Spatial proteomics identifies JAKi as treatment for a lethal skin disease.

Toxic epidermal necrolysis (TEN) is a fatal drug-induced skin reaction triggered by common medications and is an emerging public health issue1-3. Patients with TEN undergo severe and sudden epidermal detachment caused by keratinocyte cell death. Although molecular mechanisms that drive keratinocyte cell death have been proposed, the main drivers remain unknown, and there is no effective therapy for TEN4-6. Here, to systematically map molecular changes that are associated with TEN and identify potential druggable targets, we utilized deep visual proteomics, which provides single-cell-based, cell-type-resolution proteomics7,8. We analysed formalin-fixed, paraffin-embedded archived skin tissue biopsies of three types of cutaneous drug reactions with varying severity and quantified more than 5,000 proteins in keratinocytes and skin-infiltrating immune cells. This revealed a marked enrichment of type I and type II interferon signatures in the immune cell and keratinocyte compartment of patients with TEN, as well as phosphorylated STAT1 activation. Targeted inhibition with the pan-JAK inhibitor tofacitinib in vitro reduced keratinocyte-directed cytotoxicity. In vivo oral administration of tofacitinib, baricitinib or the JAK1-specific inhibitors abrocitinib or upadacitinib ameliorated clinical and histological disease severity in two distinct mouse models of TEN. Crucially, treatment with JAK inhibitors (JAKi) was safe and associated with rapid cutaneous re-epithelialization and recovery in seven patients with TEN. This study uncovers the JAK/STAT and interferon signalling pathways as key pathogenic drivers of TEN and demonstrates the potential of targeted JAKi as a curative therapy.

Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

Identification of bacteria-derived HLA-bound peptides in melanoma.

A variety of species of bacteria are known to colonize human tumours1-11, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment12-14. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.

A DNA replication-independent function of pre-replication complex genes during cell invasion in C. elegans.

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.

Size-exclusion chromatography combined with DIA-MS enables deep proteome profiling of extracellular vesicles from melanoma plasma and serum.

Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC-DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200 µL of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.

gExcite: a start-to-end framework for single-cell gene expression, hashing, and antibody analysis.

SUMMARY: Recently, CITE-seq emerged as a multimodal single-cell technology capturing gene expression and surface protein information from the same single cells, which allows unprecedented insights into disease mechanisms and heterogeneity, as well as immune cell profiling. Multiple single-cell profiling methods exist, but they are typically focused on either gene expression or antibody analysis, not their combination. Moreover, existing software suites are not easily scalable to a multitude of samples. To this end, we designed gExcite, a start-to-end workflow that provides both gene and antibody expression analysis, as well as hashing deconvolution. Embedded in the Snakemake workflow manager, gExcite facilitates reproducible and scalable analyses. We showcase the output of gExcite on a study of different dissociation protocols on PBMC samples. AVAILABILITY AND IMPLEMENTATION: gExcite is open source available on github at https://github.com/ETH-NEXUS/gExcite_pipeline. The software is distributed under the GNU General Public License 3 (GPL3).

Robust dimethyl-based multiplex-DIA doubles single-cell proteome depth via a reference channel.

Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single-cell samples, without losing proteomic depth. Lys-N digestion enables five-plex quantification at MS1 and MS2 level. Because the multiplexed channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this and confidently quantifies twice as many proteins per single cell compared to our previous work (Brunner et al, PMID 35226415), while our workflow currently allows routine analysis of 80 single cells per day. Finally, we combined mDIA with spatial proteomics to increase the throughput of Deep Visual Proteomics seven-fold for microdissection and four-fold for MS analysis. Applying this to primary cutaneous melanoma, we discovered proteomic signatures of cells within distinct tumor microenvironments, showcasing its potential for precision oncology.

Simultaneous targeted and discovery-driven clinical proteotyping using hybrid-PRM/DIA.

