Zhi-Hong Chen

Relative Abundance of EGFR Mutations Predicts Benefit From Gefitinib Treatment for Advanced Non–Small-Cell Lung Cancer

PURPOSE Our aim was to determine whether abundance of epidermal growth factor receptor (EGFR) mutations in tumors predicts benefit from treatment with EGFR-tyrosine kinase inhibitors (TKIs) for advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS We detected EGFR mutations in 100 lung cancer samples using direct DNA sequencing and amplification refractory mutation system (ARMS). Mutation-positive tumors by both methods carried high abundance of EGFR mutations. Tumors that were mutation positive by ARMS but mutation negative by direct DNA sequencing harbored low abundance of EGFR mutations. Mutation-negative tumors by both methods carried wild-type EGFR. All patients received gefitinib treatment. The correlation between EGFR mutation abundance and clinical benefit from gefitinib treatment was analyzed. RESULTS Of 100 samples, 51 and 18 harbored high and low abundances of EGFR mutations, respectively; 31 carried wild-type EGFR. Median progression-free survival (PFS) was 11.3 (95% CI, 7.4 to 15.2) and 6.9 months (95% CI, 5.5 to 8.4) in patients with high and low abundances of EGFR mutations, respectively (P = .014). Median PFS of patients with low abundance of EGFR mutations was significantly longer than that of those with wild-type tumors (2.1 months; 95% CI, 1.0 to 3.2; P = .010). Objective response rates (ORRs) were 62.7%, 44.4%, and 16.1%, and overall survival (OS) rates were 15.9 (95% CI, 13.4 to 18.3), 10.9 (95% CI, 2.7 to 19.1), and 8.7 months (95% CI, 4.6 to 12.7) for patients with high abundance of EGFR mutations, low abundance of EGFR mutations, and wild-type EGFR, respectively. The difference between patients with high and low abundances of EGFR mutations was not significant regarding ORR and OS. CONCLUSION The relative EGFR mutation abundance could predict benefit from EGFR-TKI treatment for advanced NSCLC.

In vitro sequence-dependent synergism between paclitaxel and gefitinib in human lung cancer cell lines

PurposeIn clinical trials, the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) administered concomitantly with first-line cytotoxicity chemotherapy failed to confer a survival benefit to patients with non-small-cell lung cancer (NSCLC). The aim of this study was to test whether paclitaxel followed by gefitinib is superior to other treatment schedules of NSCLC cell lines and to clarify the underlying mechanisms.MethodsHuman lung cancer cell lines with wild-type and mutant-type EGFR genes were used as in vitro models to define the differential effects of various schedules of paclitaxel with gefitinib treatment on cell growth, signaling pathway, and cell cycle distribution.ResultsSequence-dependent antiproliferative effects differed between EGFR-TKI-resistant and EGFR-TKI-sensitive lung cancer cell lines. Exposure to paclitaxel resulted in an increased pEGFR level. This increase in phosphorylation was inhibited by subsequent exposure to gefitinib, whereas during the reverse sequence, the inhibition effect was reduced. After paclitaxel exposure, a higher level of pEGFR was observed in mitotic than in interphase cells. The sequence of paclitaxel followed by gefitinib resulted in greater anti-VEGF activity than did the reverse sequence. We confirmed that gefitinib arrested cells in G1, and paclitaxel arrested them in S phase. The sequence of paclitaxel followed by gefitinib arrested cells in G1, whereas the reverse sequence arrested cells in S and G2 phases.ConclusionsThese findings suggest that the sequence of paclitaxel followed by gefitinib may be superior to other sequences in treating NSCLC cell lines. Our results also provide molecular evidence to support clinical treatment strategies for patients with lung cancer.

RRM1 single nucleotide polymorphism -37C→A correlates with progression-free survival in NSCLC patients after gemcitabine-based chemotherapy

BackgroundThe ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.MethodsPBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.ResultsRRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (P = 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (P = 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (P = 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.ConclusionsThe RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells. Methods We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry. Results In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels. Conclusion Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.

BCL11A overexpression predicts survival and relapse in non-small cell lung cancer and is modulated by microRNA-30a and gene amplification.

