Biomechanical activation of keloid fibroblasts promotes lysosomal remodelling and exocytosis.

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. While biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts (NDFs) and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix (ECM) stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fibre formation, and focal adhesion assembly. Elevated acto-myosin contractility in KDFs disrupted the normal mechanical regulation of ECM deposition and conferred resistance myosin inhibitors. Transcriptional profiling identified mechanically-regulated pathways in NDFs and KDFs, including the actin cytoskeleton, Hippo signalling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on acto-myosin contractility. However, KDFs displayed marked changes in lysosome organisation and an increase in lysosomal exocytosis, which was mediated by acto-myosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to ECM mechanics and promotes lysosomal exocytosis.

Similar local recurrence and survival in patients with T1 radial growth phase melanoma on head and neck treated with 5 or 10 mm margins: A retrospective study.

BACKGROUND: Melanoma guidelines recommend surgical excision with 10 mm margins for T1 melanomas (invasive melanomas with Breslow thickness ≤1 mm), including those in radial growth phase, which are without metastatic potential; however, such margins may be problematic on head-and-neck. OBJECTIVE: We compared outcomes of wide (10 mm margins) versus narrow (5 mm margins) excisions in patients with radial growth phase T1 melanoma on head-and-neck including face. METHODS: We retrospectively examined 610 consecutive patients excised with wide versus narrow margins, from 2001 to 2018, at six European centres. In all cases, radial growth phase, and clear margins with 5 or 10 mm of clearance, were ascertained histologically. Multivariable models investigated associations of margins and other factors with overall survival and local recurrence. RESULTS: Three hundred and sixteen (51.8%) patients received wide excision, 219 (69.3%) with primary wound closure, 97 (30.7%) with reconstruction; 294 (48.2%) patients received narrow excision, 264 (89.8%) with primary wound closure, 30 (10.2%) with reconstruction (p < 0.001). Median follow-ups were 88 months (wide) and 187 months (narrow) (inter-quartile ranges 43-133 and 79-206, respectively). Ten-year overall survival (95% confidence interval) was 96.7% (94.2%-99.3%) in wide and 98.2% (96.4%-100%) in narrow patients. Ten-year local recurrence incidence was 6.4% (4.1%-10.1%) in wide and 7.8% (5.3%-11.6%) in narrow groups. Lentigo maligna melanoma subtype appeared associated with increased risk of local recurrence in narrow versus wide patients (15.0% vs. 7.5%; p = 0.190). CONCLUSIONS: Narrower excision margins for T1 radial growth phase melanoma are not associated with worse overall survival (hazard ratio 0.97, p = 0.996) or increased local recurrence (subdistribution hazard ratio: 0.87; p = 0.751) compared to wider margins, and may be safely applied to such lesions, although caution may be required in the presence of lentigo maligna melanoma.

Growth and Viability of Cutaneous Squamous Cell Carcinoma Cell Lines Display Different Sensitivities to Isoform-Specific Phosphoinositide 3-Kinase Inhibitors.

Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.

Analysis of Sentinel Node Biopsy and Clinicopathologic Features as Prognostic Factors in Patients With Atypical Melanocytic Tumors.

BACKGROUND: Atypical melanocytic tumors (AMTs) include a wide spectrum of melanocytic neoplasms that represent a challenge for clinicians due to the lack of a definitive diagnosis and the related uncertainty about their management. This study analyzed clinicopathologic features and sentinel node status as potential prognostic factors in patients with AMTs. PATIENTS AND METHODS: Clinicopathologic and follow-up data of 238 children, adolescents, and adults with histologically proved AMTs consecutively treated at 12 European centers from 2000 through 2010 were retrieved from prospectively maintained databases. The binary association between all investigated covariates was studied by evaluating the Spearman correlation coefficients, and the association between progression-free survival and all investigated covariates was evaluated using univariable Cox models. The overall survival and progression-free survival curves were established using the Kaplan-Meier method. RESULTS: Median follow-up was 126 months (interquartile range, 104-157 months). All patients received an initial diagnostic biopsy followed by wide (1 cm) excision. Sentinel node biopsy was performed in 139 patients (58.4%), 37 (26.6%) of whom had sentinel node positivity. There were 4 local recurrences, 43 regional relapses, and 8 distant metastases as first events. Six patients (2.5%) died of disease progression. Five patients who were sentinel node-negative and 3 patients who were sentinel node-positive developed distant metastases. Ten-year overall and progression-free survival rates were 97% (95% CI, 94.9%-99.2%) and 82.2% (95% CI, 77.3%-87.3%), respectively. Age, mitotic rate/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss were factors affecting prognosis in the whole series and the sentinel node biopsy subgroup. CONCLUSIONS: Age >20 years, mitotic rate >4/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss proved to be worse prognostic factors in patients with ATMs. Sentinel node status was not a clear prognostic predictor.

