[Show abstract][Hide abstract] ABSTRACT: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a metastasis suppressor in bladder and possibly other cancers. This protein is a member of a family of proteins that maintain Rho GTPases in the cytoplasm and inhibit their activation and function. To understand the mechanism of metastasis suppression, we compared effects of RhoGDI1 and RhoGDI2. Despite showing much stronger inhibition of metastasis, RhoGDI2 is a weak inhibitor of Rho GTPase membrane targeting and function. However, point mutants that increase or decrease the affinity of RhoGDI2 for GTPases abolished its ability to inhibit metastasis. Surprisingly, metastasis suppression correlates with increased rather than decreased Rac1 activity. These data show that RhoGDI2 metastasis inhibition works through Rho GTPases but via a mechanism distinct from inhibition of membrane association.
[Show abstract][Hide abstract] ABSTRACT: Metastatic melanomas are generally resistant to chemotherapy and radiation, even when wild-type for p53. These tumors often grow in small nests where many of the cells have little contact with extracellular matrix (ECM). Previous work showed that M21 melanomas undergo apoptosis in response to chemotherapy when cells are adherent to ECM but not in suspension. Thus, reduced integrin-dependent adhesion to ECM could mediate therapy resistance. The goal of this study was to test whether stimulation of integrin signaling could increase chemotherapeutic efficacy.
Colony forming assays and survival assays were used to test the responses of melanoma lines in vitro. Severe combined immunodeficient mice with subcutaneous human melanomas received chemotherapy with or without reagents that stimulate integrin signaling; tumor volume was then monitored over time.
Clonal growth assays confirmed that M21 cells showed reduced sensitivity to the chemotherapeutic drug 1-beta-D-arabinofuranosylcytosine (araC). When five additional primary melanoma lines were screened, 80% showed higher sensitivity when adherent compared with suspended. Subcutaneous M21 tumors in vivo showed minimal ECM between tumor cells. To evaluate the importance of integrin signaling in chemoresistance in this model, mice were treated with araC, with or without the multivalent snake venom disintegrin contortrostatin or the activating anti-beta1 integrin antibody TS2/16. Although araC, TS2/16, or contortrostatin alone had little effect on M21 tumor growth, combining araC with either integrin signaling reagents strongly reduced growth (P = 0001).
Loss of integrin-mediated adhesion is rate limiting for therapeutic response in this model. Combining chemotherapy with reagents that stimulate integrin signaling may therefore provide a new approach to therapy.
No preview · Article · Nov 2008 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Most deaths from urinary bladder cancer are owing to metastatic disease. A reduction in Rho GDP Dissociation Inhibitor 2 (RhoGDI2) protein has been associated with increased risk of metastasis in patients with locally advanced bladder cancer, whereas in animal models, RhoGDI2 reconstitution in cells without expression results in lung metastasis suppression. Recently, we noted an inverse correlation between tumor RhoGDI2 and Neuromedin U (NMU) expression, suggesting that NMU might be a target of the lung metastasis suppressor effect of RhoGDI2. Here we evaluated whether NMU is regulated by RhoGDI2 and is functionally important in tumor progression. We used small interfering RNA knockdown of endogenous RhoGDI2 in poorly tumorigenic and non-metastatic human bladder cancer T24 cells and observed increased NMU RNA expression. Although NMU overexpression did not increase the monolayer growth of T24 or related T24T poorly metastatic human bladder cancer cells, it did augment anchorage-independent growth for the latter. Overexpression of NMU in T24 and T24T cells significantly promoted tumor formation of both cell lines in nude mice, but did not alter the growth rate of established tumors. Furthermore, NMU-overexpressing xenografts were associated with lower animal body weight than control tumors, indicating a possible role of NMU in cancer cachexia. NMU overexpression in T24T cells significantly enhanced their lung metastatic ability. Bioluminescent in vivo imaging revealed that lung metastases in T24T grew faster than the same tumors in the subcutaneous microenvironment. In conclusion, NMU is a RhoGDI2-regulated gene that appears important for tumorigenicity, lung metastasis and cancer cachexia, and thus a promising therapeutic target in cancer.