BackgroundAberrant activation of the proto-oncogene B-cell lymphoma/leukemia 11A (BCL11A) has been implicated in the pathogenesis of leukemia and lymphoma. However, the clinical significance of BCL11A in non-small cell lung cancer (NSCLC) remains unknown.ResultsWe examined BCL11A expression at the protein and mRNA levels in a cohort (n = 114) of NSCLC patients and assessed the relationship between BCL11A expression and clinicopathological parameters. Data from array-based Comparative Genomic Hybridization (aCGH) and microRNA transfection experiments were integrated to explore the potential mechanisms of abnormal BCL11A activation in NSCLC. Compared to adjacent non-cancerous lung tissues, BCL11A expression levels were specifically upregulated in NSCLC tissues at both the mRNA (t = 9.81, P < 0.001) and protein levels. BCL11A protein levels were higher in patients with squamous histology (χ2 = 15.81, P = 0.001), smokers (χ2 = 8.92, P = 0.004), patients with no lymph node involvement (χ2 = 5.14, P = 0.029), and patients with early stage disease (χ2 = 3.91, P = 0.048). A multivariate analysis demonstrated that in early stage NSCLC (IA–IIB), BCL11A was not only an independent prognostic factor for disease-free survival (hazards ratio [HR] 0.24, 95% confidence interval [CI] 0.12-0.50, P < 0.001), but also for overall survival (HR = 0.23, 95% CI 0.09-0.61, P = 0.003). The average BCL11A expression level was much higher in SCC samples with amplifications than in those without amplifications (t = 3.30, P = 0.023). Assessing functionality via an in vitro luciferase reporter system and western blotting, we found that the BCL11A protein was a target of miR-30a.ConclusionsOur results demonstrated that proto-oncogene BCL11A activation induced by miR-30a and gene amplification may be a potential diagnostic and prognostic biomarker for effective management of this disease.

Everolimus synergizes with gefitinib in non-small-cell lung cancer cell lines resistant to epidermal growth factor receptor tyrosine kinase inhibitors

PurposeEpidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is considered as one of the most important treatments for patients with advanced non-small-cell lung cancer (NSCLC). However, not all patients benefit from this therapy because of primary or acquired resistance, both of which are usually caused by the activation of alternative signaling pathways. Thus, a combination of different signaling pathway inhibitors is a promising strategy. We used the mammalian target of rapamycin (mTOR) inhibitor everolimus in combination with gefitinib in NSCLC cell lines to analyze the efficacy of this combination regimen and the underlying molecular mechanism.MethodsAcquired gefitinib-resistant cell lines, together with EGFR wild-type and mutant primary gefitinib-resistant NSCLC cell lines, were treated with everolimus alone, gefitinib alone, or the combination of the two drugs, and the effects were evaluated using cell proliferation assays. The effects of everolimus and gefitinib on the EGFR pathway in NSCLC cell lines were determined by Western blot analysis.ResultsCombined treatment resulted in synergistic antitumor effects in gefitinib-resistant cells A549 and H1975. The combination index (CI) of cells increased with increasing dose of everolimus. Everolimus demonstrated no apparent inhibition of phosphorylated Akt (p-Akt) and phosphorylated p44/42 MAPK (p-MAPK) in H1650 cells. Additionally, in gefitinib-resistant cell lines, the combination of gefitinib and everolimus not only showed stronger inhibition of phosphorylated mTOR and phosphorylated p70S6K expression than either drug alone but also reduced the levels of p-Akt and p-MAPK in both cell lines.ConclusionsOur data showed that the combination of everolimus and gefitinib exhibits dose-dependent synergism in primary and acquired gefitinib-resistant NSCLC cells. Thus, a preclinical rationale exists for the use of everolimus to enhance the efficacy of gefitinib in EGFR-TKI-resistant patients with NSCLC.

Molecular mechanism of the schedule-dependent synergistic interaction in EGFR-mutant non-small cell lung cancer cell lines treated with paclitaxel and gefitinib

BackgroundChemotherapy combined concurrently with TKIs produced a negative interaction and failed to improve survival when compared with chemotherapy or TKIs alone in the treatment of non-small cell lung cancer (NSCLC). The present study investigated the sequence-dependent interaction between paclitaxel and gefitinib and clarified the underlying mechanism.MethodsThe effects on cell proliferation, EGFR signaling pathway, and TGFα expression were evaluated in a panel of human NSCLC cell lines harboring EGFR mutations with three different combination sequences: sequential treatment with paclitaxel followed by gefitinib (T→G), sequential treatment with gefitinib followed by paclitaxel (G→T), or concomitant treatment (T + G).ResultsThe sequence-dependent anti-proliferative effects differed between EGFR-TKI-sensitive and -resistant cell lines carrying EGFR mutations. A synergistic anti-proliferative activity was obtained with paclitaxel treatment followed by gefitinib in all cell lines, with mean CI values of 0.63 in Hcc827, 0.54 in PC-9, 0.81 in PC-9/GR, and 0.77 in H1650 cells for the T→G sequence. The mean CI values for the G→T sequence were 1.29 in Hcc827, 1.16 in PC-9, 1.52 in PC-9/GR, and 1.5 in H1650 cells. The mean CI values for T+G concomitant treatment were 0.88 in Hcc827, 0.91 in PC-9, 1.05 in PC-9/GR, and 1.18 in H1650 cells. Paclitaxel produced a dose-dependent increase in EGFR phosphorylation. Paclitaxel significantly increased EGFR phosphorylation compared with that in untreated controls (mean differences: +50% in Hcc827, + 56% in PC-9, + 39% in PC-9/GR, and + 69% in H1650 cells; p < 0.05). The T→G sequence produced significantly greater inhibition of EGFR phosphorylation compared with the opposite sequence (mean differences: -58% in Hcc827, -38% in PC-9, -35% in PC-9/GR, and -30% in H1650 cells; p < 0.05). Addition of a neutralizing anti-TGFα antibody abolished paclitaxel-induced activation of the EGFR pathway in PC-9 and H1650 cells. Sequence-dependent TGFα expression and release are responsible for the sequence-dependent EGFR pathway modulation.ConclusionThe data suggest that the sequence of paclitaxel followed by gefitinib is an appropriate treatment combination for NSCLC cell lines harboring EGFR mutations. Our results provide molecular evidence to support clinical treatment strategies for patients with lung cancer.

