This report offers genetic proof of the hypothesis that RhoB alteration is central to the antineoplastic mechanism engaged by FTI treatment. Previous work implicated the increased expression of RhoB-GG elicited by FTIs as a sufficient cause for growth inhibition in transformed rodent cells and human carcinoma cell lines (9
). In this study, we showed that RhoB-GG is necessary for FTI-induced apoptosis and crucial for antitumor efficacy in a xenograft model. The results indicated that RhoB-GG also has a necessary role for efficient growth inhibition by FTI under anchorage-dependent conditions. However, RhoB-GG was dispensable for inhibition of anchorage-independent growth, implying that if RhoB is absent the alteration of another protein(s) (possibly H-Ras in this model) could also provide a sufficient event for this process. One question addressed here was whether the engineered RhoB-GG construction previously used to establish RhoB-GG as a sufficient condition for growth inhibition and apoptosis by FTI accurately mimicked a bona fide cellular RhoB-GG (the construction had included a small number of RhoA-derived residues). The finding that loss of cellular RhoB-GG significantly blunts the FTI response argues against the concern that the engineered construct acted through a nonspecific rather than specific gain-of-function mechanism. Some differences in the apoptotic potency of exogenous engineered RhoB-GG versus that of the endogenous isoform were noted. Nevertheless the data supported the conclusion that RhoB-GG has proapoptotic activity in transformed cells.
Despite the close structural similarity between RhoB-GG and RhoA, it was clear that RhoA could not complement losses in RhoB-GG, because RhoA was expressed similarly regardless of rhoB
genotype. As mentioned above, the exact basis for the growth-inhibitory and apoptotic properties of RhoB-GG is unclear, but we favor the interpretation that mislocalization of this isoform in FTI-treated cells (25
) is key to understanding its action. We previously suggested that loss of the farnesylated RhoB isoform (RhoB-F) may have some role in the FTI response but this study argues strongly against this possibility. In summary, our observations provide direct evidence that RhoB-GG elevation by FTIs is not only sufficient but also necessary for apoptosis and a robust antineoplastic response.
A significant finding of this study was that the apoptotic defect of −/− cells treated with FTIs was correlated with a loss of antitumor efficacy in vivo. This observation implied that RhoB-GG was needed to mediate efficient tumor suppression because it was needed to drive apoptosis. One question concerns the strength of the apoptotic response of E1A- plus Ras-transformed MEFs in vivo, which differed from the more-limited apoptosis that occurs in other xenograft models that have been published. To our knowledge, this is the first study in which oncogene-transformed primary cells were tested. Primary cells have greater sensitivity to apoptosis, especially anoikis (which may be crucial in in vivo settings), than established lines which have been examined previously in xenograft assays. Moreover, this greater sensitivity is retained in oncogene-transformed primary cells (31
). In addition, since E1A “epithelializes” cells (13
), the more pronounced apoptotic response to FTI that was seen in the MEF model might reflect a sensitization of the model to anoikis, which is characteristic of epithelial cells but not fibroblasts. In any case, while accentuated, apoptosis of murine tumors in response to FTI is not unique, insofar as it has been seen in a variety of transgenic models (3
Dominant inhibitory genetic methods have argued that Rho functions are required for efficient transformation of Rat1 cells by activated H-Ras (38
), but we found that rhoB
null MEFs were fully susceptible to cotransformation by adenovirus E1A plus activated H-Ras. This susceptibility could mean either that RhoB is dispensable in this context or that E1A complements a RhoB requirement. An additional possibility is that RhoB may have a Janus or modifier role, perhaps determined by positive- or negative-acting cell surface receptors relevant to different cellular contexts. In any case, it is clear that RhoB has nonredundant functions, insofar as other ubiquitous and even highly related Rho proteins such as RhoA cannot complement the effects of RhoB deficiency. To further investigate the function of RhoB in cell transformation and growth regulation, we are generating 3T3 cells for analysis as well as comparing the susceptibilities of rhoB
null mice to 7,12-dimethyl-benz[a]anthracene-induced skin carcinogenesis, which involves Ras activation as the initiating step (41
Concerning the relevance of genetic context, it is worth noting that mutant K-Ras and mutant H-Ras have similar abilities to sensitize fibroblasts to apoptosis by FTIs (10
). Therefore, we believe that the proapoptotic action of FTI and RhoB-GG is unlikely to be a specific feature of H-Ras in the model. Nevertheless, it will be interesting to learn whether loss of RhoB compromises the FTI response in K-Ras-transformed cells and cells with other genetic backgrounds. K-Ras- plus E1A-transformed MEFs might be expected to differ in growth suppression but to remain similarly susceptible to apoptosis, given the observations of Suzuki and colleagues with K-Ras-transformed NRK cells (44
). The effects of rhoB
deletion on apoptosis in different genetic backgrounds will be important since levels of reliance on Rho signaling pathways may differ in such backgrounds. Crossing rhoB
nullizygous animals with animals carrying various oncogenes is one way in which FTI susceptibilities to apoptosis, growth suppression, and tumorigenesis could be addressed in future work.
