6 which can be constitutively activated by K-Ras mutation, we determined whether inactivation of MEK would restore the antitumor effects of PQIP or OSI-906 (a PQIP derivative currently undergoing a phase clinical trial in our institute in NSCLC cell lines Tangeretin carry- ing mutant K-Ras ). We found that cotargeting of MEK, either with a small-molecule MEK inhibitor (PD98059 or U06) or with adenovirus expressing the dominant- negative form of MEK (Ad-MEK-DN), significantly enhanced the effects of PQIP on cell viability (Fig. 4A) and anchorage-independent colony-forming ability (Fig. 4B) in representative mutant K-Ras resistant cell lines. Furthermore, the percentage of apoptotic cells was signifi- cantly increased by the combined treatment  .

These results suggest that inactivation of MEK augments the apoptotic activities of PQIP in NSCLC cells carrying mutant K-Ras . We finally evaluated the combined effects of Dexamethasone OSI-906 and U06 in vivo. The mice treated with ve- hicle or OSI-906 alone showed similar H6B K-Ras tu- mor growth (Fig. 4D). Pharmacologic inhibition of MEK by administration of U06 dramatically augmented the effects of OSI-906 on the growth of the tumors. On day 8 after the first dose, the mean tumor volume for mice that received combined OSI-906 and U06 was significantly smaller than the mean tumor volume for mice that received vehicle, OSI-906 alone, or U06 alone. IHC staining of Ki67 and cleaved caspase-3 in the tumors dem- onstrated that the combined treatment induced a decrease in cell proliferation in association with an increase in cell apoptosis in vivo.

Taken together, these findings underscore the pivotal role of activation of the MEK/Erk pathway through K-Ras mutation in the primary resist- ance of NSCLC cells to IGF-R TKIs. DISCUSSION In the present study, we elucidate potential predictive markers of response of NSCLC cells to IGF-R TKIs. We show that: ) the expression of IGF-R/IR in NSCLC specimens is positively associated with a history of tobacco smoking, squamous cell carcinoma, WT EGFR , and mu- tant K-Ras ; ) somatic mutation of EGFR, which Formononetin 485-72-3 confers addiction to the EGFR signaling pathway, induces a lack of primary response to IGF-R TKIs in NSCLC cells; and 3) K-Ras mutation causes increased production of IGF- and activation of the IGF-R pathway but induces resistance to IGF-R TKIs. Moreover, our findings pro- vide a proof of principle that targeted inactivation of IGF- R by a TKI, in combination with MEK inhibition, can achieve a favorable outcome in the treatment of NSCLC patients with a history of tobacco smoking and mutant K-Ras .

Several preclinical and clinical studies have shown encouraging therapeutic efficacy of EGFR TKI in NSCLC with mutant EGFR ,3 ; however, the limited response rates to EGFR TKIs underscore the need to 6 Cancer Month 00, 0 6 K-Ras Mutation in NSCLC and buy Formononetin IGF-R/Kim et al Figure 3. Mutant K-Ras is a determining factor of insulinlike growth factor receptor (IGF-R) tyrosine kinase inhibitor sensitivity of nonsmall cell lung cancer (NSCLC) cells. (A) The secreted IGF- level from NSCLC cells was enhanced by mutant K- Ras . H6B-GFP or H6B–K- Ras cells (H6B cells stably transduced with retrovirus expressing Green Fluorescence Protein (GFP) or mutant K-Ras , respectively) were cultured in % fetal bovine serum for 3 days. The conditioned media (CM) from H6B-GFP and H6B– K-Ras cells were applied to an IGF- enzyme-linked immunosorbent assay.

Hierarchical clustering of protein expression data from H6B- GFP and H6B– K-Ras cells is shown (n ¼ 3 each). Reverse-phase protein array data are shown for 3 protein features. The data are presented in a matrix format: rows represent individual protein features, and obstetricians 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.