紫铆因靶向EGFR和AuroraA/B信号通路抑制非小细胞肺癌的研究

紫铆因抗多种肿瘤的生物学活性已被广泛研究,但其抑制非小细胞肺癌(non-small cell lung cancer,NSCLC)的分子机制还不清楚。研究中,通过MTS和软琼脂集落实验检测紫铆因对非小细胞肺癌细胞停泊依赖和停泊非依赖增殖的影响,利用免疫印迹检测紫铆因处理后非小细胞肺癌表皮生长因子受体(epidermal growth factor receptor,EGFR)信号通路的磷酸化状态,同时采用流式细胞术检测紫铆因对非小细胞肺癌细胞周期演进的影响。研究发现紫铆因剂量依赖性抑制A549和H1650细胞增殖,在抑制EGFR信号通路磷酸化活化的同时下调EGFR总蛋白及EGFR在胞膜和胞核的表达,而且紫铆因下调AuroraA/B和H3-S10磷酸化,促进A549细胞G2/M期阻滞。结果表明紫铆因可通过靶向EGFR和AuroraA/B信号通路抑制非小细胞肺癌。     

Digoxin Suppresses Tumor Malignancy through Inhibiting Multiple Src-Related Signaling Pathways in Non-Small Cell Lung Cancer

Non-small cell lung cancer is the predominant type of lung cancer, resulting in high mortality worldwide. Digoxin, a cardiac glycoside, has recently been suggested to be a novel chemotherapeutic agent. Src is an oncogene that plays an important role in cancer progression and is therefore a potential target for cancer therapy. Here, we investigated whether digoxin could suppress lung cancer progression through the inhibition of Src activity. The effects of digoxin on lung cancer cell functions were investigated using colony formation, migration and invasion assays. Western blotting and qPCR assays were used to analyze the mRNA and protein expression levels of Src and its downstream proteins, and a cell viability assay was used to measure cellular cytotoxicity effects. The results of the cell function assays revealed that digoxin inhibited the proliferation, invasion, migration, and colony formation of A549 lung cancer cells. Similar effects of digoxin were also observed in other lung cancer cell lines. Furthermore, we found that digoxin significantly suppressed Src activity and its protein expression in a dose- and time-dependent manner as well as reduced EGFR and STAT3 activity. Our data suggest that digoxin is a potential anticancer agent that may suppress lung cancer progression through inhibiting Src and the activity of related proteins.     

Deguelin Action Involves c-Met and EGFR Signaling Pathways in Triple Negative Breast Cancer Cells

Background

Treatment of breast cancer patients with antiestrogens and aromatase inhibitor(s) or Herceptin have shown significant success in steroid receptor positive or Her-2+ breast cancers respectively. However, choice of treatments for breast cancer patients with negative status for estrogen, progesterone receptors and HER2/neu is limited. As a result, search for appropriate therapy regimen for these triple negative breast cancers (TNBC) has become a major focus of investigations for many laboratories. Recently, Deguelin, a natural product isolated from African plant Mundulea sericea (Leguminossae) has shown both antiproliferative actions in various cancers including breast as well as chemoprenventive activity against carcinogen induced experimental cancers. In this report we evaluated efficacy and mechanism of action of Deguelin in triple negative breast cancer cell lines.

Methods/Findings

In vitro, Deguelin in a dose and time dependent manner inhibited the growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells. Deguelin (2 or 4 mg/kg body weight), when injected intraperitoneally, reduced the in vivo tumor growth of MDA-MB-231 cells transplanted subcutaneously in athymic mice. Moreover it was nontoxic as evident from daily observations on mobility, food and water consumption and comparison of bodyweight and other visceral organ weights with those in control animals at the termination of the study. The western blot analyses and immunostaining studies indicated that the deguelin effects may be mediated through EGFR-PAKT/c-Met p-ERK and NF-κB by down regulating their downstream targets such as p-STAT3, c-Myc, Survivin.

Conclusion/Significance

These results suggest that Deguelin may have a significant therapeutic value for the treatment of TNBC patients.     

Resistance to ROS1 Inhibition Mediated by EGFR Pathway Activation in Non-Small Cell Lung Cancer

The targeting of oncogenic ‘driver’ kinases with small molecule inhibitors has proven to be a highly effective therapeutic strategy in selected non-small cell lung cancer (NSCLC) patients. However, acquired resistance to targeted therapies invariably arises and is a major limitation to patient care. ROS1 fusion proteins are a recently described class of oncogenic driver, and NSCLC patients that express these fusions generally respond well to ROS1-targeted therapy. In this study, we sought to determine mechanisms of acquired resistance to ROS1 inhibition. To accomplish this, we analyzed tumor samples from a patient who initially responded to the ROS1 inhibitor crizotinib but eventually developed acquired resistance. In addition, we generated a ROS1 inhibition-resistant derivative of the initially sensitive NSCLC cell line HCC78. Previously described mechanisms of acquired resistance to tyrosine kinase inhibitors including target kinase-domain mutation, target copy number gain, epithelial-mesenchymal transition, and conversion to small cell lung cancer histology were found to not underlie resistance in the patient sample or resistant cell line. However, we did observe a switch in the control of growth and survival signaling pathways from ROS1 to EGFR in the resistant cell line. As a result of this switch, ROS1 inhibition-resistant HCC78 cells became sensitive to EGFR inhibition, an effect that was enhanced by co-treatment with a ROS1 inhibitor. Our results suggest that co-inhibition of ROS1 and EGFR may be an effective strategy to combat resistance to targeted therapy in some ROS1 fusion-positive NSCLC patients.     

