Epidermal growth factor receptor and K-Ras mutations and resistance of lung cancer to insulin-like growth factor 1 receptor tyrosine kinase inhibitors

Abstract

Background: Most patients with nonsmall cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The authors investigated the involvement of insulinlike growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF-1R TKIs.

Methods: Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in an NSCLC tissue microarray. The authors analyzed the antitumor effects of an IGF-1R TKI (PQIP or OSI-906), either alone or in combination with a small-molecular inhibitor (PD98059 or U0126) or with siRNA targeting K-Ras or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K-Ras mutations.

Results: pIGF-1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant K-Ras, and wild-type (WT) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NSCLC cells with WT EGFR and WT K-Ras but not in those with mutations in these genes. Introduction of mutant K-Ras attenuated the effects of IGF-1R TKIs on NSCLC cells expressing WT K-Ras. Conversely, inactivation of MEK restored sensitivity to IGF-TKIs in cells carrying mutant K-Ras.

Conclusions: The mutation status of both EGFR and K-Ras could be a predictive marker of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs.

Figure 1. Response of NSCLC cell lines carrying mut EGFR to treatment with PQIP

(A) The IGF-1R signaling pathway is activated by IGF-1 in mut EGFR cells. (B) PQIP inhibits IGF-1R phosphorylation, shown in a subset of mut EGFR cell lines. (C) PQIP does not affect cell viability or anchorage-independent colony formation, shown in 3 mut EGFR cell lines. Columns, means of 3 independent experiments; bars, standard deviation.

Figure 2. K-Ras mutation is associated with poor response to the IGF-1R TKI PQIP in wt EGFR NSCLC cells

(A) Role of IGF signaling in NSCLC cells with mut K-Ras (except H1299;N-Ras, listed in black) and wt K-Ras (listed in blue). The effect of PQIP on the survival/proliferation of NSCLC cells was examined (1% FBS in culture medium). Each result is displayed as a colored box; shades of blue represent the cell number relative to the control, as a percentage. The most resistant cells are at the top and the most sensitive at the bottom. AC, adenocarcinoma ;BAC, bronchioalveolar carcinoma; SQ, squamous cell carcinoma; Large, large-cell carcinoma,. (B) Relative expression of IGF-1R, IR, and pIGF-1R/IR in a panel of NSCLC cell lines. (C) Comparison of relative sensitivity to PQIP treatment between the mut and wt K-Ras groups. Relative cell viability decreased by ≥5% in response to treatment with 1 µM PQIP in each group. . (D) (top) H460 and H157 cells, which carry mut K-Ras, were transfected with K-Ras siRNA or Scr (scrambled)siRNA and then seeded in 96-well microplates. The cells were treated with PQIP for 3 days in RPMI containing 1% FBS and then subjected to MTT assay. (bottom) A549 and H596 cells, which carry mut and wt K-Ras, respectively, were treated with the indicated concentrations of PQIP for 6 hours in the absence of FBS and then stimulated with IGF-1 for 30 minutes.

Figure 3. Mut K-Ras is a determining factor of IGF-1R TKI sensitivity of NSCLC cells

(A) Secreted IGF-1 level from NSCLC cells was enhanced by mut K-Ras. H226B-GFP or H226B-K-Ras cells (H226B cells stably transduced with retrovirus expressing GFP or mut K-Ras, respectively) were cultured in 1% FBS for 3 days. The conditioned media (CM) from H226B-GFP and H226B-K-Ras cells were applied to an IGF-1 ELISA. (B) Hierarchical clustering of protein expression data from H226B-GFP and H226B-K-Ras cells (n = 3 each). Reverse-phase protein array data are shown for 131 protein features, The data are presented in a matrix format: rows represent individual protein features, and columns represent individual samples. Each cell in the matrix represents the expression level of a protein feature in an individual cell sample. Red and green reflect relatively high and low expression levels, respectively. A total of 50 proteins showed differences with P < 0.05 by Student’s t test; of these, the molecules related to Akt signaling and Ras/MAPK signaling are shown in the bottom panels. The data were analyzed by using the Cluster and TreeView programs. (C) Molecular changes of the IGF-1R/Akt pathway by mut K-Ras and PQIP. H226B cells transfected with GFP or mut K-Ras were treated with PQIP (1 µM), and the cells were harvested at the indicated time point. (D) Mut K-Ras enhanced the resistance of NSCLC cells to PQIP. Relative cell survival after PQIP treatment for 3 days is shown. Mut K-Ras blunted the sensitivity of 226B and H596 cells to PQIP. Columns, means of 3 independent experiments; bars, standard deviation.

Figure 4. Co-targeting of IGF-1R and Ras signaling overrides the resistance to IGF-1R TKI driven by the K-Ras mutation in vitro and in vivo

(A) Relative cell survival of mut Ras, resistant NSCLC cells after treatment with an IGF-1R TKI (PQIP, 5 µM), a MEK inhibitor (PD98059 [10 µM] or U0126 [2 µM or 1 µM]), or both. (B) Effect of combined IGF-1R and MEK inhibition on anchorage-independent colony-forming ability of NSCLC cells with mut Ras. The indicated NSCLC cells seeded in soft agar were treated with PQIP (1 µM), U0126 (2 µM), or both. Ad-MEK-DN, dominant negative form MEK expressing adenovirus ; Ad-EV, empty adenovirus. The relative colony numbers are shown. (C) Synergistic apoptotic effect of combined treatment with PQIP (5 µM) and U0126 (1 µM) on apoptosis of mut K-Ras-H157 NSCLC cells. The active form of caspase-3 was stained, and the percentage of apoptotic cells is plotted. (D) Mice bearing H226B-K-Ras xenograft tumors (2 tumors per mouse, 4 or 5 mice per group) were treated with vehicle (control), OSI-906 (40 mg/kg, once a day), U0126 (4 mg/kg, every other day), or both OSI-906 and U0126 as indicated. Day 0 represents the first day of drug treatment. Data are means and 95% confidence intervals. (E) IHC staining of the xenograft tumors in (D) with Ki67 and cleaved caspase-3 antibody was performed, and results are shown for at least 4 tumors in each group.