Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function

Abstract

RhoGDI2 is a suppressor of metastasis in human bladder cancer. Although diminished RhoGDI2 expression in tumors is associated with decreased patient survival, normal expression in some metastatic tumors led us to wonder whether other mechanisms regulate RhoGDI2 function. Protein interaction analysis identified Src as a novel RhoGDI2 interaction partner. Gene expression profiling and immunohistochemistry of human tumors revealed that Src levels diminish as a function of bladder cancer stage. In addition, diminished Src levels and RhoGDI2 levels appear mutually exclusive in individual tumors, indicating that both genes are likely involved in the same signaling pathway leading to metastasis suppression. Studies confirmed that activated Src kinase binds and phosphorylates RhoGDI2 in vitro and vivo. Mutagenesis revealed that Tyr-153 and, to a lesser degree, Tyr-24 were the primary Src phosphorylation sites. Phosphorylation decreased the amount of Rac1 in RhoGDI2 complexes and increased RhoGDI2 association with cell membranes. Stable expression of phosphomimetic Tyr-153 RhoGDI2 in metastatic human bladder cancer cell lines had no effect on primary tumor growth but suppressed metastasis more potently than WT RhoGDI2. These data suggest that phosphorylation by Src enhances RhoGDI2 metastasis suppression and that loss of Src relieves metastasis suppression in tumor cells that maintain RhoGDI2 expression. Our findings also suggest caution in using Src inhibitors in the hope of delaying progression in patients with bladder cancer.bladder neoplasms ͉ guanine nucleotide dissociation inhibitors ͉ neoplasm metastasis ͉ src-family kinases B ladder cancer is common in the United States (1). Most deaths from this disease are due to metastases, commonly to lung and liver. RhoGDI2 (LyGDI/D4GDI) has been shown to be a metastasis suppressor in animal models of bladder cancer and to be lost in many metastatic human tumors (2, 3). RhoGDI2 belongs to a family of related proteins that includes RhoGDI1 and RhoGDI3 (reviewed in ref. 4). RhoGDI1 is the most widely expressed and the most intensely studied. Previously, it was thought that RhoGDI2 expression was confined to cells of hematopoietic origin, but our studies showed it is expressed in a wider range of tissues and cell types (5). Much less is known about RhoGDI3. Its expression is highest in brain, lung, and testis and, in contrast to RhoGDI1 and RhoGDI2, it is not found in the cytoplasm, but is associated with vesicular membranes. RhoGDIs are thought to inhibit the activation of small GTPases of the Rho family by sequestering the GTPases in the cytosol and blocking activation by guanine nucleotide exchange factors (GEFs) (4). Previous work identified RhoA, Rac1, and Rac2 as targets of RhoGDI2-mediated inactivation (6, 7), suggesting that RhoGDI2's function overlaps that of RhoGDI1.Although RhoGDI2 expression is reduced in many metastatic bladder cancers, Ϸ35% of patients with moderate or high levels of RhoGDI2 protein still developed metastatic disease. Although factors unrelated to...

Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity

“…In addition to genes involved in EMT, preclinical studies suggest that RHO-GDP dissociation inhibitor 2 (RHOGDI2), the activity of which is regulated by SRC, is a metastasis suppressor in MIBC 173 174 . In contrast to most tumour types, the expression and activation of SRC is highest in NMIBC [174][175][176] , compatible with the activity of RHOGDI2 in these tumours. Further work on the consequences of loss of RHOGDI2 expression implicated endothelin 1 (EDN1) and versican (VCAN) as promoters of an inflammatory environment involving macrophages and the chemokine (C-C motif) ligand 2 (CCL2)-chemokine (C-C motif) receptor 2 (CCR2) signalling axis that is permissive for metastasis 177 178 .…”

Dasatinib Is Preclinically Active against Src-Overexpressing Human Transitional Cell Carcinoma of the Urothelium with Activated Src Signaling

“…Intriguingly, higher stages seemed to numerically express Src less commonly than lower stages, but statistically significant differences were not found, probably due to the small numbers of tumors per stage. Src expression decreased with higher TCC tumor stage in another recent study, which suggests that its inhibition may be more relevant in patients with lower tumor stages (27). Although the level of activated Src signaling (i.e., p-Src) in human tumor specimens is of great interest, we did not perform these studies due to the lack of fresh or frozen tumor tissue.…”

“…However, there is no evidence supporting the expression of other key targets of dasatinib, Kit and Abl, in TCC. Intriguingly, a recent preclinical study suggests the beneficial antimetastatic effect of Src in TCC with the finding that phosphorylation by Src enhanced metastasis suppression by RhoGDI2 (27). Therefore, the further validation of Src as a molecular target at least in a subset of patients with TCC is warranted.…”

Microenvironmental Influences on Metastasis Suppressor Expression and Function during a Metastatic Cell’s Journey

“…RhoGDI2 belongs to a small family of proteins acting as RhoGTPase inhibitors by reducing dissociation of GDP from Rho proteins. Src interacts with and phosphorylates RhoGDI2 to enhance the metastasis suppressive effects [109]. Recently, Said et al showed that RhoGDI2-deficient disseminated tumor cells secrete several soluble factors (i.e., ECM molecules, versican, CCL2 and IL6) that stimulate macrophage recruitment [110][111][112].…”