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

North-south asymmetry of the high-latitude thermospheric density:IMF BY effect

Previous studies have established that the y component of the interplanetary magnetic field (IMF By) plays a role in the north-south asymmetry of the high-latitude plasma convection and wind. The effect of the positive/negative IMF By in the Northern Hemisphere resembles the effect that the negative/positive IMF By would have in the Southern Hemisphere. In this study, we demonstrate that the IMF By effect can also contribute to the hemispheric asymmetry of the thermospheric density. We use high-accuracy air drag measurements from the CHAllenging Minisatellite Payload (CHAMP) satellite and SuperMAG AE index during the period 2001–2006 to examine the response of the high-latitude thermospheric density to geomagnetic activity. Our statistical analysis reveals that the density response at 400 km is greater in the Southern Hemisphere under positive IMF By conditions, and greater in the Northern Hemisphere under negative IMF By conditions. The results suggest that the IMF By effect needs to be taken into account in upper atmospheric modeling for an accurate description of high-latitude densities during periods of enhanced geomagnetic activity

miR-515-5p controls cancer cell migration through MARK4 regulation

Here, we show that miR-515-5p inhibits cancer cell migration and metastasis. RNA-seq analyses of both oestrogen receptor receptor-positive and receptor-negative breast cancer cells overexpressing miR-515-5p reveal down-regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR-515-5p inhibits MARK4 directly 3' UTR interaction and that MARK4 knock-down mimics the effect of miR-515-5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR-515-5p, suggesting miR-515-5p mediates this process through MARK4 down-regulation. Furthermore, miR-515-5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR-515-5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR-515-5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR-515-5p/MARK4 regulation in cell migration and metastasis across two common cancers

B-RAF and N-RAS Mutations Are Preserved during Short Time In Vitro Propagation and Differentially Impact Prognosis

In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients

Differential Protein Expression in Honeybee (Apis mellifera L.) Larvae: Underlying Caste Differentiation

Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on epigenetics and can help us to gain new insight into the time of departure as well as caste trajectory influencing elements at the molecular level

A three-drug nanoscale drug delivery system designed for preferential lymphatic uptake for the treatment of metastatic melanoma

Metastatic melanoma has a high mortality rate due to lymphatic progression of the disease. Current treatment is surgery followed by radiation and intravenous chemotherapy. However, drawbacks for current chemotherapeutics lie in the fact that they develop resistance and do not achieve therapeutic concentrations in the lymphatic system. We hypothesize that a three-drug nanoscale drug delivery system, tailored for lymphatic uptake, administered subcutaneously, will have decreased drug resistance and therefore offer better therapeutic outcomes. We prepared and characterized nanoparticles (NPs) with docetaxel, everolimus, and LY294002 in polyethyleneglycol-block-poly(ε-caprolactone) (PEG-PCL) polymer with different charge distributions by modifying the ratio of anionic and neutral end groups on the PEG block. These NPs are similarly sized (~48nm), with neutral, partially charged, or fully charged surface. The NPs are able to load ~2mg/mL of each drug and are stable for 24h. The NPs are assessed for safety and efficacy in two transgenic metastatic melanoma mouse models. All the NPs were safe in both models based on general appearance, weight changes, death, and blood biochemical analyses. The partially charged NPs are most effective in decreasing the number of melanocytes at both the proximal (sentinel) lymph node (LN) and the distal LN from the injection site. The neutral NPs are efficacious at the proximal LN, while the fully charged NPs have no effect on either LNs. Thus, our data indicates that the NP surface charge and lymphatic efficacy are closely tied to each other and the partially charged NPs have the highest potential in treating metastatic melanoma

A novel AKT3 mutation in melanoma tumours and cell lines

Recently, a rare activating mutation of AKT1 (E17K) has been reported in breast, ovarian, and colorectal cancers. However, analogous activating mutations in AKT2 or AKT3 have not been identified in any cancer lineage. To determine the prevalence of AKT E17K mutations in melanoma, the most aggressive form of skin cancer, we analysed 137 human melanoma specimens and 65 human melanoma cell lines for the previously described activating mutation of AKT1, and for analogous mutations in AKT2 and AKT3. We identified a single AKT1 E17K mutation. Remarkably, a previously unidentified AKT3 E17K mutation was detected in two melanomas (from one patient) as well as two cell lines. The AKT3 E17K mutation results in activation of AKT when expressed in human melanoma cells. This represents the first report of AKT mutations in melanoma, and the initial identification of an AKT3 mutation in any human cancer lineage. We have also identified the first known human cell lines with naturally occurring AKT E17K mutations

Ultralow-frequency modulation of whistler-mode wave growth

Measurements from ground-based magnetometers and riometers at auroral latitudes have demonstrated that energetic (~30-300keV) electron precipitation can be modulated in the presence of magnetic field oscillations at ultra-low frequencies. It has previously been proposed that an ultra-low frequency (ULF) wave would modulate field and plasma properties near the equatorial plane, thus modifying the growth rates of whistler-mode waves. In turn, the resulting whistler-mode waves would mediate the pitch-angle scattering of electrons resulting in ionospheric precipitation. In this paper, we investigate this hypothesis by quantifying the changes to the linear growth rate expected due to a slow change in the local magnetic field strength for parameters typical of the equatorial region around 6.6RE radial distance. To constrain our study, we determine the largest possible ULF wave amplitudes from measurements of the magnetic field at geosynchronous orbit. Using nearly ten years of observations from two satellites, we demonstrate that the variation in magnetic field strength due to oscillations at 2mHz does not exceed ±10% of the background field. Modifications to the plasma density and temperature anisotropy are estimated using idealised models. For low temperature anisotropy, there is little change in the whistler-mode growth rates even for the largest ULF wave amplitude. Only for large temperature anisotropies can whistler-mode growth rates be modulated sufficiently to account for the changes in electron precipitation measured by riometers at auroral latitudes

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma

β-Catenin Signaling Increases during Melanoma Progression and Promotes Tumor Cell Survival and Chemoresistance

Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy