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During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes., Virginia and D.K. Ludwig Fund for Cancer Research (Graduate Student Fellowship), National Institutes of Health (U.S.) (GM58801), Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology, David H. Koch Institute for Integrative Cancer Research at MIT (NCI Core Grant P30-CA14051)

Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis., Virginia and D.K. Ludwig Fund for Cancer Research (Postdoctoral fellowship), King's College London (Overseas Research PhD Studentship (KORS)), National Cancer Institute (U.S.) (U54-CA112967), National Cancer Institute (U.S.) (U54-CA163109), Ludwig Center for Molecular Oncology at MIT, David H. Koch Institute for Integrative Cancer Research at MIT (Support Grant P30-CA14051), National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051), Biotechnology and Biological Sciences Research Council (Great Britain) (BB/F011431/1), Biotechnology and Biological Sciences Research Council (Great Britain) (BB/J000590/1), Biotechnology and Biological Sciences Research Council (Great Britain) (BB/N000226/1), Wellcome Trust (London, England) (082907/Z/07/Z)

The growth of blood vessels is crucial for organ growth in the embryo and repair of wounded tissues in the adult. An imbalance in this process contributes to numerous malignant, inflammatory, ischemic, infectious and immune disorders (Ferrara et al., 2003). Postnatal neovascularization occurs through the recruitment of progenitor cells and angiogenesis. Integrins are heterodimeric cell surface molecules and are the main receptors for extracellular matrix proteins. Regulation of integrin activation is crucial during embryonic development and during adult life. Dysregulation of integrin activity leads to severe diseases. In this study, we have demonstrated that Rap1, a small GTPase regulating integrin activity, and its GEF Epac1 are expressed in both EPC and endothelial cells. Moreover, the pharmacological activator of Epac activates the small GTPase Rap1 in progenitor cells. In parallel the angiogenic growth factors VEGF and bFGF activate Rap1 in endothelial cells. In addition, the regulation of Rap1 activity in EPC and in endothelial cells plays an important role in the regulation of migration and adhesion to matrix proteins, by regulating the activity of different integrins, a mechanism known as integrin inside‐out signaling. Furthermore, regulation of Rap1 activity affects probably indirectly through outside‐in signaling of integrins the activity of several and crucial proteins such PKB/Akt and focal adhesion kinase in endothelial cells. In line with these results, we have demonstrated that Rap1 activity affect angiogenesis, homing of EPC to ischemic tissues and thereby postnatal neovascularization. The understanding how Rap1 regulates integrin activity in endothelial cells is still not completely clear, for example we have demonstrated that the known effectors of Rap1 mediating the increase of integrin activity in T and B cells, such as RAPL and RIAM are, respectively, either not increasing integrin activity or not expressed in endothelial cells. We a, Die Bildung von neuen Gefäßen im adulten Organismus spielt eine wichtige Rolle in physiologischen und pathologischen Prozessen. Die postnatale Gefäßneubildung wird durch die Angiogenese, Vaskulogenese und Arteriogenese vermittelt. Integrine sind heterodimere Transmembranproteine, die die Adhäsion an Matrix‐Proteine und interzelluläre Adhäsion vermitteln können. Integrin‐vermittelte Signale sind wichtig für die Migration, Proliferation und Differenzierung von Zellen. Die Aktivierung von Integrinen wird durch die Regulation der Konformation und Lokalisation auf der Zelloberfläche vermittelt. Rap1 ist eine kleine GTPase der Ras‐Familie. Rap1 spielt eine wichtige Rolle in der Regulation der Integrine in vielen zellulären Systemen. Zu Beginn der vorliegenden Dissertation war die Funktion von Rap1a und Rap1b für die Endothelzellbiologie unbekannt, daher war das primäre Ziel; die Rolle der GTPase Rap1 in der Angiogenese und Vaskulogenese und deren Bedeutung für die Regulation von Integrinen aufzuklären. Zusammenfassend wurde in der vorliegenden Arbeit die Rolle von Rap1 für die Funktion von Endothelzellen und für die Gefäßneubildung in vivo und in vitro untersucht. Rap1a und Rap1b sind essentiell für das angiogene Sprouting, Migration, Adhäsion und Integrin‐ Aktivitätsregulation von maturen Endothelzellen. Als funktioneller Mediator des Rap1‐abhängigen angiogenen Sproutings konnte zudem Regulator of Adhesion and cell Polarization enriched in Lymphoid tissues (RAPL) nachgewiesen werden. Weiterhin zeigen diese Daten, dass Rap1a ein wichtiges Signalmolekül für die Vermittlung der in vivo Gefäßneubildung nach Ischämie ist. Rap1 ist dabei nicht nur für die Funktion von maturen Endothelzellen wichtig, sondern spielt auch als Integrinregulator eine relevante Rolle in Progenitorzellen. Eine exogene Aktivierung des Epac/Rap1‐Systems stimulierte die Adhäsion und Migration von Progenitorzellen in vitro und förderte die Rekrutierung von Progenitorze