Mafa-dependent GABAergic activity promotes mouse neonatal apneas.

While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.

MITF activity is regulated by a direct interaction with RAF proteins in melanoma cells.

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 mouse 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 BRAF/MITF and CRAF/MITF complexes were also observed in the cytosol of NRAS-mutated mouse melanoma cells. The interaction occurs through the kinase domain of RAF proteins. Importantly, endogenous BRAF/MITF complexes were also detected in BRAF-mutated human melanoma cells. 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.

Interplay Between Diabetes and Pancreatic Ductal Adenocarcinoma and Insulinoma: The Role of Aging, Genetic Factors, and Obesity.

Epidemiologic analyses have shed light on an association between type 2 diabetes (T2D) and pancreatic ductal adenocarcinoma (PDAC). Recent data also suggest a potential relationship between T2D and insulinoma. Under rare circumstances, type 1 diabetes (T1D) can also be implicated in tumorigenesis. The biological mechanisms underlying such relationships are extremely complex. Some genetic factors contributing to the development of T2D are shared with pancreatic exocrine and endocrine tumors. Obesity and overweight can also contribute to the initiation and severity of T2D, while aging may influence both endocrine and exocrine tumors. Finally, pharmacological treatments of T2D may have an impact on PDAC. On the other hand, some treatments for insulinoma can trigger diabetes. In the present minireview, we discuss the cellular and molecular mechanisms that could explain these interactions. This analysis may help to define new potential therapeutic strategies.

An autocrine ActivinB mechanism drives TGFß/Activin signaling in Group 3 medulloblastoma.

Medulloblastoma (MB) is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFß/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. We showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation. Beside genetic alterations, we demonstrated that an ActivinB autocrine stimulation is responsible for pathway activation in a subset of Group 3 MB characterized by high PMEPA1 levels. Importantly, Galunisertib, a kinase inhibitor of the cognate receptors currently tested in clinical trials for Glioblastoma patients, showed efficacy on orthotopically grafted MB-PDX. Our data demonstrate that the TGFß/Activin pathway is active in a subset of Group 3 MB and can be therapeutically targeted.

Translational control of tumor immune escape via the eIF4F-STAT1-PD-L1 axis in melanoma.

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Here, we show that the eukaryotic translation initiation complex, eIF4F, which binds the 5' cap of mRNAs, regulates the surface expression of interferon-γ-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies.

NRL and CRX Define Photoreceptor Identity and Reveal Subgroup-Specific Dependencies in Medulloblastoma.

Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.

NRAS-driven melanoma: A RAF can hide another.

Using mouse genetics, we recently showed that BRAF has a critical role in initiation of NRAS-driven melanoma that cannot be compensated by CRAF. In contrast, RAF proteins display compensatory functions in fully established tumors and ARAF can sustain proliferation in the absence of BRAF and CRAF, highlighting an addiction to RAF signaling in NRAS-driven melanoma.

RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma.

NRAS and its effector BRAF are frequently mutated in melanoma. Paradoxically, CRAF but not BRAF was shown to be critical for various RAS-driven cancers, raising the question of the role of RAF proteins in NRAS-induced melanoma. Here, using conditional ablation of Raf genes in NRAS-induced mouse melanoma models, we investigate their contribution in tumour progression, from the onset of benign tumours to malignant tumour maintenance. We show that BRAF expression is required for ERK activation and nevi development, demonstrating a critical role in the early stages of NRAS-driven melanoma. After melanoma formation, single Braf or Craf ablation is not sufficient to block tumour growth, showing redundant functions for RAF kinases. Finally, proliferation of resistant cells emerging in the absence of BRAF and CRAF remains dependent on ARAF-mediated ERK activation. These results reveal specific and compensatory functions for BRAF and CRAF and highlight an addiction to RAF signalling in NRAS-driven melanoma.

PFKFB4 controls embryonic patterning via Akt signalling independently of glycolysis.

How metabolism regulators play roles during early development remains elusive. Here we show that PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4), a glycolysis regulator, is critical for controlling dorsal ectoderm global patterning in gastrulating frog embryos via a non-glycolytic function. PFKFB4 is required for dorsal ectoderm progenitors to proceed towards more specified fates including neural and non-neural ectoderm, neural crest or placodes. This function is mediated by Akt signalling, a major pathway that integrates cell homeostasis and survival parameters. Restoring Akt signalling rescues the loss of PFKFB4 in vivo. In contrast, glycolysis is not essential for frog development at this stage. Our study reveals the existence of a PFKFB4-Akt checkpoint that links cell homeostasis to the ability of progenitor cells to undergo differentiation, and uncovers glycolysis-independent functions of PFKFB4.

