Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Abstract

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Rasmediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Rasmediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.Mutational activation of the KRAS oncogene is the most prevalent genetic alteration associated with essentially 100% of pancreatic ductal adenocarcinomas (PDACs) 3 (1). Therefore, considerable effort has been made to develop inhibitors of K-Ras for PDAC treatment. Currently, most of these efforts are focused on inhibitors of K-Ras downstream effector signaling, with numerous inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)-AKT pathway currently under clinical evaluation (2). However, recent studies suggest that additional Ras effector pathways may also play significant roles in Ras-mediated oncogenesis. In particular, there is increasing evidence for the importance of Ral guanine nucleotide exchange factors (RalGEFs), activators of the Ral (Ras-like) small GTPases (3), in cancer growth (4).Ral GTPases function as GDP/GTP-regulated binary switches that regulate extracellular stimulus-regulated signaling networks that control a diversity of cellular processes, including exocytosis, endocytosis, activation of transcription factors, and actin cytoskeletal reorganization (4). Surprisingly, despite their significant sequence identity (82%) and interaction with a common set of effectors, the related RalA and RalB isoforms serve very distinct functions in oncogenesis. Importantly, Chien and White (5) found that RalA is necessary for tumor cell anchorage-independent growth, whereas RalB is necessary for tumor but no...

The RalB Small GTPase Mediates Formation of Invadopodia through a GTPase-Activating Protein-Independent Function of the RalBP1/RLIP76 Effector

“…Our previous studies established a critical role for the RalGEFRalB pathway in PDAC Matrigel invasion in vitro and/or metastasis as determined in the tail vein lung colonization assay (36,64). Based on these observations, we speculated that Ral GTPases may be involved in PDAC invadopodium formation.…”

The RAS-RAL axis in cancer: evidence for mutation-specific selectivity in non-small cell lung cancer

“…Approximately 85% of the amino acid sequences of these two isoforms are identical [21] . RAL GTPase can be activated by six GEFs (RALGEFs), RALGDS, RGL, RGL2/Rlf, RGL3, RALGPS1, and RALGPS2, and inactivated by two GAPs, RALGAP1, and RALGAP2 [22,23] . Four RALGEFs (RALGDS, RGL, RGL2, and RGL3) are known to directly interact with the effector binding region of GTPbound RAS and are thus important for RAS-mediated tumorigenesis [22] .…”

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

“…Like Ras, Ral small GTPases function as molecular on-off switches and cycle between inactive GDP-and active GTPbound states. The cycle is catalyzed by Ral-selective guanine nucleotide exchange factors (RalGEFs) 3 and GTPase-activating proteins (RalGAPs), which accelerate slow intrinsic exchange and GTPase activities, respectively (4). Ral GTPases are activated downstream of Ras oncoproteins.…”