

We have previously demonstrated that human heat shock protein 90 (HSP90), an intracellular self protein, is the target of cellular and humoral autoimmune responses in patients with carotid atherosclerosis. In this study, we evaluated in vitro whether oxidative stress, a feature of atherosclerotic plaque, alters HSP90 expression in endothelial cells, thus inducing surface localization of this molecule and whether the antioxidant compound 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC) is able to prevent oxidative stress-induced alterations of HSP90 localization. By the use of flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and semiquantitative reverse-transcription polymerase chain reaction, we demonstrated that exposure of human umbilical vein endothelial cells (HUVEC) to the prooxidant compound H2O2 upregulated HSP90 surface expression and reduced its secretion without altering HSP90 gene expression and intracytoplasmic protein levels. Pretreatment of HUVEC with 7,8-DHMC prevented H2O2-induced alterations of HSP90 cellular distribution and secretion. Our results suggest that the strong oxidative conditions of atherosclerotic plaques promote the upregulation of HSP90 surface expression on endothelial cells, thus rendering the protein a possible target of autoimmune reactions. The antioxidant 7,8-DHMC, by preventing oxidative-stress-triggered HSP90 surface upregulation, may be useful to counteract possible autoreactive reactions to HSP90.

The Nutraceutical Dehydrozingerone and Its Dimer Counteract Inflammation- and Oxidative Stress-Induced Dysfunction of In Vitro Cultured Human Endothelial Cells: A Novel Perspective for the Prevention and Therapy of Atherosclerosis.

Atherosclerosis is characterized by endothelial dysfunction, mainly induced by inflammation and oxidative stress. Increased reactive oxygen species (ROS) production together with increased adhesion molecules and thrombogenic tissue factor (TF) expression on endothelial cells has a key role in proatherogenic mechanisms. Therefore downmodulation of these molecules could be useful for reducing the severity of inflammation and atherosclerosis progression. Dehydrozingerone (DHZ) is a nutraceutical compound with anti-inflammatory and antioxidant activities. In this study we evaluated the ability of DHZ and its symmetric dimer to modulate hydrogen peroxide- (H2O2-) induced ROS production in human umbilical vein endothelial cells (HUVEC). We also evaluated intercellular adhesion molecule- (ICAM-) 1, vascular cell adhesion molecule- (VCAM-) 1, and TF expression in HUVEC activated by tumor necrosis factor- (TNF-) α. HUVEC pretreatment with DHZ and DHZ dimer reduced H2O2-induced ROS production and inhibited adhesion molecule expression and secretion. Of note, only DHZ dimer was able to reduce TF expression. DHZ effects were in part mediated by the inhibition of the nuclear factor- (NF-) κB activation. Overall, our findings demonstrate that the DHZ dimer exerts a potent anti-inflammatory, antioxidant, and antithrombotic activity on endothelial cells and suggest potential usefulness of this compound to contrast the pathogenic mechanisms involved in atherosclerosis progression.