Members of the Rho family of GTP-binding proteins act as molecular switches, cycling between inactive GDP-bound and active GTP-bound states [1]. The conversion between these states is regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP to increase Rho activation [2], and GTPase-accelerating proteins (GAPs) that increase the rate of Rho-catalyzed hydrolysis of the bond between β and γ PO₄ groups in GTP by providing a catalytic “arginine-finger” resulting in Rho inactivation [3], [4]. In their GTP-bound active state, information is conveyed downstream of Rho family proteins through interactions with effector proteins whose intrinsic activity, subcellular localization and/or association in multimeric protein complexes are altered so as to affect signaling flux [5], [6]. Although best known as central regulators of the actin cytoskeleton and cell morphology [7], Rho GTPases influence a myriad of additional biological processes such as cell proliferation via actin-dependent and actin-independent mechanisms [8]. In contrast to the relatively frequent incidence of activating mutations in Ras genes (K-Ras, H-Ras and N-Ras [9]), there are no published reports of activating Rho mutations in human tumors. However, numerous lines of research indicate that Rho proteins do indeed contribute to cancer [5], [6], [10].

Amongst the Rho family, the most thoroughly-characterized are RhoA, Rac1 and Cdc42. To some extent this reflects historical factors and yet in many cases these specific proteins do appear to be vitally important for a number of functions. Early studies using in vitro cell culture model systems revealed a contribution of Rac1 to cell cycle regulation [11] and in Ras-mediated transformation [12], [13]. However, given that the reagents used to inhibit endogenous protein signaling were dominant-negative versions of Rac1 that are encumbered by concerns about their specificity, the conclusions of these studies may not be adequately robust [14]. Finally, evidence for a dependence of Ras on Rac1 for biological functions accompanied by evidence of Ras-induced Rac1 activation in vivo has remained elusive.

Genetically-modified mouse models that allow for the conditional and tissue-selective deletion or activation of specific signaling proteins are now readily available [15]. In this study, we made use of a strain of mice containing a mutant K-Ras allele (K-Ras^G12D) that can be conditionally activated by Cre-recombinase mediated excision of a floxed transcription termination (LSL) cassette [16]. It had previously been shown that K-Ras driven lung tumors were dependent upon Rac1. However, whether Rac1 was actually activated by K-Ras or merely necessary at a basal level of activity for K-Ras induced effects was not determined [17]. By crossing LSL-K-Ras^G12D mice with those expressing a Cre-estrogen receptor (Cre:ER) fusion protein under the transcriptional regulation of the Cytokeratin 14 (K14) promoter, mutant K-Ras^G12D expression was induced in murine skin, which resulted in highly penetrant and rapid development of benign hyperplastic oral papillomas. Using a conformation sensitive monoclonal antibody specific for the GTP-bound form of Rac1, we found that these growths had high levels of active Rac1. Genetically removing one Rac1 allele significantly impaired K-Ras induced oral papilloma growth and promoted survival while reducing levels of active Rac1 within the papillomas. These data demonstrate that there is a general dependence of K-Ras on Rac1 to drive proliferation in epithelial tissues in vivo and reveal that this requirement is a reflection of Rac activation by K-Ras. Given that inhibitors are being developed to target Rac effector proteins such as PAK kinases [18], [19] or to block Rac activation [20], these results provide compelling target validation of Rac1 in Ras-driven cancers.

The original intent of this study was to create a genetically modified mouse model in with mutant oncogenic K-Ras^G12D would be conditionally expressed following the activation of Cre:ER protein by topical 4HT administration. However, it appears that activity of the gene product from the K14-Cre:ER transgene was insufficiently repressed in the absence of ligand, resulting in spontaneous deletion of the LSL cassette and subsequent papilloma development. The effects were likely the consequence of relatively rare events, with the potency of K-Ras^G12D in driving keratinocyte hyperproliferation making each occurrence abundantly evident.

The link between Ras and Rac was first proposed following observations that microinjection of recombinant Ras protein produced membrane ruffles which could be blocked by a form of Rac1 deficient in GTP binding said to act as a “dominant-negative” inhibitor [22], [23]. The requirement of Rac1 activity for Ras-mediated oncogenesis was initially reported in 1995 [12], [13], using transformation of NIH 3T3 mouse fibroblasts as the model system and dominant-negative Rac1 as the inhibitor. Further support for Ras activation of Rac came from pull-down assays in which the relatively greater affinity of effector protein Rac-binding domains (RBD) for GTP-bound over GDP-bound was used to selectively enrich for active Rac1 from cell lysates [24], [25]_.

Despite the level of interest in the association of Rac activity with mutant Ras in cancer, there is no data showing that Ras-driven tumors from either mouse or clinical studies are associated with elevated Rac1 activity. One reason is that the methods used to demonstrate Ras-induced Rac activation in vitro (e.g. membrane ruffling, pull-downs) cannot be directly converted to use with FFPE tumor samples. A pull-down reagent comprised of the Pak1-RBD was used as an affinity probe in immunohistochemistry experiments to demonstrate Rac activation in FFPE fixed mouse mammary tumors driven by Neu and TGFβ1 [26]. However, given that there have been few attempts to replicate this method (e.g. reference [27]), it does not appear to be easily duplicated. As a result, it would be highly advantageous if a method were developed to detect active Rac1 in tumors, which could be readily deployed using standard immunohistochemistry techniques. We found that an activation-state sensitive Rac1-specific monoclonal antibody performed as a versatile reagent that could immunoprecipitate active Rac1 and detect active Rac1 by immunofluorescence in PDGF-stimulated NIH 3T3 fibroblasts. More importantly, in FFPE sections this antibody was able to detect increased active Rac1 in K-Ras driven oral papillomas relative to normal tissue, which was reduced by the loss of one Rac1 allele. Interestingly, loss of one Rac1 allele did not reduce Rac1 activation within papillomas by 50%, consistent with the requirement for a threshold level of Rac1 activation for papillomagenesis. Our observation that the reduction in Rac1 activation was relatively subtle in Rac^Wt/- tumors compared to Rac^Wt/Wt tumors, may reflect a positive selection for cells with the greatest levels of activated Rac. In addition, the reduced tumor growth may, at least in part, be a consequence of a lower proportion of cells in the Rac^Wt/- tumors being permissive for K-Ras driven hyperproliferation, resulting in an apparent lag that delayed tumor growth. The findings presented in this study could be used to help define the tumor types and potential patient population that would benefit from therapies that target Rac activation or downstream effector signaling pathways.