(A) Negative feedback regulators differentially expressed between clinical LUADs with or without EGFR or KRAS mutations (as indicated in green or blue, respectively, in the third and second horizontal bars). Expression levels for the indicated genes as determined by RNA-seq were compared between LUAD tumors with (n = 107, red) and without (n = 123, black) KRAS or EGFR mutations. In the heatmap, red indicates high relative expression and blue, low expression. Significance, as determined by two-tailed unpaired t-test with Bonferroni multiple testing correction, is indicated as the –log2(p-value). The significance threshold was set at a p-value < 0.01 and is indicated by the dotted line. Only DUSP6 surpassed this threshold. (B) DUSP6 is the main negative feedback regulator upregulated in LUADs with EGFR or KRAS mutations. Box plots show levels of DUSP6 RNA from samples in A. LUADs with EGFR or KRAS mutations (n = 107) express DUSP6 at higher levels than do LUADs with wildtype KRAS and EGFR (n = 123) in the TCGA dataset. (C) Validation of increased DUSP6 expression in LUADs with mutated KRAS or EGFR. In an independent internal dataset from the BCCA, LUADs with EGFR or KRAS mutations (n = 54) demonstrated higher expression of DUSP6 compared to LUADs in which both EGFR and KRAS were wild-type (n = 29) and to normal lung tissues (n = 83). (D) Dusp6 is upregulated in the lungs of mice with tumors induced by mutant EGFR or Kras transgenes. Tumor-bearing lung tissues from mice expressing EGFR or Kras oncogenes produce higher levels of Dusp6 RNA than do normal lung controls or tumor-bearing lungs from mice with a MYC transgene. (E) Increased DUSP6 RNA is specific to cells with oncogenic signaling through RAS. Human primary epithelial cells expressing a HRAS oncogene (n = 10 biological replicates) express DUSP6 at higher levels than control cells producing GFP (n = 10 biological replicates) whereas cells expressing known oncogenes other than RAS genes (MYC, SRC, B-Catenin, and E2F-3) do not. (F) DUSP6 RNA levels increase in PC9, H358 and H1975 cells expressing mutant KRAS. Dox was added to induce either GFP or the KRASG12V oncogene for 24 hr; DUSP6 RNA was measured by qPCR. (G–I) DUSP6 expression is associated with P-ERK levels. (G) LUADs with EGFR or KRAS mutations (n = 107) have higher P-ERK levels, but not P-p38 or P-JNK levels, than LUADs with wildtype KRAS and EGFR (n = 123) in the TCGA dataset. (H) LUADs with the highest DUSP6 RNA levels (n = 46) demonstrated higher P-ERK levels, but not P-p38 or P-JNK levels, than LUADs with the lowest DUSP6 RNA levels (n = 46). (I) DUSP6 RNA levels correlate with the levels of P-ERK in LUADs (n = 182). Pearson correlation coefficient (r) and p-value are indicated. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS = Not Significant.