Understanding signaling cascades in melanoma

Understanding regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now appreciated that melanoma is the result of complex changes in multiple signaling pathways that affect growth control, metabolism, motility and the ability to escape cell death programs. Here we review the major signaling pathways currently known to be deregulated in melanoma with an implication to its development and progression. Among these pathways are Ras, B-Raf, MEK, PTEN, phosphatidylinositol-3 kinase (PI3Ks) and Akt which are constitutively activated in a significant number of melanoma tumors, in most cases due to genomic change. Other pathways discussed in this review include the [Janus kinase/signal transducer and activator of transcription (JAK/STAT), transforming growth factor-beta pathways which are also activated in melanoma, although the underlying mechanism is not yet clear. As a paradigm for remodeled signaling pathways, melanoma also offers a unique opportunity for targeted drug development.Fil: Lopez Bergami, Pablo Roberto. Sanford-burnham Medical Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Fitchmann, B. Sanford-burnham Medical Research Institute; Estados UnidosFil: Ronai, Ze´ev. Sanford-burnham Medical Research Institute; Estados Unido

Ultrasound attenuation in gap-anisotropic systems

Transverse ultrasound attenuation provides a weakly-coupled probe of momentum current correlations in electronic systems. We develop a simple theory for the interpretation of transverse ultrasound attenuation coefficients in systems with nodal gap anisotropy. Applying this theory we show how ultrasound can delineate between extended-s and d-wave scenarios for the cuprate superconductors.Comment: Uuencode file: 4 pages (Revtex), 3 figures. Some references adde

Hydrogels for 3D neural tissue models: understanding cell-material interactions at a molecular level.

The development of 3D neural tissue analogs is of great interest to a range of biomedical engineering applications including tissue engineering of neural interfaces, treatment of neurodegenerative diseases and in vitro assessment of cell-material interactions. Despite continued efforts to develop synthetic or biosynthetic hydrogels which promote the development of complex neural networks in 3D, successful long-term 3D approaches have been restricted to the use of biologically derived constructs. In this study a poly (vinyl alcohol) biosynthetic hydrogel functionalized with gelatin and sericin (PVA-SG), was used to understand the interplay between cell-cell communication and cell-material interaction. This was used to probe critical short-term interactions that determine the success or failure of neural network growth and ultimately the development of a useful model. Complex primary ventral mesencephalic (VM) neural cells were encapsulated in PVA-SG hydrogels and critical molecular cues that demonstrate mechanosensory interaction were examined. Neuronal presence was constant over the 10 day culture, but the astrocyte population decreased in number. The lack of astrocytic support led to a reduction in neural process outgrowth from 24.0 ± 1.3 μm on Day 7 to 7.0 ± 0.1 μm on Day 10. Subsequently, purified astrocytes were studied in isolation to understand the reasons behind PVA-SG hydrogel inability to support neural network development. It was proposed that the spatially restrictive nature (or tight mesh size) of PVA-SG hydrogels limited the astrocytic actin polymerization together with a cytoplasmic-nuclear translocation of YAP over time, causing an alteration in their cell cycle. This was confirmed by the evaluation of p27/Kip1 gene that was found to be upregulated by a twofold increase in expression at both Days 7 and 10 compared to Day 3, indicating the quiescent stage of the astrocytes in PVA-SG hydrogel. Cell migration was further studied by the quantification of MMP-2 production that was negligible compared to 2D controls, ranging from 2.7 ± 2.3% on Day 3 to 5.3 ± 2.9% on Day 10. This study demonstrates the importance of understanding astrocyte-material interactions at the molecular level, with the need to address spatial constraints in the 3D hydrogel environment. These findings will inform the design of future hydrogel constructs with greater capacity for remodeling by the cell population to create space for cell migration and neural process extension

Pig α₁-Acid Glycoprotein: Characterization and First Description in Any Species as a Negative Acute Phase Protein.

The serum protein α1-acid glycoprotein (AGP), also known as orosomucoid, is generally described as an archetypical positive acute phase protein. Here, porcine AGP was identified, purified and characterized from pooled pig serum. It was found to circulate as a single chain glycoprotein having an apparent molecular weight of 43 kDa by SDS-PAGE under reducing conditions, of which approximately 17 kDa were accounted for by N-bound oligosaccharides. Those data correspond well with the properties of the protein predicted from the single porcine AGP gene (ORM1, Q29014 (UniProt)), containing 5 putative glycosylation sites. A monoclonal antibody (MAb) was produced and shown to quantitatively and specifically react with all microheterogenous forms of pig AGP as analyzed by 2-D electrophoresis. This MAb was used to develop an immunoassay (ELISA) for quantification of AGP in pig serum samples. The adult serum concentrations of pig AGP were in the range of 1-3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2-5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase protein

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naive cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGF beta/SMAD (transforming growth factor-beta/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naive cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.Fapesp-grant number 2012/04194-1, 2013/05172-4, 2014/24400-0 and 2015/10821-7, CNPq-grant number 150447/2013-2 and 471512/2013-3 and PRODOC-grant no 3193-32/2010. Work in the lab of KS Smalley was supported by the National Institutes of Health grants R01 CA161107, R21 CA198550, and Skin SPORE grant P50 CA168536info:eu-repo/semantics/publishedVersio

DNA damage remodels the MITF interactome to increase melanoma genomic instability

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11–RAD50–NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation

Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity

Phenotypic and metabolic heterogeneity within tumors is a major barrier to effective cancer therapy. How metabolism is implicated in specific phenotypes and whether lineage-restricted mechanisms control key metabolic vulnerabilities remain poorly understood. In melanoma, downregulation of the lineage addiction oncogene microphthalmia-associated transcription factor (MITF) is a hallmark of the proliferative-to-invasive phenotype switch, although how MITF promotes proliferation and suppresses invasion is poorly defined. Here, we show that MITF is a lineage-restricted activator of the key lipogenic enzyme stearoyl-CoA desaturase (SCD) and that SCD is required for MITFHigh melanoma cell proliferation. By contrast MITFLow cells are insensitive to SCD inhibition. Significantly, the MITF-SCD axis suppresses metastasis, inflammatory signaling, and an ATF4-mediated feedback loop that maintains de-differentiation. Our results reveal that MITF is a lineage-specific regulator of metabolic reprogramming, whereby fatty acid composition is a driver of melanoma phenotype switching, and highlight that cell phenotype dictates the response to drugs targeting lipid metabolism

The transcription factor Sox5 modulates Sox10 function during melanocyte development

The transcription factor Sox5 has previously been shown in chicken to be expressed in early neural crest cells and neural crest-derived peripheral glia. Here, we show in mouse that Sox5 expression also continues after neural crest specification in the melanocyte lineage. Despite its continued expression, Sox5 has little impact on melanocyte development on its own as generation of melanoblasts and melanocytes is unaltered in Sox5-deficient mice. Loss of Sox5, however, partially rescued the strongly reduced melanoblast generation and marker gene expression in Sox10 heterozygous mice arguing that Sox5 functions in the melanocyte lineage by modulating Sox10 activity. This modulatory activity involved Sox5 binding and recruitment of CtBP2 and HDAC1 to the regulatory regions of melanocytic Sox10 target genes and direct inhibition of Sox10-dependent promoter activation. Both binding site competition and recruitment of corepressors thus help Sox5 to modulate the activity of Sox10 in the melanocyte lineage