GGCX mutations in a patient with overlapping pseudoxanthoma elasticum/cutis laxa-like phenotype.

Pseudoxanthoma elasticum (PXE) is a multisystem disorder characterized by ectopic mineralization of connective tissues with primary manifestations in the skin, eyes and the cardiovascular system. The classic forms of PXE are caused by mutations in the ABCC6 gene encoding the ABCC6 protein, expressed primarily in the liver. Cutis laxa (CL) manifests with loose and sagging skin with loss of recoil. In 2009 we investigated a 19-year-old patient with overlapping cutaneous features of PXE and CL, together with alpha thalassaemia. Genetic analysis failed to identify pathogenic mutations in ABCC6. More recently we developed a gene-targeted panel of next-generation sequencing technology. This panel has 29 genes, 22 of which, including ABCC6 and GGCX, are associated with ectopic mineralization phenotypes. Mutation analysis in the patient identified two heterozygous GGCX mutations: c.200_201delTT in exon 2 and c.763G>A, p.V255M in exon 7. The GGCX gene encodes a γ-glutamyl carboxylase necessary for activation of blood coagulation factors in the liver. The p.V255M mutation was previously reported to result in reduced γ-glutamyl carboxylase activity in vitro, while the c.200_201delTT mutation is novel. Previous studies reported that mutations in GGCX cause overlapping PXE/CL skin phenotypes in association with or without multiple vitamin K-dependent coagulation factor deficiency. Our patient had loose redundant skin, moderate-to-severe angioid streaks and characteristic calcification of elastic structures in the mid dermis, consistent with PXE/CL overlap, but no coagulation abnormalities. Our studies expand the GGCX mutation landscape in patients with PXE-like phenotypes.

A quantitative approach to histopathological dissection of elastin-related disorders using multiphoton microscopy.

BACKGROUND: Multiphoton microscopy (MPM) is a novel imaging technology that has recently become applicable for diagnostic purposes. The use of (near) infrared light in MPM allows for deep tissue imaging. In addition, this modality exploits the autofluorescent nature of extracellular matrix fibres within the skin. OBJECTIVES: To quantitate the structure and abundance of elastic fibres in human dermis in three dimensions utilizing autofluorescent signals generated by MPM for the objective examination of elastin-related skin disorders. METHODS: Cross-sections of skin samples from elastin-related disorders were analysed by MPM and correlated to histopathology. In situ visualization of elastic fibres by MPM was conducted by en face imaging of ex vivo skin samples through the intact epidermis. Image analysis software was used to quantify elastic fibres in three dimensions. RESULTS: Based on the MPM-detected elastin-specific autofluorescence, we developed the Dermal Elastin Morphology Index (DEMI), calculated as the ratio of elastic fibre surface area and volume. This enabled objective three-dimensional quantification of elastic fibres. Quantitative scoring of sun-damaged skin using DEMI correlated with qualitative histopathological grading of the severity of solar elastosis. Furthermore, this approach was applied to changes in elastic fibre architecture in other disorders, such as pseudoxanthoma elasticum (PXE), PXE-like syndrome, elastofibroma, focal dermal elastosis, anetoderma, mid-dermal elastolysis and striae distensae. We imaged elastic fibres in intact ex vivo skin imaged en face through the epidermis, indicating that this approach could be used in vivo. CONCLUSIONS: MPM has the potential for noninvasive in vivo visualization of elastic fibres in the dermis with near histological resolution. DEMI allows objective assessment of elastic fibres to support diagnosis and monitoring of disease progress or therapy of elastin-related skin disorders.

Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles.

Certain properties of the highly specialized synaptic transmitter vesicles are shared by constitutively occurring vesicles. We and others have thus identified a cDNA in various nonneuroendocrine cell types of rat and human that is related to synaptophysin, one of the major synaptic vesicle membrane proteins, which we termed pantophysin. Here we characterize the gene structure, mRNA and protein expression, and intracellular distribution of pantophysin. Its mRNA is detected in murine cell types of nonneuroendocrine as well as of neuroendocrine origin. The intron/exon structure of the murine pantophysin gene is identical to that of synaptophysin except for the last intron that is absent in pantophysin. The encoded polypeptide of calculated mol wt 28,926 shares many sequence features with synaptophysin, most notably the four hydrophobic putative transmembrane domains, although the cytoplasmic end domains are completely different. Using antibodies against the unique carboxy terminus pantophysin can be detected by immunofluorescence microscopy in both exocrine and endocrine cells of human pancreas, and in cultured cells, colocalizing with constitutive secretory and endocytotic vesicle markers in nonneuroendocrine cells and with synaptophysin in cDNA-transfected epithelial cells. By immunoelectron microscopy, the majority of pantophysin reactivity is detected at vesicles with a diameter of < 100 nm that have a smooth surface and an electron-translucent interior. Using cell fractionation in combination with immunoisolation, these vesicles are enriched in a light fraction and shown to contain the cellular vSNARE cellubrevin and the ubiquitous SCAMPs in epithelial cells and synaptophysin in neuroendocrine or cDNA-transfected nonneuroendocrine cells and neuroendocrine tissues. Pantophysin is therefore a broadly distributed marker of small cytoplasmic transport vesicles independent of their content.

