Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome

Abstract

Background: Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non-small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting.

Methods: We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan-Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided.

Results: Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor-dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers.

Conclusions: Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.

Figures

Figure 1

Mutant KRas-Gly12Cys and mutant KRas-Gly12Val and response in refractory non–small cell lung cancer (NSCLC). Kaplan–Meier plots of progression-free survival (PFS) for NSCLC patients in the BATTLE trial by tumor KRas mutation for (A) all treatments and (B) for sorafenib-treated patients. n = a/b indicates “a” total number of events in “b” patients in each category. Data were analyzed by the log-rank test. P values are two-sided. C) Cluster analysis of microarray data from patients treated in BATTLE trial of genes that most accurately define the differences between mutant KRas-Gly12Cys or mutant KRas-Gly12Val, and other mutant KRas tumors. The false discovery rate was chosen as 0.3. Red dots indicate genes known to be involved in cell cycle regulation. Panel A: The numbers at risk at 3, 6, and 12 months were 4, 1, 0 for Cys/Val; 10, 2, 0 for other; and 67, 29, 6 for wild-type KRas groups, respectively. The corresponding PFS (95% confidence intervals [CIs]) were 0.17 (95% CI = 0.07 to 0.41), 0.04 (95% CI = 0.01 to 0.28), and NA (not applicable) for the Cys/Val group; 0.53 (95% CI = 0.34 to 0.81), 0.11 (95% CI = 0.03 to 0.39), and NA for the other group; and 0.41 (95% CI = 0.34 to 0.49), 0.18 (95% CI = 0.13 to 0.25), and 0.05 (95% CI = 0.02 to 0.11) for the wild-type KRas group. Panel B: The numbers at risk at 3, 6, and 12 months were 2, 0, 0 for Cys/Val; 6, 1, 0, for other; and 29, 12, 1, for wild-type KRas groups, respectively. The corresponding PFS (95% CIs) were 0.18 (95% CI = 0.05 to 0.64), NA (NA) for the Cys/Val group; 0.67 (95% CI = 0.42 to 1.00), 0.11 (95% CI = 0.02 to 0.71), and NA for the other group; and 0.49 (95% CI = 0.38 to 0.63), 0.20 (95% CI = 0.12 to 0.33), and 0.04 (95% CI = 0.01 to 0.21) for the wild-type KRas group.

Figure 2

Mek and Akt signaling in non–small cell lung cancer (NSCLC) cell lines expressing mutant or wild-type KRas. Reverse-phase protein array levels of (A) phospho-Mek (ser217), (B) phospho-p38 (thr180), and (C) phospho-Akt (ser473) for a panel of 67 NSCLC cell lines expressing mutant or wild-type KRas. KRas 12/13 C or V = cell lines with mutant KRas-Gly12Cys or mutant KRas-Gly13Cys (n = 13) or mutant KRas-Gly12Val (n = 1); other = cell lines with other mutant KRas (n = 9), wt = cell lines with wild-type KRas (n = 45). Horizontal bars indicate the mean value for the group. P values are two-sided (Wilcoxon rank test). D) Immunoblot analysis of a smaller panel of 11 NSCLC cell lines expressing mutant KRas with different codon 12 amino acid substitutions or wild-type KRas with Gly (G) at codon 12. C = Cys; D = Asp; R = Arg; S = Ser; V = Val.

Figure 3

Signaling pathway activation in immortalized human bronchial epithelial cells with short hairpin RNA knockdown of p53 (HBECsiP53) stably transfected with KRAS expression plasmids. HBECsiP53 cells stably transfected with empty vector (vector), or vector coding for wild-type KRas (WT), mutant KRas-Gly12Cys (G12C), or mutant KRas-Gly12Asp (G12D) were grown on a plastic surface for 100 hrs (A) or in soft agarose for 4 weeks to assess anchorage-independent growth (B). Values are the mean of three experiments; error bars represent 95% confidence intervals. P values are two-sided (analysis of variance). C) Immunoblot analysis of Akt and Mapk pathway activation in the transfected HBECsiP53 cell lines. D) Pull-down assay for active RalA and RalB in the transfected HBECsiP53 cell lines. The experiments were repeated at least three times with similar results.