Targeting p63 Upregulation Abrogates Resistance to MAPK Inhibitors in Melanoma.

Targeting the MAPK pathway by combined inhibition of BRAF and MEK has increased overall survival in advanced BRAF-mutant melanoma in both therapeutic and adjuvant clinical settings. However, a significant proportion of tumors develop acquired resistance, leading to treatment failure. We have previously shown p63 to be an important inhibitor of p53-induced apoptosis in melanoma following genotoxic drug exposure. Here, we investigated the role of p63 in acquired resistance to MAPK inhibition and show that p63 isoforms are upregulated in melanoma cell lines chronically exposed to BRAF and MEK inhibition, with consequent increased resistance to apoptosis. This p63 upregulation was the result of its reduced degradation by the E3 ubiquitin ligase FBXW7. FBXW7 was itself regulated by MDM2, and in therapy-resistant melanoma cell lines, nuclear accumulation of MDM2 caused downregulation of FBXW7 and consequent upregulation of p63. Consistent with this, both FBXW7-inactivating mutations and MDM2 upregulation were found in melanoma clinical samples. Treatment of MAPK inhibitor-resistant melanoma cells with MDM2 inhibitor Nutlin-3A restored FBXW7 expression and p63 degradation in a dose-dependent manner and sensitized these cells to apoptosis. Collectively, these data provide a compelling rationale for future investigation of Nutlin-3A as an approach to abrogate acquired resistance of melanoma to MAPK inhibitor targeted therapy. SIGNIFICANCE: Upregulation of p63, an unreported mechanism of MAPK inhibitor resistance in melanoma, can be abrogated by treatment with the MDM2 inhibitor Nutlin-3A, which may serve as a strategy to overcome resistance.

Factors Affecting Sentinel Node Metastasis in Thin (T1) Cutaneous Melanomas: Development and External Validation of a Predictive Nomogram.

PURPOSE: Thin melanomas (T1; ≤ 1 mm) constitute 70% of newly diagnosed cutaneous melanomas. Regional node metastasis determined by sentinel node biopsy (SNB) is an important prognostic factor for T1 melanoma. However, current melanoma guidelines do not provide clear indications on when to perform SNB in T1 disease and stress an individualized approach to SNB that considers all clinicopathologic risk factors. We aimed to identify determinants of sentinel node (SN) status for incorporation into an externally validated nomogram to better select patients with T1 disease for SNB. PATIENTS AND METHODS: The development cohort comprised 3,666 patients with T1 disease consecutively treated at the Istituto Nazionale Tumori (Milan, Italy) between 2001 and 2018; 4,227 patients with T1 disease treated at 13 other European centers over the same period formed the validation cohort. A random forest procedure was applied to the development data set to select characteristics associated with SN status for inclusion in a multiple binary logistic model from which a nomogram was elaborated. Decision curve analyses assessed the clinical utility of the nomogram. RESULTS: Of patients in the development cohort, 1,635 underwent SNB; 108 patients (6.6%) were SN positive. By univariable analysis, age, growth phase, Breslow thickness, ulceration, mitotic rate, regression, and lymphovascular invasion were significantly associated with SN status. The random forest procedure selected 6 variables (not growth phase) for inclusion in the logistic model and nomogram. The nomogram proved well calibrated and had good discriminative ability in both cohorts. Decision curve analyses revealed the superior net benefit of the nomogram compared with each individual variable included in it as well as with variables suggested by current guidelines. CONCLUSION: We propose the nomogram as a decision aid in all patients with T1 melanoma being considered for SNB.

