FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. METHODS: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Follow-up RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. RESULTS: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. CONCLUSIONS: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. LAY SUMMARY: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.

Usefulness of an immunohistochemical score in advanced pancreatic neuroendocrine tumors treated with CAPTEM or everolimus.

BACKGROUND: Pancreatic neuroendocrine tumors are rare neoplasms for which few predictive and/or prognostic biomarkers have been validated. Our previous work suggested the potential of the combined expression of N-myc downstream-regulated gen-1 (NDRG-1), O6-methylguanine DNA methyltransferase (MGMT) and Pleckstrin homology-like domain family A member 3 (PHLDA-3) as prognostic factors for relapse and survival. METHODS: In this new multicenter study we evaluated immunohistochemistry expression in 76 patients with advanced PanNET who were treated with capecitabine-temozolomide or everolimus. Based on the immunohistochemistry panel, an immunohistochemistry prognostic score (IPS) was developed. RESULTS: In patients treated with capecitabine and temozolomide, low IPS was an independent prognostic factor for progression-free-survival and overall-survival. Similar findings were observed with highest IPS for overall-survival in patients treated with everolimus. CONCLUSION: From our knowledge, it is the first time that a simple IPS could be useful to predict outcome for patients with metastatic pancreatic neuroendocrine tumors treated with everolimus or capecitabine and temozolomide.

Novel Insights into the Role of the Mineralocorticoid Receptor in Human Glioblastoma.

The majority of glioblastoma (GBM) patients require the administration of dexamethasone (DEXA) to reduce brain inflammation. DEXA activates the glucocorticoid receptor (GR), which can consequently crosstalk with the mineralocorticoid receptor (MR). However, while GR signaling is well studied in GBM, little is known about the MR in brain tumors. We examined the implication of the MR in GBM considering its interplay with DEXA. Together with gene expression studies in patient cohorts, we used human GBM cell lines and patient-derived glioma stem cells (GSCs) to assess the impact of MR activation and inhibition on cell proliferation, response to radiotherapy, and self-renewal capacity. We show that in glioma patients, MR expression inversely correlates with tumor grade. Furthermore, low MR expression correlates with poorer survival in low grade glioma while in GBM the same applies to classical and mesenchymal subtypes, but not proneural tumors. MR activation by aldosterone suppresses the growth of some GBM cell lines and GSC self-renewal. In GBM cells, the MR antagonist spironolactone (SPI) can promote proliferation, radioprotection and cooperate with DEXA. In summary, we propose that MR signaling is anti-proliferative in GBM cells and blocks the self-renewal of GSCs. Contrary to previous evidence obtained in other cancer types, our results suggest that SPI has no compelling anti-neoplastic potential in GBM.

Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells.

BACKGROUND: The importance of the genetic background of cancer cells for the individual susceptibility to cancer treatments is increasingly apparent. In melanoma, the existence of a BRAF mutation is a main predictor for successful BRAF-targeted therapy. However, despite initial successes with these therapies, patients relapse within a year and have to move on to other therapies. Moreover, patients harbouring a wild type BRAF gene (including 25% with NRAS mutations) still require alternative treatment such as chemotherapy. Multiple genetic parameters have been associated with response to chemotherapy, but despite their high frequency in melanoma nothing is known about the impact of BRAF or NRAS mutations on the response to chemotherapeutic agents. METHODS: Using cell proliferation and DNA methylation assays, FACS analysis and quantitative-RT-PCR we have characterised the response of a panel of NRAS and BRAF mutant melanoma cell lines to various chemotherapy drugs, amongst them dacarbazine (DTIC) and temozolomide (TMZ) and DNA synthesis inhibitors. RESULTS: Although both, DTIC and TMZ act as alkylating agents through the same intermediate, NRAS and BRAF mutant cells responded differentially only to DTIC. Further analysis revealed that the growth-inhibitory effects mediated by DTIC were rather due to interference with nucleotide salvaging, and that NRAS mutant melanoma cells exhibit higher activity of the nucleotide synthesis enzymes IMPDH and TK1. Importantly, the enhanced ability of RAS mutant cells to use nucleotide salvaging resulted in resistance to DHFR inhibitors. CONCLUSION: In summary, our data suggest that the genetic background in melanoma cells influences the response to inhibitors blocking de novo DNA synthesis, and that defining the RAS mutation status could be used to stratify patients for the use of antifolate drugs.

Drug-Induced Reorganisation of Lipid Metabolism Limits the Therapeutic Efficacy of Ponatinib in Glioma Stem Cells.

