Ral GTPase promotes metastasis of pancreatic ductal adenocarcinoma via elevation of TGF-ß1 production.

Pancreatic ductal adenocarcinoma (PDAC), caused by activating mutations in K-Ras, is an aggressive malignancy due to its early invasion and metastasis. Ral GTPases are activated downstream of Ras and play a crucial role in the development and progression of PDAC. However, the underlying mechanisms remain unclear. In this study, we investigated the mechanism of Ral-induced invasion and metastasis of PDAC cells using RalGAPß-deficient PDAC cells with highly activated Ral GTPases. Array analysis and ELISA revealed increased expression and secretion of TGF-ß1 in RalGAPß-deficient PDAC cells compared to control cells. Blockade of TGF-ß1 signaling suppressed RalGAPß deficiency-enhanced migration and invasion in vitro and metastasis in vivo to levels similar to controls. Phosphorylation of c-Jun N-terminal kinase, a repressor of TGF-ß1 expression, was decreased by RalGAPß deficiency. These results indicate that Ral contributes to invasion and metastasis of PDAC cells by elevating autocrine TGF-ß1 signaling at least in part by decreasing c-Jun N-terminal kinase activity.

The RAL signaling network: Cancer and beyond.

The RAL proteins RALA and RALB belong to the superfamily of small RAS-like GTPases (guanosine triphosphatases). RAL GTPases function as molecular switches in cells by cycling through GDP- and GTP-bound states, a process which is regulated by several guanine exchange factors (GEFs) and two heterodimeric GTPase activating proteins (GAPs). Since their discovery in the 1980s, RALA and RALB have been established to exert isoform-specific functions in central cellular processes such as exocytosis, endocytosis, actin organization and gene expression. Consequently, it is not surprising that an increasing number of physiological functions are discovered to be controlled by RAL, including neuronal plasticity, immune response, and glucose and lipid homeostasis. The critical importance of RAL GTPases for oncogenic RAS-driven cellular transformation and tumorigenesis still attracts most research interest. Here, RAL proteins are key drivers of cell migration, metastasis, anchorage-independent proliferation, and survival. This chapter provides an overview of normal and pathological functions of RAL GTPases and summarizes the current knowledge on the involvement of RAL in human disease as well as current therapeutic targeting strategies. In particular, molecular mechanisms that specifically control RAL activity and RAL effector usage in different scenarios are outlined, putting a spotlight on the complexity of the RAL GTPase signaling network and the emerging theme of RAS-independent regulation and relevance of RAL.

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.

Down-regulation of RalGTPase-Activating Protein Promotes Colitis-Associated Cancer via NLRP3 Inflammasome Activation.

BACKGROUND & AIMS: Ral guanosine triphosphatase-activating protein α2 (RalGAPα2) is the major catalytic subunit of the negative regulators of the small guanosine triphosphatase Ral, a member of the Ras subfamily. Ral regulates tumorigenesis and invasion/metastasis of some cancers; however, the role of Ral in colitis-associated cancer (CAC) has not been investigated. We aimed to elucidate the role of Ral in the mechanism of CAC. METHODS: We used wild-type (WT) mice and RalGAPα2 knockout (KO) mice that showed Ral activation, and bone marrow chimeric mice were generated as follows: WT to WT, WT to RalGAPα2 KO, RalGAPα2 KO to WT, and RalGAPα2 KO to RalGAPα2 KO mice. CAC was induced in these mice by intraperitoneal injection of azoxymethane followed by dextran sulfate sodium intake. Intestinal epithelial cells were isolated from colon tissues, and we performed complementary DNA microarray analysis. Cytokine expression in normal colon tissues and CAC was analyzed by quantitative polymerase chain reaction. RESULTS: Bone marrow chimeric mice showed that immune cell function between WT mice and RalGAPα2 KO mice was not significantly different in the CAC mechanism. RalGAPα2 KO mice had a significantly larger tumor number and size and a significantly higher proportion of tumors invading the submucosa than WT mice. Higher expression levels of matrix metalloproteinase-9 and matrix metalloproteinase-13 were observed in RalGAPα2 KO mice than in WT mice. The expression levels of interleukin 1ß, NLRP3, apoptosis associated speck-like protein containing a CARD, and caspase-1 were apparently increased in the tumors of RalGAPα2 KO mice compared with WT mice. NLRP3 inhibitor reduced the number of invasive tumors. CONCLUSIONS: Ral activation participates in the mechanism of CAC development via NLRP3 inflammasome activation.