B-Raf and C-Raf are required for melanocyte stem cell self-maintenance.

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.

Skin tumors induced by sorafenib; paradoxic RAS-RAF pathway activation and oncogenic mutations of HRAS, TP53, and TGFBR1.

PURPOSE: The emergence of skin tumors in patients treated with sorafenib or with more recent BRAF inhibitors is an intriguing and potentially serious event. We carried out a clinical, pathologic, and molecular study of skin lesions occurring in patients receiving sorafenib. EXPERIMENTAL DESIGN: Thirty-one skin lesions from patients receiving sorafenib were characterized clinically and pathologically. DNA extracted from the lesions was screened for mutation hot spots of HRAS, NRAS, KiRAS, TP53, EGFR, BRAF, AKT1, PI3KCA, TGFBR1, and PTEN. Biological effect of sorafenib was studied in vivo in normal skin specimen and in vitro on cultured keratinocytes. RESULTS: We observed a continuous spectrum of lesions: from benign to more inflammatory and proliferative lesions, all seemingly initiated in the hair follicles. Eight oncogenic HRAS, TGFBR1, and TP53 mutations were found in 2 benign lesions, 3 keratoacanthomas (KA) and 3 KA-like squamous cell carcinoma (SCC). Six of them correspond to the typical UV signature. Treatment with sorafenib led to an increased keratinocyte proliferation and a tendency toward increased mitogen-activated protein kinase (MAPK) pathway activation in normal skin. Sorafenib induced BRAF-CRAF dimerization in cultured keratinocytes and activated CRAF with a dose-dependent effect on MAP-kinase pathway activation and on keratinocyte proliferation. CONCLUSION: Sorafenib induces keratinocyte proliferation in vivo and a time- and dose-dependent activation of the MAP kinase pathway in vitro. It is associated with a spectrum of lesions ranging from benign follicular cystic lesions to KA-like SCC. Additional and potentially preexisting somatic genetic events, like UV-induced mutations, might influence the evolution of benign lesions to more proliferative and malignant tumors.

Alternative splicing in oncogenic kinases: from physiological functions to cancer.

Among the 518 protein kinases encoded by the human kinome, several of them act as oncoproteins in human cancers. Like other eukaryotic genes, oncogenes encoding protein kinases are frequently subjected to alternative splicing in coding as well as noncoding sequences. In the present paper, we will illustrate how alternative splicing can significantly impact on the physiological functions of oncogenic protein kinases, as demonstrated by mouse genetic model studies. This includes examples of membrane-bound tyrosine kinases receptors (FGFR2, Ret, TrkB, ErbB4, and VEGFR) as well as cytosolic protein kinases (B-Raf). We will further discuss how regular alternative splicing events of these kinases are in some instances implicated in oncogenic processes during tumor progression (FGFR, TrkB, ErbB2, Abl, and AuroraA). Finally, we will present typical examples of aberrant splicing responsible for the deregulation of oncogenic kinases activity in cancers (AuroraB, Jak2, Kit, Met, and Ron).

Dual function of MyD88 in RAS signaling and inflammation, leading to mouse and human cell transformation.

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.

MafA transcription factor identifies the early ret-expressing sensory neurons.

Dorsal root ganglia proceed from the coalescence of cell bodies of sensory neurons, which have migrated dorsoventrally from the delaminating neural crest. They are composed of different neuronal subtypes with specific sensory functions, including nociception, thermal sensation, proprioception, and mechanosensation. In contrast to proprioceptors and thermonociceptors, little is known about the molecular mechanisms governing the early commitment and later differentiation into mechanosensitive neurons. This is mainly due to the absence of specific molecular markers for this particular cell type. Using knockout mice, we identified the bZIP transcription factor MafA as the first specific marker of a subpopulation of "early c-ret" positive neurons characterized by medium-to-large diameters. This marker will allow further functional characterization of these neurons.

B-raf alternative splicing is dispensable for development but required for learning and memory associated with the hippocampus in the adult mouse.

The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.

Gene expression signature associated with BRAF mutations in human primary cutaneous melanomas.

