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We report a novel function of RUVBL1 molecule in pancreatic cancer cells. Previous reports describe that RUVBL1 belongs to the family of AAA+ ATPases that associate with chromatin-remodelling complexes and have important roles in transcriptional regulation, the DNA damage response, telomerase activity and cellular transformation. We show that knockdown of RUVBL1 inhibited the motility and invasiveness of pancreatic cancer cells. RUVBL1 localized in the cytoplasm bound filamentous actin (F-actin) in cell protrusions, and increased concentration of monomeric globular-actin (G-actin) in cell protrusions of migrating pancreatic cancer cells. Cytoplasmic RUVBL1 functioned in additional formation of actin filaments in cell protrusions. Consequently, cytoplasmic RUVBL1 contributed to the formation of membrane protrusions by promoting peripheral actin polymerization. Our results imply that these RUVBL1-actin interactions could enhance the invasive properties of pancreatic cancer cells.

We report that Binder of Arl Two (BART) plays a role in inhibiting cell invasion by regulating the activity of the Rho small guanosine triphosphatase protein Rac1 in pancreatic cancer cells. BART was originally identified as a binding partner of ADP-ribosylation factor-like 2, a small G protein implicated as a regulator of microtubule dynamics and folding. BART interacts with active forms of Rac1, and the BART-Rac1 complex localizes at the leading edges of migrating cancer cells. Suppression of BART increases active Rac1, thereby increasing cell invasion. Treatment of pancreatic cancer cells in which BART is stably knocked down with a Rac1 inhibitor decreases invasiveness. Thus, BART-dependent inhibition of cell invasion is likely associated with decreased active Rac1. Suppression of BART induces membrane ruffling and lamellipodial protrusion and increases peripheral actin structures in membrane ruffles at the edges of lamellipodia. The Rac1 inhibitor inhibits the lamellipodia formation that is stimulated by suppression of BART. Our results imply that BART regulates actin-cytoskeleton rearrangements at membrane ruffles through modulation of the activity of Rac1, which, in turn, inhibits pancreatic cancer cell invasion.

A novel function for the binder of Arl two (BART) molecule in pancreatic cancer cells is reported. BART inhibits invasiveness of pancreatic cancer cells through binding to a Ca(2+)-dependent, phosphorylated, guanosine triphosphatase (GTPase) membrane fusion protein, annexin7 (ANX7). A tumor suppressor function for ANX7 was previously reported based on its prognostic role in human cancers and the cancer-prone mouse phenotype ANX7(+/-). Further investigation demonstrated that the BART-ANX7 complex is transported toward cell protrusions in migrating cells when BART supports the binding of ANX7 to the protein kinase C (PKC) isoform PKCα. Recent evidence has suggested that phosphorylation of ANX7 by PKC significantly potentiates ANX7-induced fusion of phospholipid vesicles; however, the current data suggest that the BART-ANX7 complex reduces PKCα activity. Knocking down endogenous BART and ANX7 increases activity of PKCα, and specific inhibitors of PKCα significantly abrogate invasiveness induced by BART and ANX7 knockdown. These results imply that BART contributes to regulating PKCα activity through binding to ANX7, thereby affecting the invasiveness of pancreatic cancer cells. Thus, it is possible that BART and ANX7 can distinctly regulate the downstream signaling of PKCα that is potentially relevant to cell invasion by acting as anti-invasive molecules.

We report that BART plays a role in inhibiting cell invasion by regulating the activity of the Rho GTPase protein RhoA in pancreatic cancer cells. BART was originally identified as a binding partner of ARL2, a small G-protein implicated as a regulator of microtubule dynamics and folding. We show that BART interacts with GTP-bound ARL2 and is required for the binding of GTP-bound ARL2 with active forms of RhoA at leading edges in migrating cancer cells. GTP-bound ARL2 inactivates RhoA and BART prevents ARL2 from regulating RhoA activity. Thus, BART binds to and functions as an inhibitor of ARL2 at leading edges of migrating cells, thereby increasing the amount of active RhoA. Treatment with the Rho inhibitor C3 exoenzyme induces cell invasion by pancreatic cancer cells to the same level as that of cells in which BART is stably knocked down by RNA interference. GTP-bound ARL2 acts as a RhoA inhibitor by a mechanism that involves the induction of actin-cytoskeleton rearrangements. We show that BART decreases actin-cytoskeleton rearrangements by inhi-biting ARL2 function and by increasing the amount of active RhoA in pancreatic cancer cells. Our results imply that BART increases active RhoA by inhibiting ARL2 function, which in turn inhibits invasiveness of cancer cells.

The regulation of mRNA stability plays an important role in the control of gene expression during cell motility and invasion. We previously reported that GTPase-activating protein [Src homology 3 (SH3) domain] binding protein (G3BP), a marker of cytoplasmic stress granules that are formed in stressed cells and regulate mRNA stability, binds and degrades the mRNA of binder of Arl two (BART) that inhibits retroperitoneal invasion and hepatic metastasis of pancreatic cancer cells. Here, we report that overexpression of the amino (N)-terminal region of G3BP, including the binding region for BART mRNA, dominant-negatively inhibits formation of the complex between endogenous G3BP and BART mRNA, and increases the expression of BART. This, in turn, inhibits the invasiveness of pancreatic cancer cells. On the other hand, the carboxy (C)-terminal region of G3BP is associated with phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that initiates stress granule assembly but does not modulate the posttranscriptional regulation of BART mRNA. N-terminal G3BP also plays a role in regulating secreted matrix metalloproteinases, transcription factors, and a variety of genes involved in cell adhesion and motility. These results suggest that N-terminal G3BP contributes to posttranscriptional regulation of cell motility and invasive capacity of pancreatic cancer., (©2011 AACR.)

We report a novel function for the CD24 molecule in pancreatic cancer cells. Intracellular CD24 is associated with stress granules that contain specific mRNAs and RNA-binding proteins that regulate mRNA stability and translation. Intracellular CD24 in stress granules is associated with G3BP, a phosphorylation-dependent endoribonuclease. The vesicles in which the CD24/G3BP complex localizes are transported toward cell protrusions in migrating cells. We show that G3BP binds to and degrades Binder of Arl Two (BART) mRNA. BART was originally identified as a binding partner of ARL2, a small G-protein implicated as a regulator of microtubule dynamics and folding. Intracellular CD24 inhibits the specific endoribonuclease activity of G3BP toward BART mRNA in stress granules. We show that knockdown of CD24 increases retroperitoneal invasion and liver metastasis of pancreatic cancer cells in an orthotopic xenograft model, and that BART also prevents retroperitoneal invasion and liver metastasis of pancreatic cancer cells. Our results imply that surface CD24 may play a role in the inhibition of cell invasion and metastasis, and that intracellular CD24 inhibits invasiveness and metastasis through its influence on the posttranscriptional regulation of BART mRNA levels via G3BP RNase activity.