The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53.

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3-/- mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/- controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell-cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.

Epigenetic Regulation of iASPP-p63 Feedback Loop in Cutaneous Squamous Cell Carcinoma.

Keratinocyte skin cancer, comprising cutaneous squamous (cSCC) and basal cell carcinoma, is the most common malignancy in the United Kingdom. P53 is frequently mutated in cSCC. iASPP is a key inhibitor of p53 and NF-κB signaling pathways and has been documented as highly expressed in several types of human cancer. We have previously identified an autoregulatory feedback loop between iASPP and p63, which is critical in epidermal homeostasis. We hypothesized a potential role for dysregulation of this axis in the pathogenesis of keratinocyte malignancies. Immunostaining of 116 cSCC clinical samples revealed increased iASPP and ΔNp63 expression, but also highlighted a significant alteration of iASPP cellular localization, with consequent deregulation of its function. Expression patterns, functionality, and gene and microRNA expression analysis were further investigated in 10 cSCC cell lines. Our data suggest that while direct effects of iASPP and p63 upon each other's expression are maintained in cSCC, epigenetic dysregulation of the feedback loop occurs at the microRNA level by a previously unreported mechanism controlling p63 expression. We demonstrate that this autoregulatory feedback loop controls cell migration in cSCC by blocking epithelial-mesenchymal transition and promoting proliferation, and provides future directions for clinical biomarker and therapeutic target discovery in cutaneous SCC.

Uncovering mechanisms of nuclear degradation in keratinocytes: A paradigm for nuclear degradation in other tissues.

Eukaryotic nuclei are essential organelles, storing the majority of the cellular DNA, comprising the site of most DNA and RNA synthesis, controlling gene expression and therefore regulating cellular function. The majority of mammalian cells retain their nucleus throughout their lifetime, however, in three mammalian tissues the nucleus is entirely removed and its removal is essential for cell function. Lens fibre cells, erythroblasts and epidermal keratinocytes all lose their nucleus in the terminal differentiation pathways of these cell types. However, relatively little is known about the pathways that lead to complete nuclear removal and about how these pathways are regulated. In this review, we aim to discuss the current understanding of nuclear removal mechanisms in these three cell types and expand upon how recent studies into nuclear degradation in keratinocytes, an easily accessible experimental model, could contribute to a wider understanding of these molecular mechanisms in both health and pathology.

Class II phosphoinositide 3-kinase C2ß regulates a novel signaling pathway involved in breast cancer progression.

It is now well established that the enzymes phosphoinositide 3-kinases (PI3Ks) have a key role in the development and progression of many cancer types and indeed PI3Ks inhibitors are currently being tested in clinical trials. Although eight distinct PI3K isoforms exist, grouped into three classes, most of the evidence currently available are focused on one specific isoform with very little known about the potential role of the other members of this family in cancer. Here we demonstrate that the class II enzyme PI3K-C2ß is overexpressed in several human breast cancer cell lines and in human breast cancer specimens. Our data indicate that PI3K-C2ß regulates breast cancer cell growth in vitro and in vivo and that PI3K-C2ß expression in breast tissues is correlated with the proliferative status of the tumor. Specifically we show that downregulation of PI3K-C2ß in breast cancer cell lines reduces colony formation, induces cell cycle arrest and inhibits tumor growth, in particular in an estrogen-dependent in vivo xenograft. Investigation of the mechanism of the PI3K-C2ß-dependent regulation of cell cycle progression and cell growth revealed that PI3K-C2ß regulates cyclin B1 protein levels through modulation of microRNA miR-449a levels. Our data further demonstrate that downregulation of PI3K-C2ß inhibits breast cancer cell invasion in vitro and breast cancer metastasis in vivo. Consistent with this, PI3K-C2ß is highly expressed in lymph-nodes metastases compared to matching primary tumors. These data demonstrate that PI3K-C2ß plays a pivotal role in breast cancer progression and in metastasis development. Our data indicate that PI3K-C2ß may represent a key molecular switch that regulates a rate-limiting step in breast tumor progression and therefore it may be targeted to limit breast cancer spread.