The standard of care for glioblastoma (GBM) involves surgery followed by adjuvant radio- and chemotherapy, but often within months, patients relapse, and this has been linked to glioma stem cells (GSCs), self-renewing cells with increased therapy resistance. The identification of the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) as key players in gliomagenesis inspired the development of inhibitors targeting these tyrosine kinases (TKIs). However, results from clinical trials testing TKIs have been disappointing, and while the role of GSCs in conventional therapy resistance has been extensively studied, less is known about resistance of GSCs to TKIs. In this study, we have used compartmentalised proteomics to analyse the adaptive response of GSCs to ponatinib, a TKI with activity against PDGFR. The analysis of differentially expressed proteins revealed that GSCs respond to ponatinib by broadly rewiring lipid metabolism, involving fatty acid beta-oxidation, cholesterol synthesis, and sphingolipid degradation. Inhibiting each of these metabolic pathways overcame ponatinib adaptation of GSCs, but interrogation of patient data revealed sphingolipid degradation as the most relevant pathway in GBM. Our data highlight that targeting lipid metabolism, and particularly sphingolipid degradation in combinatorial therapies, could improve the outcome of TKI therapies using ponatinib in GBM.

Cancer Cell-Intrinsic Alterations Associated with an Immunosuppressive Tumor Microenvironment and Resistance to Immunotherapy in Lung Cancer.

Despite the great clinical success of immunotherapy in lung cancer patients, only a small percentage of them (<40%) will benefit from this therapy alone or combined with other strategies. Cancer cell-intrinsic and cell-extrinsic mechanisms have been associated with a lack of response to immunotherapy. The present study is focused on cancer cell-intrinsic genetic, epigenetic, transcriptomic and metabolic alterations that reshape the tumor microenvironment (TME) and determine response or refractoriness to immune checkpoint inhibitors (ICIs). Mutations in KRAS, SKT11(LKB1), KEAP1 and TP53 and co-mutations of these genes are the main determinants of ICI response in non-small-cell lung cancer (NSCLC) patients. Recent insights into metabolic changes in cancer cells that impose restrictions on cytotoxic T cells and the efficacy of ICIs indicate that targeting such metabolic restrictions may favor therapeutic responses. Other emerging pathways for therapeutic interventions include epigenetic modulators and DNA damage repair (DDR) pathways, especially in small-cell lung cancer (SCLC). Therefore, the many potential pathways for enhancing the effect of ICIs suggest that, in a few years, we will have much more personalized medicine for lung cancer patients treated with immunotherapy. Such strategies could include vaccines and chimeric antigen receptor (CAR) cells.

The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma.

Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death. To identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA beta-oxidation‒related genes. In patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium-chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of nonadherent melanoma cells. Our study identifies targeting the FA-based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the antimetastatic activity of the Food and Drug Administration‒approved drug ranolazine carries translational potential.

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy.

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma.

Oncogenic alterations reveal key strategies for precision oncology in melanoma treatment.

Background: Molecular profiling with next-generation sequencing (NGS) has been applied in multiple solid tumors, including melanomas, to identify potential drug targets. However, the association between clinical outcomes and the molecular alterations has not yet been fully clarified. Methods: A total of 108 patients with melanoma were included in this study, 95 of whom had both sequencing data and clinical outcomes were collected. We analyzed the genetic alterations of 108 malignant melanoma patients using the OncoCare panel, which covers 559 genes. Results: A model was also established to predict side effects through a combination analysis of clinical data and somatic variants, yielding an area under the receiver operating characteristic curve (AUROC) score of 0.8. We also identified epidermal growth factor receptor (EGFR) mutation was excellent predictor for progression-free survival (PFS) for patient who received immunotherapy (log-rank P=0.01), while tumor mutation burden (TMB) was found to not be significantly associated with PFS (log-rank P=0.87). Combining clinical features with genetic analysis, we found that patients carrying both DNA POLD1/ALOX12B or POLD1/PTPRT mutations had a significantly lower survival rate. Conclusions: Overall, these results demonstrate the benefits of applying NGS clinical panels and shed light on future directions of personalized therapeutics for the treatment of melanoma.

Sentinel node tumor burden in cutaneous melanoma. Survival with competing risk analysis and influence in relapses and non-sentinel node status: retrospective cohort study with long follow-up in a Spanish population.