Melanocytes: from morphology to application.

Melanocytes in human skin are intricately regulated by keratinocytes and the surrounding stroma. The development of melanoma is thought to arise from disrupted melanocyte homeostasis. It is now known that microenvironment plays a major role in maintenance of cellular homeostasis and can contribute to tumor initiation and tumor progression. Historically, melanocyte studies have been performed in two-dimensional culture systems, and often with melanocytes cultured in the absence of keratinocytes. Here we present the biological basis for the use of organotypic, three-dimensional model systems in the study of melanoma, and highlight the features of the most utilized organotypic model systems.

In vivo overexpression of Emi1 promotes chromosome instability and tumorigenesis.

Cell cycle genes are often aberrantly expressed in cancer, but how their misexpression drives tumorigenesis mostly remains unclear. From S phase to early mitosis, EMI1 (also known as FBXO5) inhibits the anaphase-promoting complex/cyclosome, which controls cell cycle progression through the sequential degradation of various substrates. By analyzing 7403 human tumor samples, we find that EMI1 overexpression is widespread in solid tumors but not in blood cancers. In solid cancers, EMI1 overexpression is a strong prognostic marker for poor patient outcome. To investigate causality, we generated a transgenic mouse model in which we overexpressed Emi1. Emi1-overexpressing animals develop a wide variety of solid tumors, in particular adenomas and carcinomas with inflammation and lymphocyte infiltration, but not blood cancers. These tumors are significantly larger and more penetrant, abundant, proliferative and metastatic than control tumors. In addition, they are highly aneuploid with tumor cells frequently being in early mitosis and showing mitotic abnormalities, including lagging and incorrectly segregating chromosomes. We further demonstrate in vitro that even though EMI1 overexpression may cause mitotic arrest and cell death, it also promotes chromosome instability (CIN) following delayed chromosome alignment and anaphase onset. In human solid tumors, EMI1 is co-expressed with many markers for CIN and EMI1 overexpression is a stronger marker for CIN than most well-established ones. The fact that Emi1 overexpression promotes CIN and the formation of solid cancers in vivo indicates that Emi1 overexpression actively drives solid tumorigenesis. These novel mechanistic insights have important clinical implications.

A stress-induced early innate response causes multidrug tolerance in melanoma.

Correction to: Oncogene (2015) 34, 4448­4459; doi:10.1038/onc.2014.372; published online 24 November 2014. In this article, published online 24 November 2014, the authors have noticed that the latest supplementary information was not used. The corrected supplementary information (Supplementary Materials) appears online together with this corrigendum. The authors would like to apologise for any inconvenience this may cause

A stress-induced early innate response causes multidrug tolerance in melanoma.

Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

Meeting report from the 7th International Melanoma Congress, Sydney, November, 2010.

The 2010 7th International Melanoma Congress sponsored by the Society for Melanoma Research and held in Sydney, Australia, was held together with the International Melanoma and Skin Cancer Centers group and the International Melanoma Pathology Study Group. As a consequence, there were over 900 registrants that included a wide range of clinicians (surgeons, medical oncologists, dermatologists) specialising in the management of melanoma as well as scientists and students carrying out laboratory-based research in melanoma. There was a general consensus that this grouping of clinicians, pathologists and scientists was mutually advantageous and plans are afoot to continue this grouping in future meetings. The meeting was dominated by the advances being made in treatment of melanoma with selective BRAF inhibitors but interest in epithelial mesenchymal transition and phenotypic changes in melanoma was apparent in many of the talks. The authors have attempted to capture many of the new developments in melanoma research but apologize to those speakers and poster presenters who had equally important findings not captured in these summaries.

Ki67 expression levels are a better marker of reduced melanoma growth following MEK inhibitor treatment than phospho-ERK levels.

The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies.

Tissue expression of the vesicle protein pantophysin.

The cell-type restricted expression of cytoplasmic microvesicle membrane protein isoforms may be a consequence of the functional adaptation of these vesicles to the execution of specialized processes in cells of different specialization. To characterize the expression of the vesicle protein pantophysin, an isoform of the synaptic vesicle proteins synaptophysin and synaptoporin, we have prepared and characterized antibodies useful for the immunological detection of pantophysin in vitro and in situ. Using these reagents, we show by immunoblot analyses that pantophysin expression is not homogeneous but differs significantly between various bovine tissues. Furthermore, these differences are not exactly paralleled by the expression of other vesicle proteins of the SCAMP (secretory carrier-associated membrane protein) and VAMP (vesicle-associated membrane protein) types that have previously been localized to pantophysin vesicles in cultured cells. By immunofluorescence microscopy, pantophysin expression is seen predominantly in non-neuroendocrine cells with pronounced membrane traffic. Pantophysin staining codistributes with SCAMP and VAMP immunoreactivities in most instances but differs in some. Remarkably, pantophysin staining in liver is restricted to cells surrounding sinusoids and is not detectable in hepatocytes, similar to that of the SCAMP and VAMP isoforms as detected by our reagents in tissue sections.