Characterisation of the Ral GTPase inhibitor RBC8 in human and mouse platelets.

The Ral GTPases, RalA and RalB, have been implicated in numerous cellular processes, but are most widely known for having regulatory roles in exocytosis. Recently, we demonstrated that deletion of both Ral genes in a platelet-specific mouse gene knockout caused a substantial defect in surface exposure of P-selectin, with only a relatively weak defect in platelet dense granule secretion that did not alter platelet functional responses such as aggregation or thrombus formation. We sought to investigate the function of Rals in human platelets using the recently described Ral inhibitor, RBC8. Initial studies in human platelets confirmed that RBC8 could effectively inhibit Ral GTPase activation, with an IC50 of 2.2 µM and 2.3 µM for RalA and RalB, respectively. Functional studies using RBC8 revealed significant, dose-dependent inhibition of platelet aggregation, secretion (α- and dense granule), integrin activation and thrombus formation, while α-granule release of platelet factor 4, Ca2+ signalling or phosphatidylserine exposure were unaltered. Subsequent studies in RalAB-null mouse platelets pretreated with RBC8 showed dose-dependent decreases in integrin activation and dense granule secretion, with significant inhibition of platelet aggregation and P-selectin exposure at 10 µM RBC8. This study strongly suggests therefore that although RBC8 is useful as a Ral inhibitor in platelets, it is likely also to have off-target effects in the same concentration range as for Ral inhibition. So, whilst clearly useful as a Ral inhibitor, interpretation of data needs to take this into account when assessing roles for Rals using RBC8.

The Small GTPase Ral orchestrates MVB biogenesis and exosome secretion.

Extracellular vesicles are novel mediators of cell-cell communication. They are present in all species and involved in physiological and pathological processes. One class of extracellular vesicles, the exosomes, originate from an endosomal compartment, the MultiVesicular Body (MVB), and are released from the cell upon fusion of the MVB with the plasma membrane. Although different molecular mechanisms have been associated with MVB biogenesis and exosome secretion, how they coordinate remains poorly documented. We recently found that the small GTPase Ral contributes to exosome release in nematodes and mammalian tumor cells. More specifically, we found that C. elegans RAL-1 is required for the biogenesis of MVBs, and later for MVB fusion with the plasma membrane. Here, we discuss our results in relationship with other factors involved in extracellular vesicle production such as the ESCRT complex and Phospholipase 1D. We propose models to explain Ral function in exosome secretion, its conservation in animals, and its possible role in tumor progression.

Ral GTPase and the exocyst regulate autophagy in a tissue-specific manner.

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress-induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation-induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context-dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch.

Nodal signals via ß-arrestins and RalGTPases to regulate trophoblast invasion.

Placentation is critical for establishing a healthy pregnancy. Trophoblasts mediate implantation and placentation and certain subtypes, most notably extravillous cytotrophoblast, are highly invasive. Trophoblast invasion is tightly regulated by microenvironmental cues that dictate placental morphology and depth. In choriocarcinomas, malignant trophoblast cells become hyperinvasive, breaching the myometrium and leading to major complications. Nodal, a member of the TGF-ß superfamily, is expressed throughout the endometrium during the peri-implantation period and in invasive trophoblast cells. Nodal promotes the invasion of numerous types of cancer cells. However, Nodal's role in trophoblast and choriocarcinoma cell invasion is unclear. Here we show that Nodal stimulates the invasion of both the non-malignant HTR-8SV/neo trophoblast and JAR choriocarcinoma cells in a dose-dependent manner. We found that endogenous ß-arrestins and Ral GTPases, key regulators of the cell cytoskeleton, are constitutively associated with Nodal receptors (ALK4 and ALK7) in trophoblast cells and that RalA is colocalized with ALK4 in endocytic vesicles. Nodal stimulates endogenous ß-arrestin2 to associate with phospho-ERK1/2, and knockdown of ß-arrestin or Ral proteins impairs Nodal-induced trophoblast and choriocarcinoma cell invasion. These results demonstrate, for the first time, that ß-arrestins and RalGTPases are important regulators of Nodal-induced invasion.