Several authors have studied the potential of sentinel lymph node (SLN) tumor burden as prognostic factor but the microscopic classifications used in different study groups were variable. We examined the prognostic role of tumor burden in SLN on melanoma specific-survival and competing causes of death. We also analysed clinical and histological factors as predictors of disease relapses and additional non sentinel lymph node (NSLN) metastases. We included all patients with cutaneous melanoma that underwent SLN biopsy between 2002 and 2012 at Complejo Hospitalario de Navarra (Spain). The study end-points were death due to melanoma, melanoma relapse and involvement of NSLN. We used Fine-Gray test for competing risk analysis. A logistic regression model was performed to predict the risk of involvement of NSLN. Between 2002 and 2012, there were 348 patients who underwent SLN biopsy in our centre (308 were eligible for the study). 26.9% patients positive SLN. 88 patients died during the follow-up period and 66 (75%) died from melanoma. The 5-year cumulative incidence of melanoma death was 15.33% (95 % CI 15.25-15.42). The cumulative probability of death from melanoma was associated with gender, histological subtype, Breslow thickness, mitotic rate, ulceration and SLN tumor burden. In multivariable analysis, Breslow thickness and SLN tumor burden remained as independent prognostic factors. SLN tumor burden appears to be an important prognostic factor. It is very important reporting these characteristics in pathological reports. More prospective studies would be necessary to analyze these variables and to be able to make recommendations in management of melanoma patients.

Tyrosine Kinase Inhibitors in Adult Glioblastoma: An (Un)Closed Chapter?

Glioblastoma (GBM) is the most common and lethal form of malignant brain tumor. GBM patients normally undergo surgery plus adjuvant radiotherapy followed by chemotherapy. Numerous studies into the molecular events driving GBM highlight the central role played by the Epidermal Growth Factor Receptor (EGFR), as well as the Platelet-derived Growth Factor Receptors PDGFRA and PDGFRB in tumor initiation and progression. Despite strong preclinical evidence for the therapeutic potential of tyrosine kinase inhibitors (TKIs) that target EGFR, PDGFRs, and other tyrosine kinases, clinical trials performed during the last 20 years have not led to the desired therapeutic breakthrough for GBM patients. While clinical trials are still ongoing, in the medical community there is the perception of TKIs as a lost opportunity in the fight against GBM. In this article, we review the scientific rationale for the use of TKIs targeting glioma drivers. We critically analyze the potential causes for the failure of TKIs in the treatment of GBM, and we propose alternative approaches to the clinical evaluation of TKIs in GBM patients.

STATegra: Multi-Omics Data Integration - A Conceptual Scheme With a Bioinformatics Pipeline.

Technologies for profiling samples using different omics platforms have been at the forefront since the human genome project. Large-scale multi-omics data hold the promise of deciphering different regulatory layers. Yet, while there is a myriad of bioinformatics tools, each multi-omics analysis appears to start from scratch with an arbitrary decision over which tools to use and how to combine them. Therefore, it is an unmet need to conceptualize how to integrate such data and implement and validate pipelines in different cases. We have designed a conceptual framework (STATegra), aiming it to be as generic as possible for multi-omics analysis, combining available multi-omic anlaysis tools (machine learning component analysis, non-parametric data combination, and a multi-omics exploratory analysis) in a step-wise manner. While in several studies, we have previously combined those integrative tools, here, we provide a systematic description of the STATegra framework and its validation using two The Cancer Genome Atlas (TCGA) case studies. For both, the Glioblastoma and the Skin Cutaneous Melanoma (SKCM) cases, we demonstrate an enhanced capacity of the framework (and beyond the individual tools) to identify features and pathways compared to single-omics analysis. Such an integrative multi-omics analysis framework for identifying features and components facilitates the discovery of new biology. Finally, we provide several options for applying the STATegra framework when parametric assumptions are fulfilled and for the case when not all the samples are profiled for all omics. The STATegra framework is built using several tools, which are being integrated step-by-step as OpenSource in the STATegRa Bioconductor package.

Understanding the Molecular Mechanism of miR-877-3p Could Provide Potential Biomarkers and Therapeutic Targets in Squamous Cell Carcinoma of the Cervix.

No therapeutic targets and molecular biomarkers are available in cervical cancer (CC) management. In other cancer types, micro-RNA-877-3p (miR-877-3p) has been associated with events relevant for CC development. Thus, we aimed to determine miR-877-3p role in CC. miR-877-3p levels were examined by quantitative-PCR in 117 cervical lesions and tumors. Effects on CC cell proliferation, migration, and invasion were evaluated upon anti-miR-877-3p transfection. miR-877-3p dependent molecular mechanism was comprehensively explored by proteomics, dual-luciferase reporter assay, western blot, and immunohistochemistry. Cervical tumors expressed higher miR-877-3p levels than benign lesions. miR-877-3p promoted CC cell migration and invasion, at least partly by modulating cytoskeletal protein folding through the chaperonin-containing T-complex protein 1 complex. Notably, miR-877-3p silencing synergized with paclitaxel. Interestingly, miR-877-3p downregulated the levels of an in silico-predicted target, ZNF177, whose expression and subcellular location significantly distinguished high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix (SCCCs). Cytoplasmic ZNF177 was significantly associated with worse progression-free survival in SCCC. Our results suggest that: (i) miR-877-3p is a potential therapeutic target whose inhibition improves paclitaxel effects; (ii) the expression and location of its target ZNF177 could be diagnostic biomarkers between HSIL and SCCC; and (iii) cytoplasmic ZNF177 is a poor-prognosis biomarker in SCCC.

