The products of the Ras proto-oncogenes as well as the retinoblastoma tumor suppressor gene have been implicated in the regulation of a variety of cellular processes. Even though both Ras and pRB have each been placed in somewhat linear pathways, it is becoming increasingly clear that the signals that govern cellular processes such as entry and exit from the cell cycle, differentiation, and apoptosis are under the control of complex regulatory networks rather than simple linear pathways. In addition, these pathways are likely to be wired differently in different tissue contexts, and in various tumor types.
Although many human tumors exhibit deregulation of pRB pathway components, in general, a relatively small subset of tumor types exhibit pRB loss. Several previous studies have led to the suggestion that activated Ras cooperates with a disruption of the RB pathway in tumorigenesis. However, in those cases, RB pathway disruption does not involve the direct loss of the pRB protein, but instead involves either loss of the Cdk inhibitor p16, amplification of cyclin D1, or mutation of cdk4 (33
). Although it is commonly accepted that this is a linear pathway, it is not entirely clear that cyclin D amplification or p16 loss is equivalent to pRB loss in oncogenesis. In fact, our findings suggest that the expression of the pRB tumor suppressor protein is important for the proliferation of human tumor cells that harbor an activated Ras pathway and is not equivalent to loss of p16 or activation of cyclin D in this setting.
It has been previously suggested that Rb
-deficient tumors have increased levels of active, GTP-bound Ras (41
), which could bypass the requirement for Ras activation in these tumors. Our results (Fig. to ) suggest an additional explanation for the absence of Ras mutations in pRB-deficient tumors since activation of Ras has an inhibitory effect on the proliferation of pRB-deficient cells. The phenomenon that tumors carrying Ras mutations typically maintain an intact Rb
gene and, in some cases, exhibit very high levels of pRB expression (2
) has been explained by experiments in which it was shown that activation of Ras results in increased levels of cyclin D1 (21
). The higher levels of cyclin D in these tumor cells would result in increased activity of cdk4 and cdk6, which according to current models inactivates pRB function. Thus, if constitutive activation of Ras results in inactivation of the pRB protein there would be no pressure to lose the Rb
gene in tumors harboring an activated Ras pathway. However, applying the same logic, one can imagine that there should be no pressure to lose p16 in tumors where cyclin D expression is up-regulated by activating mutations in the Ras pathway; nevertheless, p16 expression is frequently lost in tumors harboring Ras mutations.
Also, we and others find that p16 overexpression (50
) (data not shown) is inhibitory when expressed in colorectal tumor cells, whereas pRB overexpression is not (Fig. ). In addition, depletion of pRB in tumors harboring Ras pathway mutations would be inconsequential, whereas our studies demonstrate that depletion of pRB surprisingly results in decreased proliferation in these tumor cells (Fig. and ). Thus, loss of p16 and loss of pRB clearly has distinct consequences for cells in the context of Ras transformation. It is also worth noting that our studies show that activated Ras, in addition to increasing the levels of cyclin D, stimulates the expression of pRB, indicating that the pool of active pRB is not significantly affected by Ras activation.
Taken together, our findings imply that although p16, cyclin D, cdk4, and pRB are largely regarded as components of the same linear pathway, loss of p16 expression or gain of cyclin D kinase activity is not equivalent to losing pRB in the context of activated Ras. Importantly, pRB is not the only cellular substrate of cyclin D-dependent kinases, and our data in murine cells suggests that the other members of the pRB family, p107 and/or p130, may be the relevant substrates in the presence of activating mutations in the Ras pathway. Notably, it has been shown that, in addition to pRB, there is a requirement for p107 and p130 in p16-induced arrest (5
), suggesting that p107 and p130 are important downstream targets in p16-induced proliferative arrest.
Although it is generally believed that the cooperativity between activated Ras and E1A (and other viral oncoproteins) is associated with their ability to bind and neutralize the actions of pRB, our data also suggest that p107 and p130 might be important targets for these viral oncoproteins in this context. Significantly, a recent study has demonstrated that simian virus 40 infection of growth-arrested monkey kidney epithelial cells results in the specific disruption of p130-E2F and p107-E2F complexes, whereas pRB-E2F complexes are not affected (66
). Previous experiments have also found evidence to suggest that the targeting of all three members of the pRB family of proteins is required for the cooperativity between Ras and simian virus 40 T antigen (49
Overall, our results are consistent with a requirement for pRB for the full transforming activity of H-RasV12
and suggest that the ability of E1A or p16 to target p107 and/or p130 is in fact its relevant oncogenic function in the context of its ability to cooperate with activated Ras. Taken together, these observations raise the possibility that in the context of an activated Ras pathway, loss of p16, similar to the expression of viral oncoproteins, is equivalent to inactivation of all three members of the pRB family of proteins (Fig. ). It may also be interesting to speculate that the requirement for pRB in Ras-mediated transformation is tumor specific and does not occur in primary cells where Ras has been show to induce premature senescence (53
). The effects we see may require mutations in other pathways, such as the p53 pathway, which is mutated in most tumor cells and extensive cross talk has been seen between the p53 and pRB tumor suppressor pathways (for a review see reference 45
). Finally, it is formally possible that there are rare tumors that harbor both Ras and Rb
mutations, but which compensate for the Rb
loss through disruption of p107 and/or p130 function or activation of oncogenes that will overcome the negative effect that losing pRB would have on these tumors.
FIG. 8. Loss of pRB protein is distinct from the “pRB pathway” disruption in the context of activated Ras. Taken together, our results suggest that the ability of viral oncoproteins and cyclin D-dependent kinases to target p107 and/or p130 is (more ...)
Our collective observations also point to a clear functional distinction between the closely related pRB and p107 proteins in the presence of activated Ras, whereas in other contexts, these proteins seem to exhibit substantial functional redundancy (14
). The pRB-mediated repression of p107 expression seen in colorectal tumor cell lines provide a potential mechanistic explanation for the requirement for pRB in Ras-mediated oncogenic transformation. Furthermore, the strong inhibitory effect that p107, but not pRB, exerts on the proliferation of tumor cells harboring an activated Ras pathway (Fig. ) suggests that p107 provides a tumor-suppressive activity in this context.
In conclusion, the finding that loss of the Rb
tumor suppressor leads to a proliferative disadvantage in tumor cells harboring activating mutations in the Ras pathway is a previously unrecognized and “counterintuitive” relationship between two of the most intensively investigated cancer genes and highlights the context-dependent nature of oncogene and tumor suppressor function. These findings are particularly surprising when considering that pRB is widely recognized as a proliferation inhibitor, and potentially explain why human tumors do not exhibit simultaneous loss of pRB and activating Ras mutations (35