Activated Cdc42 mutants are capable of transforming immortalized fibroblasts, suggesting a role for Cdc42 in oncogenesis 
. While mutations in Cdc42 have not been reported in human cancer, upregulation of Cdc42 protein expression or activity has been reported in a variety of tumor types and in some instances have been correlated with poor prognosis 
. However, how Cdc42 activity contributes to the transformed phenotype and cooperates with other oncogenic events has not been rigorously explored. Furthermore, additional studies are needed to assess the consequences and feasibility of targeting Cdc42 signaling in distinct tumorigenic and tumor maintenance contexts. Here, we report that Cdc42 is critical for oncogenic Ras-driven cell transformation and tumor growth. Consistent with studies using dominant-negative Cdc42 mutants 
, we found that the loss of Cdc42 dramatically inhibits the ability of HRasV12 to promote cellular transformation, as determined by soft agar colony formation. However, in contrast to dominant-negative studies, we reveal that Cdc42 loss results in a significant reduction in cell growth, attributable to a G1 phase cell cycle arrest. Interestingly, this observation is consistent with a previous report that suggested a requirement Cdc42 for progression into S phase by dominant negative mutant expression 
. The current work is significant because it unambiguously demonstrates the essential role of Cdc42 activity in Ras-mediated transformation and tumorigenesis by a genetic means, as previous approaches utilizing overexpression of a mutant Cdc42 may very well introduce non-specific artifacts due to cross-reactivity of dominant negative Cdc42 mutant with multiple upstream Rho GEFs, thus affectingother Rho GTPases and related signaling networks. In addition to studies carried out in rodent cells, transformation of human fibroblasts with HRasV12 is also inhibited upon dominant negative Cdc42 expression. Interestingly, expression of angiogenic factors was downregulated upon Cdc42 inhibition in this model 
. However, while this effect may account for differences in tumorigenesis in vivo
, it does not explain in vitro
changes in cell growth observed upon Cdc42 inhibition.
Interestingly, the level of Cdc42 activity was found to be significantly enhanced upon HRasV12 expression compared to non-transformed or c-Myc transformed cells, suggesting that Cdc42 can be specifically activated by oncogenic Ras. Consistent with the idea that oncogenic Ras signaling renders cells “addicted” to Cdc42 activation, HRasV12-transformed cells are exceedingly sensitive to Cdc42 deletion, while non-transformed cells or cells transformed by c-Myc overexpression are minimally affected by Cdc42 loss. This further suggests that Cdc42 dependence is restricted to certain oncogenic contexts, such as those induced upon oncogenic H-Ras activation.
In addition to changes in cell growth, we observed a dramatic change in the morphology of HRasV12-transformed cells upon Cdc42 loss, which correlated with a reduction in the activation of the focal adhesion protein, FAK. One recent study by Zheng et. al, suggested that phosphorylation of FAK downstream of Ras is inhibited to promote Ras-induced cell migration and that the reduction in phosphorylated FAK requires the activity of Cdc42 
. Interestingly, we also see a decrease in p-FAK levels following Ras transformation (); however p-FAK levels are largely absent following Cdc42 deletion. This seeming discrepancy could possibly reflect a dosing effect, as the previous study utilizes an siRNA-mediated silencing of Cdc42, which often leaves residual protein, while our targeted gene deletion approach eliminates Cdc42 signaling to a point which is not conducive to continued signaling to FAK. Alternatively, this could represent a cell-type specific effect. However, it is worth noting that, while the mechanism proposed by Zheng et al.
for Cdc42-induced FAK inhibition involved activation of ERK signaling downstream of active Cdc42, we did not observe any appreciable disruption of either MEK or ERK activation upon Cdc42 loss (Figure S4
). This observation is consistent with previous reports that demonstrated activation of JNK signaling, but not ERK signaling, upon Cdc42 activation 
While changes in the phosphorylation status of MEK and ERK were not observed in HRasV12-expressing cells following Cdc42 loss, reduction in the levels of p-Akt was evident, suggesting that Cdc42 depletion may compromise the activation of PI3K signaling downstream of activated Ras. This is consistent with reports that GTP-bound Cdc42 can interact with the p85 subunit of PI3K to modulate PI3K activity 
. Recently, Ras was reported to associate with Cdc42 at endomembranes, while Ras molecules restricted to the plasma membrane were able to maintain signaling to Raf 
. It is tempting to hypothesize that Cdc42 may modulate Ras-mediated transformation by altering signaling to PI3K at endomembranes. However, complementing Cdc42 deficient cells with a constitutively active Akt was only able to partially restore proliferation of Cdc42 deficient cells, indicating that additional mechanism may exist by which disruption of Cdc42 signaling hinders Ras-induced cell growth. Aside from discrete signaling activities of Ras localized at various cellular compartments, restriction of Ras localization also appears to impact the ability of Ras to promote transformation 
. To this end, whether Cdc42 loss impacts subcellular localization of oncogenic Ras has not been explored. Interestingly, Cdc42 has been previously shown to modulate EGFR-induced transformation by regulating its localization and turnover 
, establishing a precedence for Cdc42-mediated changes in transformation following modulation of oncogene localization and stability.
Understanding the signaling events necessary for the transforming activities of Ras is crucial for the development of novel Ras-targeted therapies. Emerging studies have begun to explore the therapeutic values of various targeting strategies outside the canonical Ras effector pathway, Raf-MEK-ERK, and emphasize the significance of identifying additional pathways either directly involved in Ras signaling or functioning as key modifiers that are required for oncogenic Ras-driven tumorigenesis 
. Whether and how Cdc42 and its related pathways contribute to Ras-mediated transformation and tumor maintenance in primary human pathologies will be an issue of further investigation. In particular, given the well appreciated cell-type specific signaling function of Cdc42 
, it can be anticipated that the proof of principle shown here may apply to specific tissue/cell types where Ras-induced transformation may be intimately dependent on Cdc42. Additionally, while the described studies specifically identify a requirement for Cdc42 in HRasV12-driven transformation, how Cdc42 might affect transformation resulting from the activation of other Ras proteins (i.e. KRAS and NRAS) commonly activated in human cancers remains to be explored.