Baccarini

The research goal of the Baccarini lab is to define the essential function of RAF and MEK in in vivo models of tissue development, remodeling, and neoplasia. To this end, they combine phenotype analysis in the context of the whole organism (in vivo) as well as of cells (ex vivo) with biochemical experiments in ablated cells to elucidate the molecular basis of a given phenotype. In the past few years, the Baccarini lab has shown that BRAF is the non-redundant activator of the ERK pathway in vivo in the developing mouse placenta (Figure 1) and in a mouse tumor model of insulinoma, in which cases it promotes angiogenesis (1,2), and in the central nervous system, where it regulates the differentiation of dorsal root ganglia (3) and of oligodendrocytes (4). In contrast, RAF1 is essential to promote survival and migration independently of its enzymatic activity as a MEK kinase (5). Instead, RAF1 binds to and negatively regulates two proapoptotic kinases [MST2 (6,7) and ASK1 (8)] and the Rho-dependent kinase ROKα, which controls cell shape, migration, and the expression of the death receptor FAS (9,10).

Given the link between the RAS/RAF/MEK/ERK pathway and tumorigenesis, we have analyzed the role of RAF in models of epithelial carcinogenesis. We have shown that RAF1 ablation prevents the development and causes the regression of RAS-driven tumors (11). In this system, RAS promotes complex formation between RAF1 and ROKα, and RAF1 operates as an endogenous inhibitor of ROKα, necessary to suppress keratinocyte differentiation (12). The effects of BRAF ablation on skin carcinogenesis are milder, and like most of the phenotypes observed in BRAF knockout tissues, depend on the reduction of MEK/ERK activation (13). Thus, BRAF and RAF1 have non-redundant functions in RAS-driven tumorigenesis.

More recently, we investigated what would happen if we removed both RAF proteins from keratinocytes in the epidermis of mice. Surprisingly, the mice developed an allergic disease similar to human atopic dermatitis. This effect is different from the side effects caused by drugs that inhibit Raf proteins. Further analysis revealed that in the epidermis, Raf are required to keep the balance between ERK signaling, promoted by BRAF, and JNK signaling, inhibited by RAF1. If both RAF are missing, excessive stress signaling in keratinocytes causes inflammation and allergy by activating immune cells both locally and systemically (14).

The RAF1-ROKα complex also plays a protumorigenic role in stroma, being essential for sprouting the vascularization of the tumors. In endothelial cells, RAF1 is required for the recruitment of ROKα to the junctions keeping endothelial cells together; therefore, at least in endothelial cells RAF1 plays the dual role of a ROKα inhibitor as well as scaffold. If the complex is not correctly localized, these junction are unstable, new vessels cannot form, and tumors cannot grow (15).

At the level of the Raf substrates, we have shown that one MEK protein, MEK1, has two unexpected essential functions as a negative regulator of RAS signaling: within the pathway, it is necessary to dim ERK signaling via the MEK2/ERK axis (16); and outside of the ERK pathway it is required to contain the strength and duration of PIP3/AKT signaling (17). Crucially, both functions depend on the phosphorylation of a site present in the proline-rich region of MEK1 but not MEK2, T292. T292 is phosphorylated by activated ERK, thereby providing negative feedback to the pathway; this fact, however, also means that MEK inhibitors prevent T292 phosphorylation and therefore increase PIP3/AKT signaling. A potential consequence of this is that tumor cells will be primed for the emergence of inhibitor resistance pathways relying on AKT signaling.

MEK1’s negative regulation of PIP3/AKT signaling requires the formation of a complex containing the large adaptor MAGI1 and the phosphatase PTEN. If this complex is not formed, PTEN is not recruited to the membrane, and is therefore unable to control PIP3 accumulation.