BACKGROUND: Clinical samples are irreplaceable, and their transformation into searchable and reusable digital biobanks is critical for conducting statistically empowered retrospective and integrative research studies. Currently, mainly data-independent acquisition strategies are employed to digitize clinical sample cohorts comprehensively. However, the sensitivity of DIA is limited, which is why selected marker candidates are often additionally measured targeted by parallel reaction monitoring. METHODS: Here, we applied the recently co-developed hybrid-PRM/DIA technology as a new intelligent data acquisition strategy that allows for the comprehensive digitization of rare clinical samples at the proteotype level. Hybrid-PRM/DIA enables enhanced measurement sensitivity for a specific set of analytes of current clinical interest by the intelligent triggering of multiplexed parallel reaction monitoring (MSxPRM) in combination with the discovery-driven digitization of the clinical biospecimen using DIA. Heavy-labeled reference peptides were utilized as triggers for MSxPRM and monitoring of endogenous peptides. RESULTS: We first evaluated hybrid-PRM/DIA in a clinical context on a pool of 185 selected proteotypic peptides for tumor-associated antigens derived from 64 annotated human protein groups. We demonstrated improved reproducibility and sensitivity for the detection of endogenous peptides, even at lower concentrations near the detection limit. Up to 179 MSxPRM scans were shown not to affect the overall DIA performance. Next, we applied hybrid-PRM/DIA for the integrated digitization of biobanked melanoma samples using a set of 30 AQUA peptides against 28 biomarker candidates with relevance in molecular tumor board evaluations of melanoma patients. Within the DIA-detected approximately 6500 protein groups, the selected marker candidates such as UFO, CDK4, NF1, and PMEL could be monitored consistently and quantitatively using MSxPRM scans, providing additional confidence for supporting future clinical decision-making. CONCLUSIONS: Combining PRM and DIA measurements provides a new strategy for the sensitive and reproducible detection of protein markers from patients currently being discussed in molecular tumor boards in combination with the opportunity to discover new biomarker candidates.

COL10A1 expression distinguishes a subset of cancer-associated fibroblasts present in the stroma of high-risk basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) is the most frequently diagnosed skin cancer and the most common malignancy in humans. Different morphological subtypes of BCC are associated with a low or high risk of recurrence and aggressiveness, but the underlying biology of how the individual subtypes arise remains largely unknown. As the majority of BCCs appear to arise from mutations in the same pathway, we hypothesized that BCC development, growth and invasive potential is also influenced by the tumour microenvironment and, in particular, by cancer-associated fibroblasts (CAFs) and the factors they secrete. OBJECTIVES: To characterize the stroma of the different BCC subtypes with a focus on CAF populations. METHODS: To investigate the stromal features of the different BCC subtypes, we used laser capture microdissection (LCM) followed by RNA sequencing (RNA-Seq). Fifteen BCC samples from five different 'pure' subtypes (i.e. superficial, nodular, micronodular, sclerosing and basosquamous; n = 3 each) were selected and included in the analysis. Healthy skin was used as a control (n = 6). The results were confirmed by immunohistochemistry (IHC). We validated our findings in two independent public single-cell RNA-Seq (scRNA-Seq) datasets and by RNAscope. RESULTS: The stroma of the different BCC subtypes were found to have distinct gene expression signatures. Nodular and micronodular appeared to have the most similar signatures, while superficial and sclerosing the most different. By comparing low- and high-risk BCC subtypes, we found that COL10A1 is overexpressed in the stroma of sclerosing/infiltrative and basosquamous but not in micronodular high-risk subtypes. Those findings were confirmed by IHC in 93 different BCC and 13 healthy skin samples. Moreover, scRNA-Seq analysis of BCCs from two independent datasets found that the COL10A1-expressing population of cells is associated with the stroma adjacent to infiltrative BCC and shows extracellular matrix remodelling features. CONCLUSIONS: We identified COL10A1 as a marker of high-risk BCC, in particular of the sclerosing/infiltrative and basosquamous subtypes. We demonstrated at the single-cell level that COL10A1 is expressed by a specific CAF population associated with the stroma of infiltrative BCC. This opens up new, tailored treatment options, and suggests COL10A1 as a new prognostic biomarker for BCC progression.

Basal cell carcinoma ('BCC' for short) is the most common type of cancer in humans. BCC occurs when a certain type of skin cell transforms in the outermost layer of the skin. This is mostly caused by a lot of exposure to sunlight. BCC can appear in different forms, or 'subtypes'. In each subtype, cancer cells grow in a specific way and are visually distinct from the surrounding tissue, known as the 'stroma'. The different subtypes of BCC can have a low or high risk of cancer recurrence and different levels of aggressiveness. We aimed to find out what the stroma is made up of in the different subtypes of BCC, focusing on the differences between low-risk and high-risk cancers. We measured gene expression in specific areas of tissue and identified one gene called 'COL10A1' as being overexpressed in the stroma of high-risk BCC, especially in two particular subtypes ('sclerosing' and 'basosquamous' BCC). We confirmed this result in BCC biopsies. We also checked other published data and found that COL10A1 is mostly expressed in a type of cell called a 'fibroblast'. Fibroblasts expressing the COL10A1 gene are present in the stroma right beside the infiltrative area of BCC. Our findings could be used to develop more personalized treatment of BCC. The stroma may be a potential anti-cancer target, as well as a new way of testing for BCC progression.

