Knockdown of oncogenic KRAS in non-small cell lung cancers suppresses tumor growth and sensitizes tumor cells to targeted therapy

Abstract

Oncogenic KRAS is found in more than 25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS-mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we conducted microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines with and without oncogenic KRAS. We found that although the mitogen-activated protein kinase pathway is significantly downregulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-epidermal growth factor receptor, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment, but may offer possibilities of combining anti-KRAS strategies with other targeted drugs.

Fig. 1

(A) Immunoblot of KRAS protein expression in H23, H1792, H358, H441, H1299 NSCLC cell lines. Thirty μg of whole cell lysate was loaded per lane and Western Blot was performed. (B) Positive correlation between KRAS copy number and the protein expression levels in NSCLC cell lines (Pearson r = 0.9636, P = 0.0083). The protein expression levels were determined by densitometry. (C) Stable knockdown of mutant KRAS protein by retroviral-mediated shRNA in NSCLC cell lines. P: parental cells, Mock: pRS control vector-infected cells, KRAS-C: pRS-KRAS-C12-infected cells, KRAS-V: pRS-KRAS-V12-infected cells. (D) Specific reduction of mutant KRAS transcripts by retroviral shRNA vectors (pRS-KRAS-C12 or pRS-KRAS-V12) but not by the pRS control vector in the NSCLC cells. BstNI digestion cuts the wild-type KRAS allele (e.g., H1299 cells) to produce a 156-bp DNA fragment whereas the mutant KRAS allele remains uncut to produce a 186-bp DNA fragment (e.g., the H2122 cell line that has a homozygous KRAS G12C mutation was used as a control).

Fig. 1

(A) Immunoblot of KRAS protein expression in H23, H1792, H358, H441, H1299 NSCLC cell lines. Thirty μg of whole cell lysate was loaded per lane and Western Blot was performed. (B) Positive correlation between KRAS copy number and the protein expression levels in NSCLC cell lines (Pearson r = 0.9636, P = 0.0083). The protein expression levels were determined by densitometry. (C) Stable knockdown of mutant KRAS protein by retroviral-mediated shRNA in NSCLC cell lines. P: parental cells, Mock: pRS control vector-infected cells, KRAS-C: pRS-KRAS-C12-infected cells, KRAS-V: pRS-KRAS-V12-infected cells. (D) Specific reduction of mutant KRAS transcripts by retroviral shRNA vectors (pRS-KRAS-C12 or pRS-KRAS-V12) but not by the pRS control vector in the NSCLC cells. BstNI digestion cuts the wild-type KRAS allele (e.g., H1299 cells) to produce a 156-bp DNA fragment whereas the mutant KRAS allele remains uncut to produce a 186-bp DNA fragment (e.g., the H2122 cell line that has a homozygous KRAS G12C mutation was used as a control).

Fig. 2

(A) The effects of KRAS knockdown on the phosphorylation of MEK, ERK, Akt, STAT3 and EGFR in the KRAS mutation-positive NSCLC cell lines. Thirty μg of whole cell lysate was loaded per lane for western blot analysis. Mock: pRS vector-infected cells, KRAS-C: pRS-KRAS-C12-infected cells, KRAS-V: pRS-KRAS-V12-infected cells. (B) The effect of KRAS knockdown on the phosphoprotein levels. The data were obtained from three independent experiments.

Fig. 2

(A) The effects of KRAS knockdown on the phosphorylation of MEK, ERK, Akt, STAT3 and EGFR in the KRAS mutation-positive NSCLC cell lines. Thirty μg of whole cell lysate was loaded per lane for western blot analysis. Mock: pRS vector-infected cells, KRAS-C: pRS-KRAS-C12-infected cells, KRAS-V: pRS-KRAS-V12-infected cells. (B) The effect of KRAS knockdown on the phosphoprotein levels. The data were obtained from three independent experiments.

Fig. 3

The effects of (A) p38 V, (B) gefitinib, and (C) cetuximab on colony formation in H23, H1792, H358 and H441 cells with KRAS knockdown (red line) or without KRAS knockdown (blue line) at various concentrations. Number of colonies in untreated cells was set at 100%. The data were obtained from six independent experiments. *, P<0.05; **, P <0.01; ***, P <0.001.

Fig. 3

The effects of (A) p38 V, (B) gefitinib, and (C) cetuximab on colony formation in H23, H1792, H358 and H441 cells with KRAS knockdown (red line) or without KRAS knockdown (blue line) at various concentrations. Number of colonies in untreated cells was set at 100%. The data were obtained from six independent experiments. *, P<0.05; **, P <0.01; ***, P <0.001.

Fig. 3

The effects of (A) p38 V, (B) gefitinib, and (C) cetuximab on colony formation in H23, H1792, H358 and H441 cells with KRAS knockdown (red line) or without KRAS knockdown (blue line) at various concentrations. Number of colonies in untreated cells was set at 100%. The data were obtained from six independent experiments. *, P<0.05; **, P <0.01; ***, P <0.001.