Constitutive Autophagy and Nucleophagy during Epidermal Differentiation.

Epidermal keratinocytes migrate through the epidermis up to the granular layer where, on terminal differentiation, they progressively lose organelles and convert into anucleate cells or corneocytes. Our report explores the role of autophagy in ensuring epidermal function providing the first comprehensive profile of autophagy marker expression in developing epidermis. We show that autophagy is constitutively active in the epidermal granular layer where by electron microscopy we identified double-membrane autophagosomes. We demonstrate that differentiating keratinocytes undergo a selective form of nucleophagy characterized by accumulation of microtubule-associated protein light chain 3/lysosomal-associated membrane protein 2/p62 positive autolysosomes. These perinuclear vesicles displayed positivity for histone interacting protein, heterochromatin protein 1α, and localize in proximity with Lamin A and B1 accumulation, whereas in newborn mice and adult human skin, we report LC3 puncta coincident with misshaped nuclei within the granular layer. This process relies on autophagy integrity as confirmed by lack of nucleophagy in differentiating keratinocytes depleted from WD repeat domain phosphoinositide interacting 1 or Unc-51 like autophagy activating kinase 1. Final validation into a skin disease model showed that impaired autophagy contributes to the pathogenesis of psoriasis. Lack of LC3 expression in psoriatic skin lesions correlates with parakeratosis and deregulated expression or location of most of the autophagic markers. Our findings may have implications and improve treatment options for patients with epidermal barrier defects.

Prediction of survival in patients with thin melanoma: results from a multi-institution study.

PURPOSE: Cutaneous melanoma incidence is increasing. Most new cases are thin (≤ 1 mm) with favorable prognoses, but survival is nonetheless variable. Our aim was to investigate new prognostic factors and construct a nomogram for predicting survival in individual patients. PATIENTS AND METHODS: Data from 2,243 patients with thin melanoma were retrieved from prospectively maintained databases at six centers. Kaplan-Meier survival and crude cumulative incidences of recurrence were estimated, and competing risks were taken into account. Multivariable Cox regression was used to investigate survival predictors. RESULTS: Median follow-up was 124 months (interquartile range, 106 to 157 months); 12-year overall survival was 85.3% (95% CI, 83.4% to 87.2%). Median times to local, regional, and distant recurrence were 79, 78, and 107 months, respectively. Relapse was significantly related to age, Breslow thickness, mitotic rate (MR), ulceration, lymphovascular invasion (LVI), and regression; incidence was lower and subgroup differences were less marked for distant metastasis than for regional relapse. The worst prognosis categories were age older than 60 years, Breslow thickness more than 0.75 mm, MR ≥ 1, presence of ulceration, presence of LVI, and regression ≥ 50%. Breslow thickness more than 0.75 mm, MR ≥ 1, presence of ulceration, and LVI (all P = .001) were significantly associated with sentinel node positivity. Age, MR, ulceration, LVI, regression, and sentinel node status were independent predictors of survival and were used to construct a nomogram to predict 12-year overall survival. The nomogram was well calibrated and had good discriminative ability (adjusted Harrell C statistic, 0.88). CONCLUSION: Our findings suggest including LVI and regression as new prognostic factors in the melanoma staging system. The nomogram appears useful for risk stratification in clinical management and for recruiting patients to clinical trials.

iASPP is a novel autophagy inhibitor in keratinocytes.