Identification of a Dexamethasone Mediated Radioprotection Mechanism Reveals New Therapeutic Vulnerabilities in Glioblastoma.

(1) Background: Despite the indisputable effectiveness of dexamethasone (DEXA) to reduce inflammation in glioblastoma (GBM) patients, its influence on tumour progression and radiotherapy response remains controversial. (2) Methods: We analysed patient data and used expression and cell biological analyses to assess effects of DEXA on GBM cells. We tested the efficacy of tyrosine kinase inhibitors in vitro and in vivo. (3) Results: We confirm in our patient cohort that administration of DEXA correlates with worse overall survival and shorter time to relapse. In GBM cells and glioma stem-like cells (GSCs) DEXA down-regulates genes controlling G2/M and mitotic-spindle checkpoints, and it enables cells to override the spindle assembly checkpoint (SAC). Concurrently, DEXA up-regulates Platelet Derived Growth Factor Receptor (PDGFR) signalling, which stimulates expression of anti-apoptotic regulators BCL2L1 and MCL1, required for survival during extended mitosis. Importantly, the protective potential of DEXA is dependent on intact tyrosine kinase signalling and ponatinib, sunitinib and dasatinib, all effectively overcome the radio-protective and pro-proliferative activity of DEXA. Moreover, we discovered that DEXA-induced signalling creates a therapeutic vulnerability for sunitinib in GSCs and GBM cells in vitro and in vivo. (4) Conclusions: Our results reveal a novel DEXA-induced mechanism in GBM cells and provide a rationale for revisiting the use of tyrosine kinase inhibitors for the treatment of GBM.

Cooperative behaviour and phenotype plasticity evolve during melanoma progression.

A major challenge for managing melanoma is its tumour heterogeneity based on individual co-existing melanoma cell phenotypes. These phenotypes display variable responses to standard therapies, and they drive individual steps of melanoma progression; hence, understanding their behaviour is imperative. Melanoma phenotypes are defined by distinct transcriptional states, which relate to different melanocyte lineage development phases, ranging from a mesenchymal, neural crest-like to a proliferative, melanocytic phenotype. It is thought that adaptive phenotype plasticity based on transcriptional reprogramming drives melanoma progression, but at which stage individual phenotypes dominate and moreover, how they interact is poorly understood. We monitored melanocytic and mesenchymal phenotypes throughout melanoma progression and detected transcriptional reprogramming at different stages, with a gain in mesenchymal traits in circulating melanoma cells (CTCs) and proliferative features in metastatic tumours. Intriguingly, we found that distinct phenotype populations interact in a cooperative manner, which generates tumours of greater "fitness," supports CTCs and expands organotropic cues in metastases. Fibronectin, expressed in mesenchymal cells, acts as key player in cooperativity and promotes survival of melanocytic cells. Our data reveal an important role for inter-phenotype communications at various stages of disease progression, suggesting these communications could act as therapeutic target.

Distinct utilization of effectors and biological outcomes resulting from site-specific Ras activation: Ras functions in lipid rafts and Golgi complex are dispensable for proliferation and transformation.

Ras proteins are distributed in different types of plasma membrane microdomains and endomembranes. However, how microlocalization affects the signals generated by Ras and its subsequent biological outputs is largely unknown. We have approached this question by selectively targeting RasV12 to different cellular sublocalizations. We show here that compartmentalization dictates Ras utilization of effectors and the intensity of its signals. Activated Ras can evoke enhanced proliferation and transformation from most of its platforms, with the exception of the Golgi complex. Furthermore, signals that promote survival emanate primarily from the endoplasmic reticulum pool. In addition, we have investigated the need for the different pools of endogenous Ras in the conveyance of upstream mitogenic and transforming signals. Using targeted RasN17 inhibitory mutants and in physiological contexts such as H-Ras/N-Ras double knockout fibroblasts, we demonstrate that Ras functions at lipid rafts and at the Golgi complex are fully dispensable for proliferation and transformation.

Phenotype plasticity as enabler of melanoma progression and therapy resistance.

