Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer

Abstract

The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common β subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.

Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer*

“…Furthermore, annexin A1 is a key regulator of pathological angiogenesis and physiological angiogenic balance (29). Attenuated expression of RALGAPA2 leads to tumor invasion and metastasis of bladder cancer (21). Gridin regulates reorganization of the actin cytoskeleton and modulation of AKT activity, which ultimately result in cancer invasion and angiogenesis (30).…”

Inhibition of Ral GTPases Using a Stapled Peptide Approach

“…Nevertheless, we have demonstrated here the important overall effect of SP1 in cells, which is to prevent Ral signaling by competing with effector binding. Furthermore, GTP-specific peptides would be particularly useful in cells that have reduced RalGAP activity, for example invasive bladder cancer cell lines (27).…”

“…However, no GEFs have been found that discriminate between the two Ral isoforms, and the one structure of a RalGEF with Ral shows that all the contacts with the GEF protein are conserved between RalA and RalB (25). Similarly, RalGAPs appear to act on both isoforms in vitro (26) and in cell lines (27).…”

Platelets Are Highly Activated in Patients of Chronic Thromboembolic Pulmonary Hypertension

“…Because complex formation of α and β subunits is essential for the GAP activity, as well as mutual protein stability, the RalGAP activity could be monitored by evaluating the amount of common RalGAPβ subunit. 19,20 To address the cause of elevated activation of RalA in patients with PAH and CTEPH, we evaluated the expression levels of RalGAPβ subunit in platelets. As shown in Figure 4, relative expression levels of platelets of non-PH patients (n=9), patients with PAH (n=11), and patients with CTEPH (n=17) were 1±0.76, 0.65±0.54, and 0.57±0.35 folds, relatively (Figure 4).…”