Fumiko Taguchi

Establishment of a human non‐small cell lung cancer cell line resistant to gefitinib

The epidermal growth factor receptor (EGFR) tyrosine‐kinase inhibitor gefitinib (Iressa®, ZD1839) has shown promising activity preclinically and clinically. Because comparative investigations of drug‐resistant sublines with their parental cells are useful approaches to identifying the mechanism of gefitinib resistance and select factors that determine sensitivity to gefitinib, we established a human non‐small cell lung carcinoma subline (PC‐9/ZD) that is resistant to gefitinib. PC‐9/ZD cells are ∼180‐fold more resistant to gefitinib than their parental PC‐9 cells and PC‐9/ZD cells do not exhibit cross‐resistance to conventional anticancer agents or other tyrosine kinase inhibitors, except AG‐1478, a specific inhibitor of EGFR. PC‐9/ZD cells also display significant resistance to gefitinib in a tumor‐bearing animal model. To elucidate the mechanism of resistance, we characterized PC‐9/ZD cells. The basal level of EGFR in PC‐9 and PC‐9/ZD cells was comparable. A deletion mutation was identified within the kinase domain of EGFR in both PC‐9 and PC‐9/ZD, but no difference in the sequence of EGFR cDNA was detected in either cell line. Increased EGFR/HER2 (and EGFR/HER3) heterodimer formations were demonstrated in PC‐9/ZD cells by chemical cross‐linking and immunoprecipitation analysis in cells unexposed to gefitinib. Exposure to gefitinib increased heterodimer formation in PC‐9 cells, but not in PC‐9/ZD cells. Gefitinib inhibits EGFR autophosphorylation in a dose‐dependent manner in PC‐9 cells but not in PC‐9/ZD cells. A marked difference in inhibition of site‐specific phosphorylation of EGFR was observed at Tyr1068 compared to other tyrosine residues (Tyr845, 992 and 1045). To elucidate the downstream signaling in the PC9/ZD cellular machinery, complex formation between EGFR and its adaptor proteins GRB2, SOS, and Shc was examined. A marked reduction in the GRB2‐EGFR complex and absence of SOS‐EGFR were observed in PC‐9/ZD cells, even though the protein levels of GRB2 and SOS in PC‐9 and PC‐9/ZD cells were comparable. Expression of phosphorylated AKT was increased in PC‐9 cells and inhibited by 0.02 μM gefitinib. But the inhibition was not significant in PC‐9/ZD cells. These results suggest that alterations of adaptor‐protein‐mediated signal transduction from EGFR to AKT is a possible mechanism of the resistance to gefitinib in PC‐9/ZD cells. These phenotypes including EGFR–SOS complex and heterodimer formation of HER family members are potential biomarkers for predicting resistance to gefitinib.

Synergistic interaction between the EGFR tyrosine kinase inhibitor gefitinib (“Iressa”) and the DNA topoisomerase I inhibitor CPT‐11 (irinotecan) in human colorectal cancer cells

Epidermal growth factor receptor [EGFR (HER1, erbB1)] is a receptor with associated tyrosine kinase activity, and is expressed in colorectal cancers and many other solid tumors. We examined the effect of the selective EGFR tyrosine kinase inhibitor (EGFR‐TKI) gefitinib (“Iressa”) in combination with the DNA topoisomerase I inhibitor CPT‐11 (irinotecan) on human colorectal cancer cells. EGFR mRNA and protein expression were detected by RT‐PCR and immunoblotting in all 7 colorectal cancer cell lines studied. Gefitinib inhibited the cell growth of the cancer cell lines in vitro with an IC50 range of 1.2–160 μM by 3,(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay. Lovo cells exhibited the highest level of protein and autophosphorylation of EGFR and were the most sensitive to gefitinib. The combination of gefitinib and CPT‐11 induced supra‐additive inhibitory effects in COLO320DM, WiDR and Lovo cells, assessed by an in vitro MTT assay. Administration of gefitinib and CPT‐11 had a supra‐additive inhibitory effect on WiDR cells and tumor shrinkage was observed in Lovo cell xenografts established in nude mice, whereas no additive effect of combination therapy was observed in COLO320DM cells. To elucidate the mechanisms of synergistic effects, the effect of CPT‐11‐exposure on phosphorylation of EGFR was examined by immunoprecipitation. CPT‐11 increased phosphorylation of EGFR in Lovo and WiDR cells in time‐ and dose‐dependent manners. This EGFR activation was completely inhibited by 5 μM gefitinib and gefitinib‐induced apoptosis was enhanced by combination with CPT‐11, measured by PARP activation although no PARP activation was induced by 5 μM CPT‐11 alone. These results suggested that these modification of EGFR by CPT‐11, in Lovo cells, is a possible mechanism for the synergistic effect of CPT‐11 and gefitinib. These findings imply that the EGFR‐TKI gefitinib and CPT‐11 will be effective against colorectal tumor cells that express high levels of EGFR, and support clinical evaluation of gefitinib in combination with CPT‐11, in the treatment of colorectal cancers.

Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib ("Iressa")-sensitive and resistant xenograft models.

ZD6474 is a novel, orally available inhibitor of vascular endothelial growth factor (VEGF) receptor‐2 (KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 has been shown to inhibit angiogenesis and tumor growth in a range of tumor models. Gefitinib (“Iressa”) is an selective EGFR tyrosine kinase inhibitor (TKI) that blocks signal transduction pathways. We examined the antitumor activity of ZD6474 in the gefitinib‐sensitive lung adenocarcinoma cell line, PC‐9, and a gefitinib‐resistant variant (PC‐9/ZD). PC‐9/ZD cells showed cross‐resistance to ZD6474 in an in vitro dye formation assay. In addition, ZD6474 showed dose‐dependent inhibition of EGFR phosphorylation in PC‐9 cells, but inhibition was only partial in PC‐9/ZD cells. ZD6474‐mediated inhibition of tyrosine residue phosphorylation (Tyr992 and Tyr1045) on EGFR was greater in PC‐9 cells than in PC‐9/ZD cells. These findings suggest that the inhibition of EGFR phosphorylation by ZD6474 can contribute a significant, direct growth‐inhibitory effect in tumor cell lines dependent on EGFR signaling for growth and/or survival. The effect of ZD6474 (12.5–50 mg/kg/day p.o. for 21 days) on the growth of PC‐9 and PC‐9/ZD tumor xenografts in athymic mice was also investigated. The greatest effect was seen in gefitinib‐sensitive PC‐9 tumors, where ZD6474 treatment (>12.5 mg/kg/day) resulted in tumor regression. Dose‐dependent growth inhibition, but not tumor regression, was seen in ZD6474‐treated PC‐9/ZD tumors. These studies demonstrate that the additional EGFR TKI activity may contribute significantly to the anti‐tumor efficacy of ZD6474, in particular in those tumors that are dependent on continued EGFR‐signaling for proliferation or survival. In addition, these results provide a preclinical rationale for further investigation of ZD6474 as a potential treatment option for both EGFR‐TKI‐sensitive and EGFR‐TKI‐resistant tumors.

Combination Therapy With Gefitinib and Rofecoxib in Patients With Platinum-Pretreated Relapsed Non–Small-Cell Lung Cancer

PURPOSE In non-small-cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) play major roles in tumorigenesis. This phase I/II study evaluated combined therapy with the EGFR tyrosine kinase inhibitor (TKI) gefitinib and the COX-2 inhibitor rofecoxib in platinum-pretreated, relapsed, metastatic NSCLC (n = 45). PATIENTS AND METHODS Gefitinib 250 mg/d was combined with rofecoxib (dose escalated from 12.5 to 25 to 50 mg/d through three cohorts, each n = 6). Because the rofecoxib maximum-tolerated dose was not reached, the 50 mg/d cohort was expanded for efficacy evaluation (n = 33). RESULTS Among the 42 assessable patients, there was one complete response (CR) and two partial responses (PRs) and 12 patients with stable disease (SD); disease control rate was 35.7% (95% CI, 21.6% to 52.0%). Median time to tumor progression was 55 days (95% CI, 47 to 70 days), and median survival was 144 days (95% CI, 103 to 190 days). In a pilot study, matrix-assisted laser desorption/ionization (MALDI) proteomics analysis of baseline serum samples could distinguish patients with an objective response from those with SD or progressive disease (PD), and those with disease control (CR, PR, and SD) from those with PD. The regimen was generally well tolerated, with predictable toxicities including skin rash and diarrhea. CONCLUSION Gefitinib combined with rofecoxib provided disease control equivalent to that expected with single-agent gefitinib and was generally well tolerated. Baseline serum proteomics may help identify those patients most likely to benefit from EGFR TKIs.

hnRNP L enhances sensitivity of the cells to KW‐2189

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in several RNA‐related biological processes. We demonstrated hnRNP L as a candidate protein of DARP (duocarmycin‐DNA adduct recognizing protein) by gel shift assay and amino acid sequencing. Stable transfectants of hnRNP L showed high sensitivity of the cells to the growth inhibitory effect of KW‐2189, a duocarmycin derivative in vitro. Immunostaining of hnRNP L demonstrated differential intracellular localization of hnRNP L among human lung cancer cell lines. A transfection study using a series of deletion mutants of hnRNP L fused to indicated that the N‐terminal portions of RRM(RNA recognition motif)1, RRM3 and RRM2 are involved in localization of hnRNP L. We identified sequences in these portions that have high homology with the sequences of known NLS (nuclear localization signal) and NES (nuclear export signal). hnRNP L is a factor that determines the sensitivities of cancer cells to the minor groove binder, and overexpression and differential intracellular localization of hnRNP L are involved in its function in lung cancer.