Figure 4

Signaling pathway activation in NSCLC cell lines expressing mutant or wild-type KRas. A) Phospho-p70 S6K (thr389) levels measured by reverse-phase protein array in the panel of 67 NSCLC cell lines grown in medium containing 10% serum. KRas 12/13 C or V = cell lines with mutant KRas-Gly12Cys or mutant KRas-Gly13Cys (n = 13) or mutant KRas-Gly12Val (n = 1); other = cell lines with other mutant KRas (n = 9), wt = cell lines with wild type KRas (n = 45). Horizontal dotted lines indicate the mean value for the group. B) Two-way hierarchical clustering of NSCLC cell lines based on their expression of phosphorylated Akt and phosphorylated signaling proteins in related signaling pathways. Mutation type is indicated by the color bar above the heatmap: 14 cell lines with mutant KRas-Gly12Cys, mutant KRas-Gly13Cys, or mutant KRas-Gly12Val (green–blue) and 14 cell lines with other mutant KRas proteins (pink). C) Immunoblot analysis of Mapk, Akt, and p70 S6 kinase activation in transfected HBECsiP53 cells treated with the mTOR inhibitor rapamycin (0.5 μM) for 16 hours HBECsiP53 cells stably transfected with empty vector (vector), or vector coding for wild-type KRas (WT), mutant KRas-Gly12Cys (G12C), or mutant KRas-Gly12Asp (G12D). D) Immunoblot analysis of rapamycin-treated NSCLC cell lines expressing mutant KRas or mutant epidermal growth factor receptor (EGFR). The type of mutation is shown in parentheses.

Figure 5

Molecular modeling of the KRas proteins. A) KRas with critical amino acid residues (depicted in stick representations) in association with phosphatidylinositol 3-kinase (PI-3-K) (depicted as a gray surface). In mutant KRas-Gly12Asp (red), the Switch II loop is pushed away from GTP (blue) by the large side chain of the Asp residue, thereby preventing GTP hydrolysis. Wild-type KRas (green) and mutant KRas-Gly12Cys (yellow) have similar conformations of Gln61 (Q61) that are open to GTP hydrolysis. Thus, in the presence of PI-3-K, mutant KRas-Gly12Asp is more firmly locked in the GTP-bound state compared with either wild-type or mutant KRas-Gly12Cys and thus constitutively active. B) KRas in association with RaLGDS (depicted as gray surfaces), the activator of RalA and RalB proteins. KRas exists as a homodimer, with Tyr32 (Y32) of one KRas molecule interacting with the γ-phosphate (blue) of another KRas molecule. The bulky side chain Asp residue in mutant KRas-Gly12Asp results in steric clashes with Y32, which impairs dimerization and, thus, binding and activation of RaLGDS. C) Box plots of predicted binding of wild-type KRas (WT), mutant KRas-Gly12Asp (G12D), and mutant KRas-Gly12Cys (G12C) to RaLGDS. The binding scores were calculated using the ZRANK program for 60 snapshot structures from the molecular dynamics simulations. The ZRANK score estimates the relative binding energy of protein–protein interactions, with a lower score indicating tighter binding. For each box, the bottom and top lines represent the 25th and 75th percentiles, respectively, and the horizontal line represents the median (50th percentile) of the ZRANK scores. The error bars represent the range from the minimum to the maximum of all ZRANK scores collected based on the 60 snapshot structures from the molecular dynamics simulations. D) Comparison of predicted binding of the various forms of KRas to RaLGDS with predicted binding of KRas to PI-3-K. The height (y-axis) of each panel represents the ratio of averaged binding of KRas to PI-3-K over binding to RaLGDS in terms of ZRANK scores. Error bars represent the standard error of the ratios (the corresponding 95% confidence intervals are 0.706 to 0.728 for the WT prediction, 0.723 to 0.751 for the G12D prediction, and 0.599 to 0.619 for the G12C prediction).

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) activation of Akt signaling (acting through PI-3-K), RalA and RalB signaling (acting through RaLGDS), and Mek signaling (acting through c-Raf). Forward transmission of signals is represented by arrows. Solid lines show established pathways, and dashed lines represent possible pathways. P70 S6 kinase (p70S6K) is activated and exerts feedback inhibition on GFR activation of Akt. The thickness of the lines indicates the strength of the feedback inhibition: weak inhibition by mutant KRas-Gly12Asp, moderate inhibition by wild-type KRas, and strong inhibition by mutant KRas-Gly12Cys.