The lab continues to focus on deciphering signaling pathways in vivo. We carry out studies in different cell lineages, given the often-contrasting evidence about the role and relevance of signal transduction pathways studied in different cellular systems. For each process, we are using several in vitro models and at least one in vivo model, to ensure the relevance and the validity of the information obtained. Manuela Baccarini is the coordinator of the PhD Program “Molecular Mechanisms of Cell Signaling” (W1220), originally funded by the Austrian National Scientific Research Fund. We offer competitive PhD projects in the areas of development, tumorigenesis, inflammation, and biogenesis of signaling complexes.

References

1. Galabova-Kovacs, G. et al. Essential role of B-Raf in ERK activation during extraembryonic development. Proc Natl Acad Sci U S A 103, 1325-30 (2006).
2. Sobczak, I. et al. B-Raf is required for ERK activation and tumor progression in a mouse model of pancreatic beta-cell carcinogenesis. Oncogene 27, 4779-87 (2008).
3. Zhong, J. et al. Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo. Nat Neurosci 10, 598-607 (2007).
4. Galabova-Kovacs, G. et al. Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development. J Cell Biol 180, 947-55 (2008).
5. Mikula, M. et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf- 1 gene. Embo J 20, 1952-1962. (2001).
6. Matallanas, D. et al. RASSF1A elicits apoptosis through an MST2 pathway directing proapoptotic transcription by the p73 tumor suppressor protein. Mol Cell 27, 962-75 (2007).
7. O'Neill, E., Rushworth, L., Baccarini, M. & Kolch, W. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306, 2267-70 (2004).
8. Yamaguchi, O. et al. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest 114, 937-43 (2004).
9. Ehrenreiter, K. et al. Raf-1 regulates Rho signaling and cell migration. J Cell Biol 168, 955-64 (2005).
10. Piazzolla, D., Meissl, K., Kucerova, L., Rubiolo, C. & Baccarini, M. Raf-1 sets the threshold of Fas sensitivity by modulating Rok-{alpha} signaling. J. Cell Biol. 171, 1013-1022 (2005).
11. Ehrenreiter, K., Kern, F., Velamoor, V., Meissl, K., Galabova-Kovacs, G., Sibilia, M., and Baccarini, M. Raf-1 addiction in Ras-induced carcinogenesis. Cancer Cell, 16, 149-160 (2009).
12. Niault, T, Sobczak, I., Meissl, K., Weitsman, G., Piazzolla, D., Maurer, G., Kern, F., Ehrenreiter, K., Hamerl, M., Moarefi, I., Leung, T., Carugo, O., Ng, T., and Baccarini, M. From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits Rok-alpha kinase activity. J Cell Biol, 187, 335-342 (2009)
13. Kern, F., Doma, E., Rupp, C., Niault, T. & Baccarini, M. Essential, non-redundant roles of B-Raf and Raf-1 in Ras-driven skin tumorigenesis. Oncogene, 32, 2483-92 (2013)                                   14. Raguz, J., Jeric, I., Niault, T., Nowacka, J.D., Kuzet, S.E., Rupp, C., Fischer, I. Biggi, S. Borsello, T., and Baccarini, M. Epidermal RAF prevents allergic skin disease. eLife; 19;5. pii: e14012. (2016)
15. Wimmer, R., Cseh, B., Maier, B., Scherrer, K. & Baccarini, M. Angiogenic sprouting requires the fine tuning of endothelial cell cohesion by the Raf-1/Rok-alpha complex. Developmental cell, 22, 158-171, (2012)
16. Catalanotti, F. et al. A MEK1-MEK2 heterodimer determines the strength and duration of the ERK signal. Nat Struct Mol Biol 16, 294-303 (2009).
17. Zmajkovicova, K., Jesenberger, V., Catalanotti, F., Baumgartner, C., Reyes, G. & Baccarini, M. MEK1 Is Required for PTEN Membrane Recruitment, AKT Regulation, and the Maintenance of Peripheral Tolerance. Molecular cell 50, 43-55, (2013).