We observed that RhoB-GG promoted apoptosis in a manner that was not correlated with inhibition of endogenous Akt activity in E1A-plus Ras-transformed MEFs. These observations were consistent with previous evidence that FTI-induced apoptosis is not associated with Akt inhibition (10
). However, this issue deserves careful examination in a variety of models because of evidence that FTIs may influence Akt activity in certain settings. The RhoB effector kinase Prk has been found to associate with and regulate the activity of Pdk1, the kinase responsible for activating Akt following its membrane recruitment (2
). For reasons that remain unclear, in FTI-treated cells RhoB-GG is mislocalized away from the endosomal site where RhoB is normally found (25
). Therefore, RhoB-GG may sequester RhoB effector signaling molecules such as Prk away from the endosome. If so, this sequestration could impact the activity of Pdk1 and Akt, due to the ability of Prk to regulate Pdk1. While this biochemical linkage must be considered tentative at this time, it is interesting that one recent study has suggested that FTI may promote apoptosis in certain human epithelial tumor cell lines by inhibiting the Akt-2 isoform (21
). In transient expression assays of COS epithelial cells, we have observed that both FTIs and RhoB-GG can inhibit Akt-1 activation by mutant Ras or epidermal growth factor (A.-X. Liu and G. C. Prendergast, submitted for publication). These observations are consistent with a potential Prk-Pdk1 connection and raise the possibility that cell type or genetic background may dictate the mechanism through which RhoB-GG acts. However, the physiological relevance of these observations remains to be established, since the weight of the evidence to date suggests that FTIs do not influence endogenous Akt-1 activity in transformed Rat1 or MEF cells under conditions where the drugs can induce apoptosis. Recognizing the above caveats, we interpret our findings to mean that RhoB-GG can mediate the proapoptotic effects of FTIs through an Akt-1-independent mechanism.
Apoptosis would be expected to be an important component of any successful clinical application of FTIs. In this regard, we note that the results of preclinical experiments and human clinical trials to date suggest strongly that FTIs will need to be applied combinatorially with other agents to achieve efficacy (i.e., cell death). We have reported that inhibitors of cell adhesion or phosphatidylinositol 3′-kinase cooperate with FTI to efficiently kill Ras-transformed cells (10
). In addition, others have shown that FTIs synergize with classical cytotoxic cancer therapeutics and irradiation (4
). Based on this and other studies, we propose that RhoB-GG provides a crucial signal(s) needed to mediate the synergistic effect of FTI when combined with other modalities. In support of this proposal, we have observed that E1A- plus Ras-transformed −/− cells are resistant to apoptosis induced by irradiation or doxorubicin treatment and that they lack susceptibility to FTI-induced sensitization to these treatments (A.-x. Liu and G. C. Prendergast, unpublished observations).
In closing, we remain intrigued by the connections between Rho and apoptosis (5
) and how the organization of integrins and the actin cytoskeleton regulated by Rho impact the antineoplastic response to FTIs. We have suggested that the means by which FTIs cause cell death ultimately involve partial or abortive responses of the transformed cell to induction of some integrin-dependent process (27
). Normal cells would be immune to this process since they already exist in a state where such processes are fully operative. Since RhoB-GG induces actin stress fiber formation, it is tempting to speculate that it acts by facilitating the organization of focal adhesions and integrins in malignant cells, leading to a conflict with oncogene-mediated signals which suppress this organization. As mentioned, since focal adhesions and integrins are already in a highly ordered state in normal cells, this model offers an appealing explanation for why FTIs do not affect normal cells (35