Distinct Variations in Gene Expression and Cell Composition across Lichen Planus Subtypes.

Lichen planus (LP) is a highly prevalent inflammatory skin disease. While various clinical subtypes have been defined, detailed comparisons of these variants are lacking. This study aimed to elucidate differences in gene expression and cellular composition across LP subtypes. Lesional skin biopsies from 28 LP patients (classical, oral, genital, and lichen planopilaris) and seven non-diseased skin controls (NDC) were analyzed. Gene expression profiling of 730 inflammation-related genes was conducted using NanoString. Immune cell compositions were assessed by multiplex immunohistochemistry. Gene expression profiles revealed unique inflammatory signatures for each LP subtype. Lichen planopilaris exhibited the most divergence, with downregulated gene expression and upregulation of complement pathway genes (C5-7), along with elevated M2 macrophages. Oral and genital LP demonstrated similar profiles with strong upregulation of TNF-related and Toll-like receptor-associated genes. Oral LP showed the highest upregulation of cytotoxicity-associated genes, as well as high numbers of CD8+ IL-17A+ (Tc17) cells (8.02%). Interferon gene signatures were strongly upregulated in oral and classical LP. The study highlights distinct differences in inflammatory gene expression and cell composition across LP subtypes, emphasizing the need for tailored therapeutic approaches.

Proteomic identification of a marker signature for MAPKi resistance in melanoma.

MAPK inhibitors (MAPKi) show outstanding clinical response rates in melanoma patients harbouring BRAF mutations, but resistance is common. The ability of melanoma cells to switch from melanocytic to mesenchymal phenotypes appears to be associated with therapeutic resistance. High-throughput, subcellular proteome analyses and RNAseq on two panels of primary melanoma cells that were either sensitive or resistant to MAPKi revealed that only 15 proteins were sufficient to distinguish between these phenotypes. The two proteins with the highest discriminatory power were PTRF and IGFBP7, which were both highly upregulated in the mesenchymal-resistant cells. Proteomic analysis of CRISPR/Cas-derived PTRF knockouts revealed targets involved in lysosomal activation, endocytosis, pH regulation, EMT, TGFß signalling and cell migration and adhesion, as well as a significantly reduced invasive index and ability to form spheres in 3D culture. Overexpression of PTRF led to MAPKi resistance, increased cell adhesion and sphere formation. In addition, immunohistochemistry of patient samples showed that PTRF expression levels were a significant biomarker of poor progression-free survival, and IGFBP7 levels in patient sera were shown to be higher after relapse.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

The A to I editing landscape in melanoma and its relation to clinical outcome.

RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies.

Establishing standardized immune phenotyping of metastatic melanoma by digital pathology.

CD8+ tumor-infiltrating T cells can be regarded as one of the most relevant predictive biomarkers in immune-oncology. Highly infiltrated tumors, referred to as inflamed (clinically "hot"), show the most favorable response to immune checkpoint inhibitors in contrast to tumors with a scarce immune infiltrate called immune desert or excluded (clinically "cold"). Nevertheless, quantitative and reproducible methods examining their prevalence within tumors are lacking. We therefore established a computational diagnostic algorithm to quantitatively measure spatial densities of tumor-infiltrating CD8+ T cells by digital pathology within the three known tumor compartments as recommended by the International Immuno-Oncology Biomarker Working Group in 116 prospective metastatic melanomas of the Swiss Tumor Profiler cohort. Workflow robustness was confirmed in 33 samples of an independent retrospective validation cohort. The introduction of the intratumoral tumor center compartment proved to be most relevant for establishing an immune diagnosis in metastatic disease, independent of metastatic site. Cut-off values for reproducible classification were defined and successfully assigned densities into the respective immune diagnostic category in the validation cohort with high sensitivity, specificity, and precision. We provide a robust diagnostic algorithm based on intratumoral and stromal CD8+ T-cell densities in the tumor center compartment that translates spatial densities of tumor-infiltrating CD8+ T cells into the clinically relevant immune diagnostic categories "inflamed", "excluded", and "desert". The consideration of the intratumoral tumor center compartment allows immune phenotyping in the clinically highly relevant setting of metastatic lesions, even if the invasive margin compartment is not captured in biopsy material.

Bioinformatics for precision oncology.

