Papers by Manuela Baccarini
Bulletin du cancer
liaison à l'ADN des complexes NF-kappaB dans les noyaux isolés de biopsie de patients atteints de... more liaison à l'ADN des complexes NF-kappaB dans les noyaux isolés de biopsie de patients atteints de ABC-DLBCL. LMP1 induit uniquement les complexes de la voie classique. Malgré l'activation de la voie alternative par l'EBV, p100 n'est pas à l'origine de dimères RelB/p52 mais exerce essentiellement une activité répressive sur les dimères RelB/p50. Dans ce contexte, RelA et RelB sont en compétition pour s'associer à p50. Les dimères RelA mais pas RelB sont associés à la fois à la survie et la prolifération des cellules B immortalisées par l'EBV. Une activité de liaison à l'ADN des complexes RelA est retrouvée dans 100 % des ABC-DLBCL. Alors que RelB n'est jamais trouvé seul, les complexes RelA et RelB sont retrouvés dans 22 % des cas. Nous concluons que la voie classique d'activation des complexes NF-kappaB RelA est la voie oncogénique majeure initiée par LMP1 dans les lymphocytes B transformés par l'EBV et est très probablement la principale cible oncogénique dans les ABC-DLBCL.
Journal of Experimental Medicine, Oct 2, 2000
bioRxiv (Cold Spring Harbor Laboratory), Feb 1, 2023
Lysosomes are highly dynamic organelles that regulate metabolic signaling pathways by recruiting ... more Lysosomes are highly dynamic organelles that regulate metabolic signaling pathways by recruiting cytosolic molecules to protein platforms on the lysosomal membrane. We performed a proximity-dependent labeling screen to identify novel proteins recruited to the LAMTOR complex, which regulates lysosome positioning and key signaling pathways such as mTORC1, AMPK, and MEK/ERK. We identified a network of proteins involved in actin remodelling, including Pleckstrin homology domain-containing family G member 3 (PLEKHG3), an actin-binding Rho guanine nucleotide exchange factor enriched in protrusions. We show that GFP-PLEKHG3 accumulates in focal adhesion sites, where it colocalizes with peripheral lysosomes. Peripheral accumulation of lysosomes concentrates PLEKHG3 below the plasma membrane, inhibits protrusion formation and limits cell motility. Thus, subcellular positioning of lysosomes impacts PLEKHG3 subcellular localization and the cell's protrusion activity, shape, and motility. The results shed new light .
The FEBS Journal, 2022
In this special interview series, we profile members of The FEBS Journal editorial board to highl... more In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus, perspectives on the journal and future directions in their field. Manuela Baccarini is Professor of Cell Signaling at the University of Vienna, Coordinator of the International PhD Program 'Signaling Mechanisms in Cellular Homeostasis' and Director of the Vienna BioCenter PhD Program, a graduate school of the University and Medical University of Vienna in collaboration with the Institute of Molecular Pathology and the Austrian Academy of Sciences, Institute for Medical Biotechnology and Gregor Mendel Institute, as well as EMBO member and corresponding member of the Austrian Academy of Sciences. She has served as an editorial board member of The FEBS Journal since 2016.
Molecular and Cellular Biology, Nov 1, 1996
Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kin... more Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me v /me v ) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me v /me v mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me v /me v cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways.
EMBO Reports, Aug 4, 2014
More than 30% of all human cancers contain activating mutations of the small G-protein RAS. As a ... more More than 30% of all human cancers contain activating mutations of the small G-protein RAS. As a result of this, RAS has been intensely studied and many efforts have been made to identify pathways that sustain RAS-driven transformation [1]. Recent studies have indicated that the transcription factor GATA2 is one of these partners in crime, but a mechanistic link between RAS and GATA2 had not been identified [2]. A paper in this issue of EMBO reports closes this gap showing that GATA2 can be activated by p38 in RAStransformed cells [3].
Infection and Immunity, Mar 1, 1999
Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical... more Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical events that follow lipopolysaccharide (LPS) treatment of macrophages or their infection by virulent and attenuated Salmonella strains. Phagocytosis as well as the secretion of invasion-associated proteins is dispensable for ERK activation by the pathogen. Furthermore, the pathways used by Salmonella and LPS to stimulate ERK are identical, suggesting that kinase activation might be solely mediated by LPS. Both stimuli activate ERK by a mechanism involving herbimycin-dependent tyrosine kinase(s) and phosphatidylinositol 3-kinase. Phospholipase D activation and stimulation of protein kinase C appear to be intermediates in this novel pathway of MEK/ERK activation.
Journal of Cell Biology, Mar 7, 2005
af kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform... more af kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-R deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-␣ to the plasma membrane. Raf-1 physically associates with Rok-␣ in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.
Journal of Cell Biology, Dec 19, 2005
blation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hyp... more blation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-␣. In this study, we show that the kinase-independent modulation of Rok-␣ signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-␣ complexes, and Rok-␣ signaling is up-regulated in Raf-1-deficient cells. This leads to A increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-␣ or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.
