Currently, the most vigorously pursued anti-Ras approaches are inhibitors of the Raf-MEK-ERK or PI3K-AKT effector signaling (6
). However, these efforts are complicated by the likelihood that Ras-mediated oncogenesis involves these and other effector pathways. In this study, we extended our previous evaluation of MEK inhibitors (25
) and concluded that KRAS
mutation status but not pERK activity may be a marker to define selumitinib resistance in CRC. Although, pAKT activity was weakly associated with inhibitor insensitivity, PIK3C
A mutation status was not. We also found Ral activation in CRC cell lines and tumors. However, in contrast to our observations in KRAS
mutant PDAC, where RalA but not RalB promoted PDAC anchorage-independent and tumorigenic growth, we found that RalA and RalB exhibited opposing roles for CRC anchorage-independent growth. These results reveal the striking cell context functional differences that these GTPases may have in KRAS
Our analyses with selumetinib reached the same conclusion as we did with other MEK1/2-selective inhibitors (25
); pERK activation did not reliably predict MEK inhibitor sensitivity. However, we did find a different pattern of sensitivity to selumetinib when compared to U0126 and CI-1040. Whereas we found previously that a subset of KRAS
mutant CRC cells did exhibit sensitivity to U0126 and CI-1040, we saw that all KRAS
mutant CRC lines were resistant to treatment with selumetinib. Perhaps this different activity reflects the more specific nature of this MEK1/2 inhibitor and different off-target activities of the other inhibitors (e.g., MEK5-ERK5 inhibition). Also, one potential caveat to our analyses is that MEK inhibitory activity was determined on adherent cultures, whereas growth inhibitory activity was determined in nonadherent three-dimensional colonies. One recent study found that KRAS
mutation status did not correlate with selumetinib sensitivity, but did find that inhibitor resistance correlated with weak ERK and/or strong AKT activity (27
). Consistent with their findings, we did find elevated pAKT in all KRAS
mutant CRC cell lines and a weak association of elevated pAKT with selumitinib resistance. Although KRAS
mutant cell lines showed partial sensitivity to PI3K inhibition, we found that concurrent PI3K inhibition did not further enhance MEK inhibitor sensitivity. Our results are consistent with another recent study where selumetinib response did not correlate with RAS
mutation or PI3K activation (41
). Our results support the need to assess the importance of other effectors in RAS
We previously observed a striking requirement for RalA but not RalB for the anchorage-independent and tumorigenic growth of PDAC cell lines (18
). In the present study, we found that RalA was also necessary for CRC anchorage-independent growth for both KRAS
mutant cell lines. Surprisingly, stable suppression of RalB caused a significant enhancement of soft agar colony size and colony forming efficiency. These results extend previous findings of striking functional differences with the related RalA and RalB isoforms (19
), and additionally reveal a significant RalB functional difference in KRAS
mutant tumor cells that arise from different tissues. While we do not have a mechanistic explanation for this cell context difference, it may reflect differences in RalB subcellular localization or posttranslational modifications, leading to different activation of effectors, in each tumor type.
The different functional roles of RalA and RalB in the growth of different tumor types complicate the issue of whether isoform-selective or pan-Ral therapeutic approaches will be the most effective. For five of six KRAS
mutant CRC cell lines, we found that concurrent suppression of both RalA and RalB resulted in statistically insignificant reduction in colony formation when compared to the control shGFP cells. These results contrast with previous studies in different cancer types where the phenotype of RalA is dominant over that of RalB (19
). These observations argue that a RalA-selective therapeutic approach may be the best approach for inhibiting the growth of CRC and PDAC cells. However, we also found that RalB was necessary for PDAC Matrigel invasion and lung colonization metastasis (18
). Whether RalB loss will promote CRC invasion and metastasis will need to be established to better understand the consequences of RalA and RalB ablation for tumor growth in the CRC patient.
Our results with sustained RalB suppression differ from previous studies where transient RalB suppression caused CRC apoptotic cell death (19
). When we evaluated transient RalB inactivation, we also observed cell death (data not shown). We suspect that with sustained suppression of RalB, compensatory events occur to offset the initial deleterious consequences of RalB loss. Consistent with this possibility, we observed a modest 1.3- to 1.5-fold increase in the steady-state level of RalA-GTP was increased by RalB suppression in KRAS
mutant CRC lines that may contribute to the enhancement of growth. However, we suspect that additional more significant compensatory events must also contribute. In contrast, we observed a 59- to 70-fold increase in RalB-GTP levels by RalA suppression in KRAS
mutant cells. Our observation that steady-state expression of constitutively activated RalB impaired CRC growth (data not shown) argues that this increase contributes to RalA suppression-associated growth inhibition. Since it is likely that targeted therapies focused on signal transduction molecules will require chronic therapy to maintain persistent suppression of target activity, we believe that our observations with sustained Ral suppression are relevant and important for understanding the potential consequences of Ral targeted therapies for CRC treatment.
In light of our observed opposing functions of sustained RalA and RalB depletion in CRC anchorage-independent growth, we were surprised to find that both RalA and RalB activities were dependent on RalBP1 binding. Since RalA and RalB exhibit different subcellular localizations, perhaps each GTPase engages RalBP1 in spatially-distinct locations, leading to distinct cellular outcomes. Interestingly, suppression of RalBP1 also reduced soft agar growth, indicating that its role in RalA function is dominant over its role in RalB function. In any case, our implication of RalBP1 in Ral-dependent oncogenesis contrasts with other studies where RalBP1 has not been involved. Furthermore, while both RalA and RalB required association with exocyst components to regulate CRC growth, RalA required association with Exo84 but not Sec5 whereas RalB required Sec5 but not Exo84 binding. One possible explanation for this result is that the differential requirements for Sec5 and Exo84 are unrelated to exocyst function. Certainly for Sec5, one exocyst independent function involves the TBK1 protein kinase (22
). Similarly, it was suggested that Exo84 also exhibits an exocyst-independent function required for growth transformation (42
). That suppression of Exo84 or Sec5 expression both reduced soft agar growth may reflect both Ral-dependent and –independent functions.
In summary, our results, while supporting the value of targeting Ral GTPases for KRAS mutant CRC, also indicate that Ral targeted therapies may need to be tailored differently for different cancers. For example, since we found that RalB was important for PDAC invasion and metastasis, a RalB-selective therapy may be ideally suited for advanced PDAC. In contrast, a RalB-selective therapy may enhance CRC tumor growth. Future studies with genetic ablation of RalA or RalB in KRAS-driven mouse models of PDAC and CRC will provide a more comprehensive understanding of the most effective approach for Ral inhibition for cancer treatment.