With the aim to correlate BRAF mutation status with gene expression in human primary cutaneous melanomas, and thus to get more insight on the consequences of BRAF mutation on cell biology, we analyzed all expression data obtained in melanomas from which DNA was extracted from the same tissue slides that were used for the expression study. A cohort of 69 frozen primary melanoma whose oligonucleotide micro-array expression data were available, were genotyped for BRAF and NRAS genes. The expression data from these melanomas were re-analyzed according to BRAF mutational status. A set of 250 probes representing 209 genes that were significantly (raw P< or =0.001) associated with BRAF mutation status was identified and 17 of these were previously shown to be implicated in cutaneous melanoma progression or pigmentation pathway-associated genes driven by the microphthalmia transcription factor (MITF). The list of 34 top probes contained no more than 1% of false discoveries with a probability of 0.95. Among the genes that differentiated most strongly between BRAF mutated and non-mutated melanomas, there were those involved in melanoma immune response such as MAGE-D2, CD63, and HSP70. These findings support the immunogenicity of BRAF(V600E), eliciting patients T-cell responses in various in vitro assays. The genes whose expression is associated with BRAF mutations are not simply restricted to the MAPK/ERK signaling but also converge to enhanced immune responsiveness, cell motility and melanosomes processing involved in the adaptative UV response.

A new MAFia in cancer.

Like JUN and FOS, the Maf transcription factors belong to the AP1 family. Besides their established role in human cancer--overexpression of the large Maf genes promotes the development of multiple myeloma--they can display tumour suppressor-like activity in specific cellular contexts, which is compatible with their physiological role in terminal differentiation. However, their oncogenic activity relies mostly on the acquisition of new biological functions relevant to cell transformation, the most striking characteristic of Maf oncoproteins being their ability to enhance pathological interactions between tumour cells and the stroma.

GSK-3-mediated phosphorylation enhances Maf-transforming activity.

The Maf oncoproteins are b-Zip transcription factors of the AP-1 superfamily. They are involved in developmental, metabolic, and tumorigenic processes. Maf proteins are overexpressed in about 50% of human multiple myelomas. Here, we show that Maf-transforming activity is controlled by GSK-3-dependent phosphorylation and that phosphorylation by GSK-3 can increase the oncogenic activity of a protein. Using microarray analysis, we identify a gene-expression subprogram regulated by GSK-3-mediated Maf phosphorylation involved in extracellular matrix remodeling and relevant to cancer progression. We also demonstrate that GSK-3 triggers MafA sequential phosphorylation on residues S61, T57, T53, and S49, inducing its ubiquitination and degradation. Paradoxically, this phosphorylation increases MafA-transcriptional activity through the recruitment of the coactivator P/CAF. We further demonstrate that P/CAF protects MafA from ubiquitination and degradation, suggesting that, upon the release of the coactivator complex, MafA becomes polyubiquitinated and degraded to allow the response to terminate.

Differential regulation of B-raf isoforms by phosphorylation and autoinhibitory mechanisms.

The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge region upstream of the kinase domain. The presence of exon 8b in the B2-Raf(8b) isoform and exon 9b in the B3-Raf(9b) isoform differentially regulates B-Raf by decreasing and increasing MEK activating and oncogenic activities, respectively. Using different cell systems, we investigated here the molecular basis of this regulation. We show that exons 8b and 9b interfere with the ability of the B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365A mutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf(8b) was more pronounced. This was correlated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf(9b) isoform was poorly phosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but the S429A mutation activated B2-Raf(8b), whereas it inhibited B3-Raf(9b). These results indicate that phosphorylation on both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mechanisms. Finally, we show that autoinhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing.

MafA transcription factor is phosphorylated by p38 MAP kinase.

Basic-leucine zipper transcription factors of the Maf family are key regulators of various developmental and differentiation processes. We previously reported that the phosphorylation status of MafA is a critical determinant of its biological functions. Using Western blot and mass spectrometry analysis, we now show that MafA is phosphorylated by p38 MAP kinase and identify three phosphoacceptor sites: threonine 113 and threonine 57, evolutionarily conserved residues located in the transcription activating domain, and serine 272. Mutation of these residues severely impaired MafA biological activity. Furthermore, we show that p38 also phosphorylates MafB and c-Maf. Together, these findings suggest that the p38 MAP kinase pathway is a novel regulator of large Maf transcription factors.