The EMBO Journal, Apr 17, 2001
The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we r... more The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we report that c-raf-1 ±/± embryos are growth retarded and die at midgestation with anomalies in the placenta and in the fetal liver. Although hepatoblast proliferation does not appear to be impaired, c-raf-1 ±/± fetal livers are hypocellular and contain numerous apoptotic cells. Similarly, the poor proliferation of Raf-1 ±/± ®broblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect. Furthermore, Raf-1-de®cient ®broblasts are more sensitive than wildtype cells to speci®c apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor-a. MEK/ERK activation is normal in Raf-1-de®cient cells and embryos, and is probably mediated by B-Raf. These results indicate that the essential function of Raf-1 is to counteract apoptosis rather than to promote proliferation, and that effectors distinct from the MEK/ERK cascade must mediate the anti-apoptotic function of Raf-1.
eLife, Jul 19, 2016
The RAS pathway is central to epidermal homeostasis, and its activation in tumors or in Rasopathi... more The RAS pathway is central to epidermal homeostasis, and its activation in tumors or in Rasopathies correlates with hyperproliferation. Downstream of RAS, RAF kinases are actionable targets regulating keratinocyte turnover; however, chemical RAF inhibitors paradoxically activate the pathway, promoting epidermal proliferation. We generated mice with compound epidermisrestricted BRAF/RAF1 ablation. In these animals, transient barrier defects and production of chemokines and Th2-type cytokines by keratinocytes cause a disease akin to human atopic dermatitis, characterized by IgE responses and local and systemic inflammation. Mechanistically, BRAF and RAF1 operate independently to balance MAPK signaling: BRAF promotes ERK activation, while RAF1 dims stress kinase activation. In vivo, JNK inhibition prevents disease onset, while MEK/ ERK inhibition in mice lacking epidermal RAF1 phenocopies it. These results support a primary role of keratinocytes in the pathogenesis of atopic dermatitis, and the animals lacking BRAF and RAF1 in the epidermis represent a useful model for this disease.
The FASEB Journal, Aug 4, 2011
Regulation of glucose homeostasis by insulin depends on pancreatic β-cell growth, survival, and f... more Regulation of glucose homeostasis by insulin depends on pancreatic β-cell growth, survival, and function. Raf-1 kinase is a major downstream target of several growth factors that promote proliferation and survival of many cell types, including the pancreatic β cells. We have previously reported that insulin protects β cells from apoptosis and promotes proliferation by activating Raf-1 signaling in cultured human islets, mouse islets, and MIN6 cells. As Raf-1 activity is critical for basal apoptosis and insulin secretion in vitro, we hypothesized that Raf-1 may play an important role in glucose homeostasis in vivo. To test this hypothesis, we utilized the Cre-loxP recombination system to obtain a pancreatic β-cell-specific ablation of Raf-1 kinase gene (RIPCre(+/+):Raf-1(flox/flox)) and a complete set of littermate controls (RIPCre(+/+):Raf-1(wt/wt)). RIPCre(+/+):Raf-1(flox/flox) mice were viable, and no effects on weight gain were observed. RIPCre(+/+):Raf-1(flox/flox) mice had increased fasting blood glucose levels and impaired glucose tolerance but normal insulin tolerance compared to littermate controls. Insulin secretion in vivo and in isolated islets was markedly impaired, but there was no apparent effect on the exocytosis machinery. However, islet insulin protein and insulin 2 mRNA, but not insulin 1 mRNA, were dramatically reduced in Raf-1-knockout mice. Analysis of insulin 2 knockout mice demonstrated that this reduction in mRNA was sufficient to impair in vivo insulin secretion. Our data further indicate that Raf-1 specifically and acutely regulates insulin 2 mRNA via negative action on Foxo1, which has been shown to selectively control the insulin 2 gene. This work provides the first direct evidence that Raf-1 signaling is essential for the regulation of basal insulin transcription and the supply of releasable insulin in vivo.
Oncogene, Apr 5, 2023
More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are ... more More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are present in 40% of colorectal cancer (CRC) in the two main CRC subgroups, MSS (Microsatellite Stable) and MSI (Microsatellite Instable). Studies in RAS-driven tumors have shown essential roles of the RAS effectors RAF and specifically of RAF1, which can be dependent or independent of RAF's ability to activate the MEK/ERK module. In this study, we demonstrate that RAF1, but not its kinase activity, plays a crucial role in the proliferation of both MSI and MSS CRC cell line-derived spheroids and patient-derived organoids, and independently of KRAS mutation status. Moreover, we could define a RAF1 transcriptomic signature which includes genes that contribute to STAT3 activation, and could demonstrate that RAF1 ablation decreases STAT3 phosphorylation in all CRC spheroids tested. The genes involved in STAT3 activation as well as STAT3 targets promoting angiogenesis were also downregulated in human primary tumors expressing low levels of RAF1. These results indicate that RAF1 could be an attractive therapeutic target in both MSI and MSS CRC regardless of their KRAS status and support the development of selective RAF1 degraders rather than RAF1 inhibitors for clinical use in combination therapies.