Malignant melanoma is notorious for its inter- and intratumour heterogeneity, based on transcriptionally distinct melanoma cell phenotypes. It is thought that these distinct phenotypes are plastic in nature and that their transcriptional reprogramming enables heterogeneous tumours both to undergo different stages of melanoma progression and to adjust to drug exposure during treatment. Recent advances in genomic technologies and the rapidly expanding availability of large gene expression datasets have allowed for a refined definition of the gene signatures that characterize these phenotypes and have revealed that phenotype plasticity plays a major role in the resistance to both targeted therapy and immunotherapy. In this Review we discuss the definition of melanoma phenotypes through particular transcriptional states and reveal the prognostic relevance of the related gene expression signatures. We review how the establishment of phenotypes is controlled and which roles phenotype plasticity plays in melanoma development and therapy. Because phenotype plasticity in melanoma bears a great resemblance to epithelial-mesenchymal transition, the lessons learned from melanoma will also benefit our understanding of other cancer types.

RAS at the Golgi antagonizes malignant transformation through PTPRκ-mediated inhibition of ERK activation.

RAS GTPases are frequently mutated in human cancer. H- and NRAS isoforms are distributed over both plasma-membrane and endomembranes, including the Golgi complex, but how this organizational context contributes to cellular transformation is unknown. Here we show that RAS at the Golgi is selectively activated by apoptogenic stimuli and antagonizes cell survival by suppressing ERK activity through the induction of PTPRκ, which targets CRAF for dephosphorylation. Consistently, in contrast to what occurs at the plasma-membrane, RAS at the Golgi cannot induce melanoma in zebrafish. Inactivation of PTPRκ, which occurs frequently in human melanoma, often coincident with TP53 inactivation, accelerates RAS-ERK pathway-driven melanomagenesis in zebrafish. Likewise, tp53 disruption in zebrafish facilitates oncogenesis driven by RAS from the Golgi complex. Thus, RAS oncogenic potential is strictly dependent on its sublocalization, with Golgi complex-located RAS antagonizing tumor development.

Activation of H-Ras in the endoplasmic reticulum by the RasGRF family guanine nucleotide exchange factors.

Recent findings indicate that in addition to its location in the peripheral plasma membrane, H-Ras is found in endomembranes like the endoplasmic reticulum and the Golgi complex. In these locations H-Ras is functional and can efficiently engage downstream effectors, but little is known about how its activation is regulated in these environments. Here we show that the RasGRF family exchange factors, both endogenous and ectopically expressed, are present in the endoplasmic reticulum but not in the Golgi complex. With the aid of H-Ras constructs specifically tethered to the plasma membrane, endoplasmic reticulum, and Golgi complex, we demonstrate that RasGRF1 and RasGRF2 can activate plasma membrane and reticular, but not Golgi-associated, H-Ras. We also show that RasGRF DH domain is required for the activation of H-Ras in the endoplasmic reticulum but not in the plasma membrane. Furthermore, we demonstrate that RasGRF mediation favors the activation of reticular H-Ras by lysophosphatidic acid treatment whereas plasma membrane H-Ras is made more responsive to stimulation by ionomycin. Overall, our results provide the initial insights into the regulation of H-Ras activation in the endoplasmic reticulum.

Targeting invasive properties of melanoma cells.

Melanoma is a skin cancer notorious for its metastatic potential. As an initial step of the metastatic cascade, melanoma cells part from the primary tumour and invade the surrounding tissue, which is crucial for their dissemination and the formation of distant secondary tumours. Over the last two decades, our understanding of both, general and melanoma specific mechanisms of invasion has significantly improved, but to date no efficient therapeutic strategy tackling the invasive properties of melanoma cells has reached the clinic. In this review, we assess the major contributions towards the understanding of the molecular biology of melanoma cell invasion with a focus on melanoma specific traits. These traits are based on the neural crest origin of melanoma cells and explain their intrinsic invasive nature. A particular emphasis is given not only to lineage specific signalling mediated by TGFß, and noncanonical and canonical WNT signalling, but also to the role of PDE5A and RHO-GTPases in modulating modes of melanoma cell invasion. We discuss existing caveats in the current understanding of the metastatic properties of melanoma cells, as well as the relevance of the 'phenotype switch' model and 'co-operativity' between different phenotypes in heterogeneous tumours. At the centre of these phenotypes is the lineage commitment factor microphthalmia-associated transcription factor, one of the most crucial regulators of the balance between de-differentiation (neural crest specific gene expression) and differentiation (melanocyte specific gene expression) that defines invasive and noninvasive melanoma cell phenotypes. Finally, we provide insight into the current evidence linking resistance to targeted therapies to invasive properties of melanoma cells.