Mouse Platelet Ral GTPases Control P-Selectin Surface Expression, Regulating Platelet-Leukocyte Interaction.

OBJECTIVE: RalA and RalB GTPases are important regulators of cell growth, cancer metastasis, and granule secretion. The purpose of this study was to determine the role of Ral GTPases in platelets with the use of platelet-specific gene-knockout mouse models. APPROACH AND RESULTS: This study shows that platelets from double knockout mice, in which both GTPases have been deleted, show markedly diminished (≈85% reduction) P-selectin translocation to the surface membrane, suggesting a critical role in α-granule secretion. Surprisingly, however, there were only minor effects on stimulated release of soluble α- and δ-granule content, with no alteration in granule count, morphology, or content. In addition, their expression was not essential for platelet aggregation or thrombus formation. However, absence of surface P-selectin caused a marked reduction (≈70%) in platelet-leukocyte interactions in blood from RalAB double knockout mice, suggesting a role for platelet Rals in platelet-mediated inflammation. CONCLUSIONS: Platelet Ral GTPases primarily control P-selectin surface expression, in turn regulating platelet-leukocyte interaction. Ral GTPases could therefore be important novel targets for the selective control of platelet-mediated immune cell recruitment and inflammatory disease.

RalBP1 is necessary for metastasis of human cancer cell lines.

RalA expression in human prostate cancer is associated with cell migration and is necessary for bone metastasis. However, the downstream effectors of RalA that mediate these functions remain unclear. Here we examined cell migration after small interfering RNA-mediated depletion of Ral effectors Ral binding protein 1 (RalBP1/RLIP), exocyst complex component 2 (Sec5), and phospholipase D1 (PLD1) and found that RalBP1 and RalA depletion inhibited cell migration to a similar extent. Stable lentivirus short hairpin interfering RNA-mediated depletion of RalA and RalBP1 in PC3 human prostate cancer cells inhibited bone metastasis after intracardiac inoculation. Depletion of RalBP1 diminished orthotopic tumor growth of PC3 cells and inhibited spontaneous metastasis from this site. Interestingly, the expression of wild-type or RalA mutants deficient in RalBP1 binding was effective at rescuing the reduced metastatic capacity of RalA-depleted PC3 cells, suggesting that RalA depletion does not reduce this solely by diminished interaction with RalBP1. To determine whether the role of RalBP1 in metastasis is relevant beyond prostate cancer, we studied the requirement of RalBP1 expression in an experimental metastasis model of human bladder cancer, a tumor type with high RalBP1 expression. Depletion of RalBP1 in UMUC3 cells resulted in decreased lung colonization while having a minimal effect on subcutaneous tumor growth. Our studies are the first to suggest that the expression of RalBP1 is necessary for human cancer cell metastasis. Furthermore, we show that the requirement for RalA expression for manifestation of this phenotype is not entirely dependent on a RalA-RalBP1 interaction.

Expression profiling of Ral-depleted bladder cancer cells identifies RREB-1 as a novel transcriptional Ral effector.

Although the monomeric GTPases RalA and RalB have been shown to regulate a variety of transcription factors, little is known regarding the differences or similarities in transcriptional programs regulated by RalA compared to RalB. Further, the association of these transcriptional pathways to human carcinogenesis and progression remains unclear. Here, we studied the role of RalA and/or RalB in transcriptional regulation by combining short interfering RNA depletion of Ral with gene expression profiling via microarray in the human bladder cancer cell line, UMUC-3. A large number of genes were found to be similarly modulated in cells with RalA and RalB depletion, suggesting that RalA and RalB impinge on overlapping transcriptional signaling pathways. However, smaller sets of genes were modulated by depletion of RalA or RalB, indicating that these closely related proteins also regulate nonoverlapping transcriptional pathways. Computational analysis of upstream sequences of genes modulated by Ral depletion identified Ras-responsive element-binding protein (RREB)-1, as a putative Ral transcriptional target, which we verified experimentally. Importantly, as a group, Ral-regulated probe sets identified here were disproportionally represented among those differentially expressed as a function of human bladder transformation. Taken together, these data strongly suggest that Ral family members mediate both common and specific transcriptional programs that are associated with human cancer and identify RREB-1 as a novel transcriptional effector of Ral.

The metastasis-associated gene CD24 is regulated by Ral GTPase and is a mediator of cell proliferation and survival in human cancer.

Ral GTPases are important mediators of transformation, tumorigenesis, and cancer progression. We recently identified the metastasis-associated protein CD24, a glycosyl phosphatidyl inositol-linked surface protein, as a downstream target of Ral signaling by profiling the expression of RalA/B-depleted bladder carcinoma cells. Because CD24 is highly expressed in bladder and many other tumor types, we sought to determine if this protein plays an essential role in maintaining the malignant phenotype. Here, we show that loss of CD24 function in cell lines derived from common tumor types is associated with decreased rates of cell proliferation, clonogenicity in soft agar, changes in the actin cytoskeleton, and induction of apoptosis. Given these phenotypes, we evaluated a human bladder cancer tissue microarray by immunohistochemistry for CD24 to determine if CD24 is a prognostic cancer biomarker. Multivariate analysis showed that increased CD24 expression correlated with shorter patient disease-free survival (P = 0.07). In conclusion, we show that CD24 is a novel and functionally relevant Ral-regulated target and a potentially important prognostic marker. We suggest that these insights may lead to future therapeutic approaches that seek to eliminate CD24 function in cancer cells.

RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells.

RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.