Growth Factors Journal, 1993
Multipotent hematopoietic progenitor cell lines (FDCP-Mix) infected with a retroviral vector expr... more Multipotent hematopoietic progenitor cell lines (FDCP-Mix) infected with a retroviral vector expressing the GM-CSF gene show functional downregulation of the GM-CSF receptor when maintained in IL-3 and activation of the receptor resulting in synchronous differentiation into mature granulocytes and macrophages on withdrawal of IL-3. This system has now been used to investigate whether or not receptors for some of the other growth factors are also influenced as a consequence of differentiation. We show here the lineage specific receptors for M-CSF, G-CSF and erythropoietin are all upregulated, regardless of whether or not differentiation is induced by GM-CSF or by other conditions. Concomitant induction of the mRNA coding for the ligands M-CSF and G-CSF, but not for erythropoietin, suggests that M-CSF and possibly G-CSF facilitate macrophage or granulocyte differentiation by an autocrine stimulation of the lineage specific receptors. FDCP-Mix mutants that are blocked in their ability to differentiate on exposure to GM-CSF, but that still require GM-CSF for proliferation, do not express increased levels of M-CSF receptor nor M-CSF. Based on these data, we suggest that expression of these lineage specific receptors is part of the intrinsic endogenous program of myeloid differentiation.
Oncogene, Jun 25, 2012
Ras-driven tumorigenesis is assumed to depend on Raf for ERK activation and proliferation; yet, a... more Ras-driven tumorigenesis is assumed to depend on Raf for ERK activation and proliferation; yet, an in vivo requirement for Raf as MEK/ERK activator in this setting has not been demonstrated to date. Here, we show that epidermis-restricted B-Raf ablation restrains the onset and stops the progression of established Ras-driven tumors by limiting MEK/ERK activation and proliferation. Concomitant elimination of B-Raf and Raf-1 enforces the abrupt regression of established tumors owing to the decrease in ERK activation and proliferation caused by B-Raf ablation combined with the ERK-independent increase in Rho-dependent kinase (Rok) signaling and differentiation triggered by Raf-1 inactivation. Thus, B-Raf and Raf-1 have non-redundant functions in Ras-driven tumorigenesis. Of note, Raf kinase inhibitors achieve impressive results in melanomas harboring oncogenic BRAF, but are ineffective against Ras-driven tumors; moreover, therapy-related skin tumors driven by a paradox ERK activation as well as primary and acquired resistance have been reported. Our results suggest that therapies targeting both Raf kinase-dependent and-independent pathways may be effective against a broader range of malignancies and reduce the risks of adverse effects and/or resistance.
Nature Communications, May 12, 2017
NRAS and its effector BRAF are frequently mutated in melanoma. Paradoxically, CRAF but not BRAF w... more 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.
International Journal of Molecular Sciences, Sep 25, 2013
The mammalian skin is the largest organ of the body and its outermost layer, the epidermis, under... more The mammalian skin is the largest organ of the body and its outermost layer, the epidermis, undergoes dynamic lifetime renewal through the activity of somatic stem cell populations. The EGFR-Ras-Raf pathway has a well-described role in skin development and tumor formation. While research mainly focuses on its role in cutaneous tumor initiation and maintenance, much less is known about Ras signaling in the epidermal stem cells, which are the main targets of skin carcinogenesis. In this review, we briefly discuss the properties of the epidermal stem cells and review the role of EGFR-Ras-Raf signaling in keratinocyte stem cells during homeostatic and pathological conditions.
Proceedings of the National Academy of Sciences of the United States of America, Nov 24, 1998
The stability of the ompA mRNA depends on the bacterial growth rate. The 5 untranslated region is... more The stability of the ompA mRNA depends on the bacterial growth rate. The 5 untranslated region is the stability determinant of this transcript and the target of the endoribonuclease, RNase E, the key player of mRNA degradation. An RNA-binding protein with affinity for the 5 untranslated region ompA was purified and identified as Hfq, a host factor initially recognized for its function in phage Q replication. The ompA RNA-binding activity parallels the amount of Hfq, which is elevated in bacteria cultured at slow growth rate, a condition leading to facilitated degradation of the ompA mRNA. In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has no affinity for the lpp transcript whose degradation, like that of bulk mRNA, is not affected by bacterial growth rate. Compatible with our results, we found that the intracellular concentration of RNase E and its associated degradosome components is independent of bacterial growth rate. Thus our results suggest a regulatory role for Hfq that specifically facilitates the ompA mRNA degradation in a growth rate-dependent manner.
Uploads
Papers by Manuela Baccarini