B-Raf and C-Raf kinases have emerged as critical players in melanoma. However, little is known about their role during development and homeostasis of the melanocyte lineage. Here, we report that knockout of B-raf and C-raf genes in this lineage results in normal pigmentation at birth with no defect in migration, proliferation, or differentiation of melanoblasts in mouse hair follicles. In contrast, the double raf knockout mice displayed hair graying resulting from a defect in cell-cycle entry of melanocyte stem cells (MSCs) and their subsequent depletion in the hair follicle bulge. Therefore, Raf signaling is dispensable for early melanocyte lineage development, but necessary for MSC maintenance.
Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation.
We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation.
AbstractSignals from the extracellular matrix are essential for the survival of many cell types. Dominant-negative mutants of two members of Rho family GTPases, Rac1 and Cdc42, mimic the loss of anchorage in primary mouse fibroblasts and are potent inducers of apoptosis. This pathway of cell death requires the activation of both the p53 tumor suppressor and the extracellular signal-regulated mitogen-activated protein kinases (Erks). Here we characterize the proapoptotic Erk signal and show that it differs from the classically observed survival-promoting one by the intensity of the kinase activation. The disappearance of the GTP-bound forms of Rac1 and Cdc42 gives rise to proapoptotic, moderate activation of the Raf-MEK-Erk cascade via a signaling pathway involving the kinases phosphatidlyinositol 3-kinase and Akt. Moreover, concomitant activation of p53 and inhibition of Akt are both necessary and sufficient to signal anoikis in primary fibroblasts. Our data demonstrate that the GTPases of the Rho family control three major components of cellular signal transduction, namely, p53, Akt, and Erks, which collaborate in the induction of apoptosis due to the loss of anchorage. ACKNOWLEDGMENTSWe are grateful to A. Brunet, P. Chavrier, A. Hall, P. Lenormand, C. Marshall, C. Norbury, S. Roche, M. Vandromme, M. White, and E. Yonish-Rouach for various plasmids used in this work and to P. Boquet for the generous gift of C. difficile toxin B. We thank Damien Gregoire for help with the anoikis experiments, Pierre Travo for help with immunofluorescence microscopy, and Bob Hipskind for invaluable comments on the manuscript.This work was supported by INSERM, CNRS, and Association pour la Recherche contre le Cancer (support given to U.H.). T.F.F. is the recipient of Career Development Award DAMD17-00-1-0214 and O.Z. is the recipient of a fellowship from Association pour la Recherche contre le Cancer.
Medulloblastoma (MB), the most frequent malignant brain tumor in children, is localized in the cerebellum. The standard care includes surgery, radiotherapy, and chemotherapy leading to an overall survival (OS) of 70–80%, but survivors suffer from severe side effects. Based on gene expression, MB is divided in four different molecular subgroups—WNT, SHH, Group 3 (G3), and Group 4 (G4)—which differ in terms of clinics, prognosis, genetic alterations, and cell of origin. The WNT group is characterized by the activation of the WNT/β-catenin signaling pathway and displays the best prognosis. The SHH group is driven by deregulation of the SHH signaling pathway and has an intermediate prognosis. G3 and G4 are less characterized. Contrary to the SHH and WNT groups, no specific alteration of a given signaling pathway has been described. G3 is the group with the worse prognosis. Few recurrent genetic alterations have been characterized including MYC amplification in less than 20% of G3 tumors. Nevertheless, all G3-MBs overexpress MYC through mechanisms not completely understood. G3-MBs also express an abnormal photoreceptor differentiation program found in the retina but not in the cerebellum during normal development. It has been shown that NRL and CRX, two master transcription factors (TF) of the photoreceptor lineage, are required for the establishment of this program as well as for G3 tumor maintenance. G4 has an intermediate prognosis, and the most frequent alteration is the overexpression of PRDM6. It has been recently proposed that this group could be driven by activation of an ERBB4-SRC signaling. Established cell lines and patient-derived xenografts (PDXs) are available to study MB. The different groups of MB have also been modeled in vivo using either genetically engineered mouse models (GEMMs) or orthotopic transplantation of mouse cerebellar progenitors modified to overexpress oncogenes and/or to inactivate tumor suppressors. Here, we provide the readers with tools and information that allow MB modeling in vivo. We describe how to purify granular cell cerebellar progenitors or PDXs and to culture them in vitro in order to modulate gene expression by lentiviral infection. We provide protocols for the retrovirus production and infection. We also describe the experimental procedures for orthotopic grafting in the cerebellum, which is used to assess how genetic modifications alter in vivo tumor formation of reinjected modified PDX cells or GCPs.
Abstract The MITF transcription factor and the RAS/RAF/MEK/ERK pathway are two interconnected main players in melanoma. Understanding how MITF activity is regulated represents a key question since its dynamic modulation is involved in the phenotypic plasticity of melanoma cells and their resistance to therapy. By investigating the role of ARAF in NRAS-driven melanoma through mass spectrometry experiments followed by a functional siRNA-based screen, we unexpectedly identified MITF as a direct ARAF partner. Interestingly, this interaction is conserved among the RAF protein kinase family since the formation of BRAF/MITF and CRAF/MITF complexes was also observed in the cytosol of NRAS-mutated melanoma cells. The interaction occurs through the kinase domain of RAF proteins and is correlated with their kinase activity level. RAF/MITF complexes modulate MITF nuclear localization by inducing an accumulation of MITF in the cytoplasm, thus negatively controlling its transcriptional activity. Taken together, our study highlights a new level of regulation between two major mediators of melanoma progression, MITF and the MAPK/ERK pathway, which appears more complex than previously anticipated.
