October 2019 Newsletter: RhoB GTPase: Tumor Promoter or Suppressor?

RhoB is a Rho-family GTPase that regulates essential physiological processes such as cell division, morphology, motility, adhesion, and intracellular transport, primarily through dynamic remodeling of the actin cytoskeleton, and whose expression and/or activity is pathologically dysfunctional in human diseases such as cancer and neurodegenerative diseases^1-3. Due to its unique C-terminal region and distinct post-translational modifications there, RhoB is localized not only to the plasma membrane (like other Rho GTPases), but also to endosomes, multivesicular bodies, and even the nucleus^1,3 (Fig. 1). Like other Rho-family GTPases, RhoB functions as a binary switch in signaling cascades, cycling between a GDP-bound, inactive state and a GTP-bound, active state. The GTP/GDP cycling is controlled by guanine nucleotide exchange factors (GEFs; activation by exchanging GDP for GTP) and GTPase-activating proteins (GAPs; inactivation by GTP hydrolysis)^1-3.

RhoB expression and/or activity is regulated by a variety of physiological stimuli. Normally expressed at low levels under steady state conditions, RhoB expression and/or activity is rapidly up-regulated by hypoxia, growth factors, inflammatory cytokines, and stress stimuli including UV radiation^1,3-8 (Fig. 1). Upon activation, RhoB regulates cellular responses to UV-induced DNA damage, apoptosis, cell cycle progression, and cell migration (and invasion in the case of cancer cells)^1,3. RhoB’s distinctive subcellular localization to membrane vesicles enables RhoB-mediated regulation of intracellular transport. Endosome-localized RhoB regulates the trafficking (and thereby function) of receptor tyrosine kinase and cytokine receptor-mediated signaling cascades (e.g., EGFR, CXCR2, TNFR) and the activities of kinases such as Src and Akt^9-13 (Fig. 1). As a result, RhoB is able to regulate a wide range of essential signaling cascades involved in cellular development, proliferation, survival, and apoptosis – all pathways important in human physiology and disease^1,3.

Figure 1. Schematic diagram of different forces in a migrating cell. Actin-based protrusions (i.e., lamellipodia) at the leading edge push the cell forward, while membrane tension physically opposes this motility. At the trailing edge, membrane tension aids in actomyosin-mediated contraction.

RhoB expression and/or activity have a paradoxical relationship with tumorigenesis, as RhoB has been proposed to be both a tumor suppressor and tumor promoter, depending on the tumor microenvironment (i.e., context-dependent, cell-type specific, and even tumor stage-dependent)^1,3. Initial recognition of RhoB’s role in tumorigenesis was its requirement in Ras-mediated fibroblast transformation¹⁴. Recent studies also support a positive role for RhoB in tumorigenesis. In renal proximal tubular cells, RhoB knockdown correlates with a significant increase in apoptosis¹⁵. Similarly, knockdown of RhoB induces cell-cycle arrest and apoptosis and impairs tumorigenic potential in multiple glioblastoma cell lines¹⁶. Additional studies report elevated RhoB expression and/or activity in breast tumors vs normal tissue¹⁷ and T-cell acute lymphoblastic leukaemia vs normal T-cells¹⁸. Furthermore, elevated RhoB in the primary tumor is predictive of a poor response to EGFR-RTK inhibitor treatment in lung cancer patients¹⁹. Increases in RhoB expression/activity also reduce survival in lung adenocarcinomas²⁰, impair the efficacy of chemoradiotherapy while enhancing metastatic potential in lung cancer cells²⁰, and seemingly protect certain cancer cell lines from radiation-induced apoptosis and mitotic cell death^21-23. These findings led to the hypothesis that RhoB is important in developing and maintaining a malignant phenotype for at least certain cancers³. Conversely, there is a rich literature describing RhoB’s ability to function as a tumor suppressor³. For instance, in vitro and in vivo models of cancer demonstrate that miRNA-mediated inhibition of RhoB mRNA expression correlates with increased cancer cell activity and/or tumorigenesis. miR-21-mediated inhibition of RhoB mRNA results in increased colorectal cancer cell proliferation, migration, and invasion, while miR-19a-mediated inhibition triggers pancreatic cancer in vitro and in vivo^1,3,24,25. Additionally, RhoB is necessary for an apoptotic response to DNA damage or taxol treatment in transformed fibroblasts²⁶, and RhoB inhibits cancer cell migration, invasion, metastasis, and tumorigenesis in in vitro and in vivo oncogenic models in a cell-type and context-dependent manner^1,3,27,28. In total, many studies report an inverse correlation between RhoB expression/activity and tumor progression in multiple types of cancers^1,3. RhoB’s opposing roles in tumorigenesis are likely determined by the type of cancer and tumor stage (e.g., initiation vs progression). The dependency on context (i.e., tumor microenvironment) both elucidates and complicates the relationship between cancers and RhoB and its potential as a therapeutic target^1,3. These diverse and seemingly conflicting data also reinforce the need to think in terms of maintaining/regulating homeostasis in RhoB activity rather than in terms of inhibiting or activating RhoB activity^1,3.

