RhoGDI2, why can't you be more like your brother? The role of RhoGDI2 and RhoGDI1 in cancer biology

The RhoGTPase enzymes contribute to cancerogenesis due to their involvement in various signaling pathways. This has garnered a lot of attention towards their key regulators, among which are three RhoGDIs (Rho protein-guanine dissociation inhibitors). Recent research demonstrates that RhoGDI 1 and 2 differ in their functions and that RhoGDI2 has dual pro- and anti-tumor properties, making it an interesting target in cancer research. This study aims to enhance our understanding of the specific role of RhoGDI2 – exploring novel areas where it acts differently compared to RhoGDI1 and why it may do so.
Here, we illustrate the different ways RhoGDI2 acts as compared to RhoGDI1, by noticing how suppressing the expression of these two proteins influences cancer cell phenotypes in various cells. RhoGDI2 silencing significantly diminished cell proliferation in U2OS and PC3 (human osteosarcoma and prostate cancer, respectively) tumor cell lines and RPE-1 (retinal pigment epithelial) cell lines as opposed to RhoGDI1 silencing. Silencing RhoGDI2 in U2OS cells also induced abnormal centrosomal amplification: we see a larger number of cells with more than 2 centrosomes, as compared to cells with RhoGDI1 silencing. Lastly, we induce the formation of primary cilia in RPE-1 cells and observe a 2-fold decrease in ciliated cells with RhoGDI2 knock-down as compared to cells with RhoGDI1 knock-down.
We hypothesize that RhoGDI2 may be dictated by key interactors that are differentially expressed in different cancers and situations. To investigate this, we performed a classical IP/MS analysis for potential RhoGDI2 interactors. We shortlisted the most probable interactors of RhoGDI2 but are presently unable to validate them due to lack of optimal antibodies. To circumvent this, we plan to exploit a novel BioID approach, a proximity-dependent labelling system to screen protein interactions as they occur in living cells.
RhoGDI1 and RhoGDI2 share a highly conserved C-terminal (75% similarity) and the N-terminals are highly divergent. We suspect that another potential criterion for RhoGDI2 to function differently than RhoGDI1 could be its highly divergent N-terminal. For this, we generated PC3 cells overexpressing either recombinant RhoGDI1 or 2, or chimeric RhoGDIs resulting from swapping their N-terminal ends (N1C2 and N2C1). A preliminary experiment with these clones suggests a specific role of the N-terminal of RhoGDI2 in the regulation of PC3 cells proliferation, preliminarily validating this hypothesis. The required tools have been generated and further experiments will enable us to understand why RhoGDI2 behaves differently than RhoGDI1 in context of cancer biology.