CYP1A1*2A polymorphism as a predictor of clinical outcome in advanced lung cancer patients treated with EGFR-TKI and its combined effects with EGFR intron 1 (CA)n polymorphism

BACKGROUND Mutations in the epidermal growth factor receptor (EGFR) have been confirmed as predictors of efficacy for EGFR-tyrosine kinase inhibitors (TKIs). We investigated whether polymorphisms of the EGFR and cytochrome P450, family 1, member A1 (CYP1A1) genes were associated with clinical outcome in NSCLC patients treated with EGFR-TKI. METHODS Genotypes for the intron 1 (CA)n repeat and R497K polymorphisms in the EGFR gene and the *2A (3801 T→C) and *2C (2455 A→G) polymorphisms in CYP1A1 gene were evaluated in 115 NSCLC patients by PCR-RFLP and DNA sequencing. Genetic polymorphisms were correlated with clinical outcomes of EGFR-TKIs. From a subgroup of patients whose tumour tissues were available, associations between somatic EGFR mutations, EGFR expression, and genomic polymorphisms were also analysed. RESULTS EGFR intron 1 (CA)n and CYP1A1*2A polymorphisms were independent predictive factors (p=0.046, p=0.011, respectively) and the latter was also a prognostic factor (p=0.001) for patients treated with EGFR-TKIs. We also observed a strong synergistic effect from two genotypes. Specifically, patients with both the T/T allele of the CYP1A1 gene and shorter intron 1 CA repeats (≤ 16 CA) of the EGFR gene showed an improved response (p=0.002) compared with patients with the T/C or C/C allele and longer intron 1 CA repeats (both alleles >16 CA). In contrast, for R497K and CYP1A1*2C, no relationship was observed with clinical outcome for patients treated with EGFR-TKIs (p=0.573; p=0.629, respectively). Both SNPs in the CYP1A1 gene showed a correlation with EGFR somatic mutations. CONCLUSIONS The findings of this study suggest that the CYP1A1*2A polymorphism is a predictor for clinical outcome in NSCLC patients treated with EGFR-TKI therapy, and combining analysis of both CYP1A1*2A and EGFR intron 1 (CA)n polymorphisms may be useful for predicting treatment outcome in NSCLC patients treated with EGFR-TKIs.

MET Expression Plays Differing Roles in Non–Small-Cell Lung Cancer Patients with or without EGFR Mutation

Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and Met inhibitors have enabled progress in the management of advanced non–small-cell lung cancer (NSCLC). However, the clinical benefits of these agents are not uniform across the NSCLC spectrum. Thus, we evaluated the prognostic effect of mesenchymal-epithelial transition (MET) expression in Asian NSCLC patients with or without EGFR mutation. Methods: Frozen tumor tissues were collected from 92 patients with surgical resection and 10 with lymph node biopsy. Mutations in exons 18–21 in the EGFR–tyrosine kinase domain and MET expression were analyzed by using sequencing and immunohistochemistry, respectively. Results: The MET overexpression rate was 51% in NSCLC patients. MET-positive patients had poorer overall survival than MET-negative patients (29.8 versus 69.1 months, &khgr;2 = 7.420, p = 0.006) in patients with wild-type EGFR. However, no statistically significant difference was found in EGFR mutant patients (35.0 versus 35.9 months, &khgr;2 = 0.114, p = 0.735). Multivariate analysis showed that stage, MET expression, and sex were independent prognostic factors in patients with wild-type EGFR (&khgr;2 = 32.896, p < 0.001). Conclusions: These results suggest that MET expression has different prognostic significance in patients with differing EGFR mutation status. Whether MET inhibitors should be given early to NSCLC patients with EGFR wild-type needs further investigation.

Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.