The recently completed BATTLE trial for patients with refractory NSCLC receiving either erlotinib, vandetanib, bexarotene and erlotinib, or sorafenib found that mutant KRAS
was not associated with overall survival in any of the treatment groups (10
). We reanalyzed the BATTLE data to examine associations between mutant KRas with specific amino acid substitutions and patient survival and found that either mutant KRas-Gly12Cys or mutant KRas-Gly12Val was associated with decreased progression-free survival compared with other mutant KRas or wild-type KRas. This is the first study to our knowledge to show an association between mutant KRas amino acid substitutions and response to molecularly targeted therapy in NSCLC.
There is already evidence that mutant KRas amino acid substitution could determine patient response. A study in colon cancer patients receiving EGFR inhibitors revealed that tumors with mutant KRas-Gly12Cys or KRas-Gly12Val are associated with rapid tumor progression and decreased patient survival compared with tumors with other mutant KRas proteins (mostly Gly12Asp) or wild-type KRas (6
). A recent study has confirmed that different amino acid–substituted mutant KRas may also affect drug sensitivity of NSCLC patients (22
Dysregulation of the cell cycle is a well-characterized feature of KRas-mediated tumorigenesis (23
). Our analysis of tumor transcriptome microarray data from patients in the BATTLE trial provides the first indication to our knowledge that cell cycle signaling differs between the different forms of mutant KRas. Specifically, we found that expression of the cell cycle regulators PLK1, cyclin B, and cyclin E was decreased in tumors with mutant KRas-Gly12Cys or KRas-Gly12Val compared with tumors with other mutant KRas proteins. Our RRPA analysis of a panel of genetically characterized NSCLC cell lines revealed that compared with cell lines expressing wild-type KRas, those with mutant KRas-Gly12Cys or Gly12Val had decreased levels of phosphorylated Akt, whereas those with other mutant KRas proteins had elevated levels of phosphorylated Akt. Transfection of immortalized, p53-deficient human bronchial epithelial HBECsiP53 cells with different mutant KRas expression plasmids also showed that overexpression of mutant KRas-Gly12Cys decreased phospho-Akt levels and increased Ral activation, whereas overexpression of mutant KRas-Gly12Asp increased phospho-Akt levels and decreased Ral activation compared with cells overexpressing wild-type KRas. Moreover, we found that HBECsiP53 cells overexpressing mutant KRas-Gly12Cys had lower Akt signaling, elevated Ral signaling, and increased anchorage-independent growth compared with HBECsiP53 cells overexpressing wild-type KRas. These findings are in agreement with a previous study (25
) showing that Ral activation preferentially induces anchorage-independent growth in human cells, whereas Akt or Mek activation has only modest effects. These findings are also consistent with previous observations that when mutant KRas proteins were expressed in the lungs of mice, mutant KRas-Gly12Asp induced Raf and Akt activation (26
), whereas mutant KRas-Gly12Cys resulted in Raf and Ral activation but minimal Akt activation regardless of the expression level of the mutant KRas protein (27
Our data showed that mTOR may mediate some of the effects of different amino acid–substituted mutant KRas. We found that many NSCLC cell lines that had minimal Akt activation showed robust activation of the mTOR effector proteins p70 S6 kinase and 4E-BP. We found that inhibition of mTOR with rapamycin in NSCLC cells with mutant KRas-Gly12Cys resulted in decreased expression of p70 S6 kinase and an increase in phospho-Akt levels. The translational regulator mTOR is activated by Akt signaling, in addition to being activated by the Mapk and Ral signaling pathways (21
). In addition, p70 S6 kinase downstream of mTOR has been shown to exert a regulatory feedback response that restricts growth factor signaling to the Akt pathway (29
). Our results suggest that PI-3-K/Akt signaling is constitutively activated by mutant KRas-Gly12Asp and not subject to mTOR inhibition, whereas in cells expressing wild-type KRas or mutant KRas-GlyG12Cys, Akt activation is growth factor dependent and inhibited by mTOR.
KRas is known to interact with different downstream effectors by undergoing large conformational changes in the switch I and switch II regions of the protein surrounding codon 12 and 13 amino acids (15
). Our molecular modeling studies showed that mutant KRas-Gly12Cys likely weakens the interaction with PI-3-K, whereas the bulky Asp of mutant KRas-Gly12Asp causes steric interference of KRas homodimer formation and RaLGDS binding, which is not seen with mutant KRas-Gly12Cys. These modeling results are in agreement with the results of our cellular studies showing that mutant KRas-Gly12Asp activated PI-3-K and Mek signaling, but not Ral, whereas mutant KRas-Gl12Cys fails to activate PI-3-K signaling.
The findings of this study showing different mechanisms for signaling through wild-type KRas, mutant KRas-Gly12Asp, and mutant KRas-Gly12Cys are summarized in . Wild-type KRas activation results in signaling through Mek. Wild-type KRas has also been shown to activate Akt and RalA/B, although the conditions that dictate which pathway will be activated remain uncertain at this time. The Akt, Mek, and RalA/B pathways have all been shown capable of activating mTOR and its effector p70 S6 kinase, which serves as a negative regulator of growth factor receptor–regulated signaling to Akt. Other KRas effectors are likely also capable of regulating p70 S6 kinase. The KRas-Gly12Asp mutation preferably activates Akt signaling, making growth factor receptor Akt activation unnecessary and p70 S6 kinase inhibition irrelevant. In contrast, the KRas-Gly12Cys mutation preferentially activates RalA/B signaling over Akt and is able to suppress Akt activation through an Akt-independent activation of p70 S6 kinase. Other KRas effectors may also play a role in the signaling, but we have not considered them here.
Figure 6 Proposed pathways of signaling by wild- type KRas, mutant KRas-Gly12Asp, and mutant KRas-Gly12Cys showing membrane growth factor receptor (GFR) or wild-type KRas (wt-KRas), mutant KRas-Gly12Asp (mut-KRas-G12D), and mutant KRas-Gly12Cys (mut-KRas-G12C) (more ...)
Our observation that the substitution of different amino acids induces heterogeneous behavior in the KRas protein resulting in different signaling outputs has profound implications for identifying and treating KRAS-driven tumors. For example, it may be necessary to use different combinations of downstream signaling inhibitors when treating tumors with different mutant KRas amino acid substitutions.
This study focused on the major forms of mutant KRas in NSCLC, namely Gly12Cys (and Gly12Val) and Gly12Asp, and on the downstream signaling pathways involving PI-3-K/Akt, Mek, and Ral. We do not know how other forms of mutant KRas activate the pathways nor do we know how other known KRas downstream pathways [eg, Tiam1/Rac, PLCϵ/PKC, and Rassf1 (9
)] may be affected by mutant KRas. Consequently, our findings that mutant KRas-Gly12Cys and mutant KRas-Gly12Val are associated with overall decreased patient progression-free survival compared with other forms of mutant KRas or wild-type KRas may only be applicable to the types of molecularly targeted agents used in the BATTLE study, which focused broadly on EGFR, VEGF, PI-3-K, and Mek signaling. Future studies will need to explore the role of mutant KRas and signaling pathways in the context of other agents in individual clinical trials.