The protein iASPP (encoded by PPP1R13L) is an evolutionarily conserved p53 inhibitor, the expression of which is often upregulated in human cancers. We have recently shown that iASPP is a crucial regulator of epidermal homeostasis. Here, we report that iASPP also acts as autophagy inhibitor in keratinocytes. Our data show that depletion of iASPP protects keratinocytes from apoptosis by modulating the expression of Noxa (also known as PMAIP1). In our model, iASPP expression can affect the fission-fusion cycle, mass and shape of mitochondria. iASPP-silenced keratinocytes display disorganization of cytosolic compartments and increased metabolic stress caused by deregulation of mTORC1 signaling. Moreover, increased levels of lipidated LC3 protein confirmed the activation of autophagy in iASPP-depleted cells. We have identified a novel mechanism modulating autophagy in keratinocytes that relies upon iASPP expression specifically reducing the interaction of Atg5-Atg12 with Atg16L1, an interaction that is essential for autophagosome formation or maturation. Using organotypic culture, we further explored the link between autophagy and differentiation, and we showed that impairing autophagy affects epidermal terminal differentiation. Our data provide an alternative mechanism to explain how epithelial integrity is maintained against environmental stressors and might also improve the understanding of the etiology of skin diseases that are characterized by defects in differentiation and DNA damage responses.

Is Mcl-1L the new anti-apoptotic effector of B-RAFV(600E) in melanoma?

A recent report has unveiled a novel mechanism by which oncogenic BRAF signalling might trigger apoptotic resistance in melanoma by selectively affecting the expression of Bcl-2 family member Mcl-1L (Exp Dermatol 2013: 22: 767). Correlation of Mcl-1 splice variants and B-RAF mutational status was determined in a panel of melanoma cell lines. In vivo validation of this mechanism, which is supported by recent literature, might provide novel therapeutic approaches such as the use of targeted Mcl-1 inhibitors to improve outcome in melanoma.

p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis.

The role of apoptosis in melanoma pathogenesis and chemoresistance is poorly characterized. Mutations in TP53 occur infrequently, yet the TP53 apoptotic pathway is often abrogated. This may result from alterations in TP53 family members, including the TP53 homologue TP63. Here we demonstrate that TP63 has an antiapoptotic role in melanoma and is responsible for mediating chemoresistance. Although p63 was not expressed in primary melanocytes, up-regulation of p63 mRNA and protein was observed in melanoma cell lines and clinical samples, providing the first evidence of significant p63 expression in this lineage. Upon genotoxic stress, endogenous p63 isoforms were stabilized in both nuclear and mitochondrial subcellular compartments. Our data provide evidence of a physiological interaction between p63 with p53 whereby translocation of p63 to the mitochondria occurred through a codependent process with p53, whereas accumulation of p53 in the nucleus was prevented by p63. Using RNA interference technology, both isoforms of p63 (TA and ΔNp63) were demonstrated to confer chemoresistance, revealing a novel oncogenic role for p63 in melanoma cells. Furthermore, expression of p63 in both primary and metastatic melanoma clinical samples significantly correlated with melanoma-specific deaths in these patients. Ultimately, these observations provide a possible explanation for abrogation of the p53-mediated apoptotic pathway in melanoma, implicating novel approaches aimed at sensitizing melanoma to therapeutic agents.

Inhibitory member of the apoptosis-stimulating proteins of the p53 family (iASPP) interacts with protein phosphatase 1 via a noncanonical binding motif.

Although kinase mutations have been identified in various human diseases, much less is known about protein phosphatases. Here, we show that all apoptosis-stimulating proteins of p53 (ASPP) family members can bind protein phosphatase 1 (PP1) via two distinct interacting motifs. ASPP2 interacts with PP1 through an RVXF PP1 binding motif, whereas the inhibitory member of the ASPP family (iASPP) interacts with PP1 via a noncanonical motif (RNYF) that is located within its Src homology 3 domain (SH3). Phe-815 is crucial in mediating iASPP/PP1 interaction, and iASPP(F815A) fails to inhibit the transcriptional and apoptotic function of p53. This study identifies iASPP as a new binding partner of PP1, interacting through a noncanonical PP1 binding motif.