Summary

Despite sharing significant amino acid sequence identity with RhoA and RhoC GTPases, RhoB exerts unique regulatory control over a wide range of cellular responses through many different signaling cascades, including control of the trafficking of other Rho-family GTPases such as Cdc42 and Rac, which themselves regulate cell migration^1,3 (Fig. 1). Importantly, RhoB’s functions in tumorigenesis cannot be predicted from how RhoA and RhoC function. Thus, understanding the different conditions and contexts under which RhoB expression/activity is altered and the relationship to tumor progression or suppression is of paramount importance³. Deciphering these unknowns could offer significant therapeutic potential in the treatment of cancer, vascular, and inflammatory diseases^1,3. To assist researchers, Cytoskeleton provides Ras and Rho-family GTPase activation assay kits, purified cytoskeletal proteins, cytoskeletal antibodies, live cell imaging reagents for cytoskeletal proteins, and Signal-Seeker kits to measure endogenous levels of post-translational modifications in cells and tissues.

References

1. Vega F.M. and Ridley A.J. 2018. The RhoB small GTPase in physiology and disease. Small GTPases. 9, 384-393.

2. Jaffe A.B. and Hall A. 2005. Rho GTPases: biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21, 247-269.

3. Ju J.A. and Gilkes D.M. 2018. Rhob: team oncogene or team tumor suppressor? Genes. DOI: 10.3390/genes9020067.

4. Skuli N. et al. 2006. Activation of RhoB by hypoxia controls hypoxia-inducible factor-1α stabilization through glycogen synthase kinase-3 in U87 glioblastoma cells. Cancer Res. 66, 482-489.

5. Wojciak-Stothard B. et al. 2012. Role of RhoB in the regulation of pulmonary endothelial and smooth muscle cell responses to hypoxia. Circ. Res. 110, 1423-1434.

6. Kroon J. et al. 2013. The small GTPase RhoB regulates TNFalpha signaling in endothelial cells. PLoS One. 8, e75031.

7. Huang G. et al. 2017. RhoB regulates the function of macrophages in the hypoxia-induced inflammatory response. Cell Mol. Immunol. 14, 265-275.

8. Ju J.A. et al. 2019. RhoB is regulated by hypoxia and modulates metastasis in breast cancer. Cancer Reports. E1164.

9. Gampel A. et al. 1999. Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB. Curr. Biol. 9, 955-958.

10. Adini I. Et al. 2003. RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development. Genes Dev. 17, 2721-2732.

11. Sandilands E. et al. 2004. RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane. Dev. Cell. 7, 855-869.

12. Fernandez-Borja M. et al. 2005. RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1. J. Cell Sci. 118, 2661-2670.

13. Neel N.F. et al. 2007. RhoB plays an essential role in CXCR2 sorting decisions. J. Cell Sci. 120, 1559-1571.

14. Prendergast G.C. et al. 1995. Critical role of Rho in cell transformation by oncogenic Ras. Oncogene. 10, 2289-2296.

15. Hutchison N. et al. 2009. Rho isoforms have distinct and specific functions in the process of epithelial to mesenchymal transition in renal proximal tubular cells. Cell Signal. 21, 1522-1531.

16. Ma Y. et al. 2015. Critical functions of RhoB in support of glioblastoma tumorigenesis. Neuro. Oncol. 17, 516-525.

17. Fritz G. et al. 2002. Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters. Br. J. Cancer. 87, 635-644.

18. Bhavsar P.J. et al. 2013. Analysis of Rho GTPase expression in T-ALL identifies RhoU as a target for Notch involved in T-ALL cell migration. Oncogene. 32, 198–208.

19. Calvayrac O. et al. 2017. The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer via an AKT-dependent mechanism. EMBO Mol. Med. 9, 238-250.

20. Luis-Ravelo D. et al. 2013. RHOB influences lung adenocarcinoma metastasis and resistance in a host-sensitive manner. Mol. Oncol. 8, 192–206.

21. Ader I. et al. 2002. RhoB controls the 24kDa FGF-2-induced radioresistance in HeLa cells by preventing post-mitotic cell death. Oncogene. 21, 5998–6006.

22. Canguilhem B. et al. 2005. RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling. J. Biol. Chem. 280, 43257–43263.

23. Milia J. et al. 2005. Farnesylated RhoB inhibits radiation-induced mitotic cell death and controls radiation-induced centrosome overduplication. Cell Death Differ. 12, 492–501.

24. Liu M. et al. 2011. miR-21 targets the tumor suppressor RhoB and regulates proliferation, invasion and apoptosis in colorectal cancer cells. FEBS Lett. 585, 2998-3005.

25. Tan Y. et al. 2015. Sp1-driven up-regulation of miR-19a decreases RHOB and promotes pancreatic cancer. Oncotarget. 6, 17391-17403.

26. Liu A. et al. 2001. RhoB is required to mediate apoptosis in neoplastically transformed cells after DNA damage. Proc. Natl. Acad. Sci. U.S.A. 98, 6192-6197.

27. Chen Z. et al. 2000. Both farnesylated and geranylgeranylated RhoB inhibit malignant transformation and suppress human tumor growth in nude mice. J. Biol. Chem. 275, 17974-17978.

28. Jiang K. et al. 2004. Akt mediates Ras downregulation of RhoB, a suppressor of transformation, invasion, and metastasis. Mol. Cell Biol. 24, 5565-5576.

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