Molecular profiling of tumor biopsies plays an increasingly important role not only in cancer research, but also in the clinical management of cancer patients. Multi-omics approaches hold the promise of improving diagnostics, prognostics and personalized treatment. To deliver on this promise of precision oncology, appropriate bioinformatics methods for managing, integrating and analyzing large and complex data are necessary. Here, we discuss the specific requirements of bioinformatics methods and software that arise in the setting of clinical oncology, owing to a stricter regulatory environment and the need for rapid, highly reproducible and robust procedures. We describe the workflow of a molecular tumor board and the specific bioinformatics support that it requires, from the primary analysis of raw molecular profiling data to the automatic generation of a clinical report and its delivery to decision-making clinical oncologists. Such workflows have to various degrees been implemented in many clinical trials, as well as in molecular tumor boards at specialized cancer centers and university hospitals worldwide. We review these and more recent efforts to include other high-dimensional multi-omics patient profiles into the tumor board, as well as the state of clinical decision support software to translate molecular findings into treatment recommendations.

Small molecule promotes ß-catenin citrullination and inhibits Wnt signaling in cancer.

Wnt (wingless)/ß-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of ß-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of ß-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or ß-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of ß-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.

NGS-pipe: a flexible, easily extendable and highly configurable framework for NGS analysis.

Motivation: Next-generation sequencing is now an established method in genomics, and massive amounts of sequencing data are being generated on a regular basis. Analysis of the sequencing data is typically performed by lab-specific in-house solutions, but the agreement of results from different facilities is often small. General standards for quality control, reproducibility and documentation are missing. Results: We developed NGS-pipe, a flexible, transparent and easy-to-use framework for the design of pipelines to analyze whole-exome, whole-genome and transcriptome sequencing data. NGS-pipe facilitates the harmonization of genomic data analysis by supporting quality control, documentation, reproducibility, parallelization and easy adaptation to other NGS experiments. Availability and implementation: https://github.com/cbg-ethz/NGS-pipe. Contact: niko.beerenwinkel@bsse.ethz.ch.

Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance.

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumours expressing the BRAF(V600E) oncoprotein. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xenograft models in which drug resistance is selected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E)→MEK→ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.

Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype.

BACKGROUND: During embryonic development Wnt family members and bone morphogenetic proteins (BMPs) cooperatively induce epithelial-mesenchymal transition (EMT) in the neural crest. Wnt and BMPs are reactivated during malignant transformation in melanoma. We previously demonstrated that the BMP-antagonist noggin blocked the EMT phenotype of melanoma cells in the neural crest and malignant invasion of melanoma cells in the chick embryo; vice-versa, malignant invasion was induced in human melanocytes in vivo by pre-treatment with BMP-2. RESULTS: Although there are conflicting results in the literature about the role of ß-catenin for invasion of melanoma cells, we found Wnt/ß-catenin signaling to be analogously important for the EMT-like phenotype of human metastatic melanoma cells in the neural crest and during invasion: ß-catenin was frequently expressed at the invasive front of human primary melanomas and Wnt3a expression was inversely correlated with survival of melanoma patients. Accordingly, cytoplasmic ß-catenin levels were increased during invasion of melanoma cells in the rhombencephalon of the chick embryo. Fibroblast derived Wnt3a reduced melanoma cell adhesion and enhanced migration, while the ß-catenin inhibitor PKF115-584 increased adhesion and reduced migration in vitro and in the chick embryonic neural crest environment in vivo. Similarly, knockdown of ß-catenin impaired intradermal melanoma cell invasion and PKF115-584 efficiently reduced liver metastasis in a chick chorioallantoic membrane model. Our observations were accompanied by specific alterations in gene expression which are linked to overall survival of melanoma patients. CONCLUSION: We present a novel role for Wnt-signaling in neural crest like melanoma cell invasion and metastasis, stressing the crucial role of embryonic EMT-inducing neural crest signaling for the spreading of malignant melanoma.

Metastatic melanoma moves on: translational science in the era of personalized medicine.

Progress in understanding and treating metastatic melanoma is the result of decades of basic and translational research as well as the development of better in vitro tools for modeling the disease. Here, we review the latest therapeutic options for metastatic melanoma and the known genetic and non-genetic mechanisms of resistance to these therapies, as well as the in vitro toolbox that has provided the greatest insights into melanoma progression. These include next-generation sequencing technologies and more complex 2D and 3D cell culture models to functionally test the data generated by genomics approaches. The combination of hypothesis generating and hypothesis testing paradigms reviewed here will be the foundation for the next phase of metastatic melanoma therapies in the coming years.