Post-Progression Survival after EGFR-TKI for Advanced Non-Small Cell Lung Cancer Harboring EGFR Mutations

Background

Non-small cell lung cancer (NSCLC) patients that harbor epidermal growth factor receptor (EGFR) mutations benefit from receiving an EGFR-tyrosine kinase inhibitor (TKI); however, post-progression survival (PPS) after EGFR-TKI treatment has not been sufficiently studied.

Methods

We retrospectively reviewed the clinical data from stage IV or recurrent NSCLC patients who harbored EGFR mutations and who received EGFR-TKI as their first-line treatment in our institute between 2009 and 2011.

Results

In total, 36 patients received EGFR-TKI treatment as their first-line therapy. Of those 36 patients, 30 experienced recurrence and were enrolled in this study. The median progression-free survival (PFS) of these patients was 8.2 months. Twelve patients received EGFR-TKI treatment beyond the diagnosis of progressive disease (PD), and 8 received second-line therapy. The PPS after EGFR-TKI treatment was 9.1 months, and survival after the termination of EGFR-TKI treatment in those patients treated with second-line chemotherapy was 13.9 months. The site of relapse was investigated and PFS in EGFR-TKI-treated patients with relapse in the brain (11.6 months) showed a trend toward a longer PFS compared with patients with relapse at other sites (8.2 months). The median PPS after EGFR-TKI treatment also showed the same trend in each group (12.9 and 9.2 months, respectively).

Conclusions

The PPS after EGFR-TKI treatment failure was 9.1 months, while the survival of patients who underwent second-line chemotherapy after the termination of EGFR-TKI treatment was 13.9 months, comparable with the overall survival of EGFR mutation-negative patients, as previously reported. The prognosis of these NSCLC patients with EGFR mutations varied according to the sites of recurrence after first-line EGFR-TKI treatment. Of particular note was the prognosis of patients with brain metastases, which tended to be better than that of patients with metastases to other sites.     

Alterations in Genes of the EGFR Signaling Pathway and Their Relationship to EGFR Tyrosine Kinase Inhibitor Sensitivity in Lung Cancer Cell Lines

Background

Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs.

Methodology/Principal Findings

We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3 HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results: 1. mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance. 2. mKRAS was associated with increased in vitro resistance to gefitinib.

Conclusions/Significance

Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.     

结/直肠癌的潜在治疗靶点

传统的结/直肠癌化疗药物,作用选择性较低,副反应明显,疗效欠佳,患者病死率居高不下.寻找新型治疗药物,十分必要.在结/直肠癌发病机制的探索过程中,研究者发现:一些分子的变化影响着疾病进程,对病情起指示作用,有可能成为治疗靶点.围绕这些分子设计药物,制定治疗方案,有望提高疗效,改善病人生存质量,降低不良反应的发生.本文就结/直肠癌的部分潜在治疗靶点:EGFR,VEGF,HADC,COX-2,PPARγ及galectin-3做一综述.     

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.     

Effects of Icotinib on Advanced Non-Small Cell Lung Cancer with Different EGFR Phenotypes

Icotinib is the first oral epidermal growth factor receptor (EGFR) tyrosine kinase receptor inhibitor, which has been proven to exert significant inhibitory effects on non-small cell lung cancer in vitro. Clinical evidence has showed that the efficacy of Icotinib on retreating advanced non-small cell lung cancer is comparable to Gefitinib. However, different phenotypes of EGFR can affect the therapeutic outcomes of EGFR tyrosine kinase receptor inhibitor. Therefore, our study focused on efficacy and safety of Icotinib in patients with advanced non-small cell lung cancer of different EGPR phenotypes. Clinical data of patients with advanced non-small cell lung cancer who received Icotinib treatment from August, 2011 to May, 2013 were retrospectively analyzed. Kaplan–Meier analysis was used for survival analysis and comparison. 18 wild-type EGFR and 51 mutant type were found in a total of 69 patients. Objective response rate of patients with mutant type EGFR was 54.9 % and disease control rate was 86.3 %. Objective response rate of wild-type patients was 11.1 % (P = 0.0013 vs mutant type), disease control rate was 50.0 % (P = 0.0017). Median progression-free survival (PFS) of mutant type and wild-type patients were 9.7 and 2.6 months, respectively (P < 0.001). Median PFS of exon 19 mutated mutant patients was 11.3 months, mean PFS of exon 21 L858R mutated mutant patients was 8.7 months (P = 0.3145). Median overall survival (OS) of EGFR mutated patients had not reached. OS time of 13 wild-type patients was 12.9 months (P < 0.001). The common adverse reactions of Icotinib included rash, diarrhea, itching skin with occurrence rates of 24.6 % (17/69), 13.0 % (9/69), and 11.6 % (8/69), respectively. Most adverse reactions were grade I–II. Icotinib has great efficacy in EGFR mutated patients, making it an optimal regimen to treat EGFR mutated patients. Furthermore, most of adverse reactions associated with Icotinib treatment were tolerable.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.     

Resistance to BET Inhibitor Leads to Alternative Therapeutic Vulnerabilities in Castration-Resistant Prostate Cancer

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Dose-Dependent Mutation Rates Determine Optimum Erlotinib Dosing Strategies for EGFR Mutant Non-Small Cell Lung Cancer Patients

Background

The advent of targeted therapy for cancer treatment has brought about a paradigm shift in the clinical management of human malignancies. Agents such as erlotinib used for EGFR-mutant non-small cell lung cancer or imatinib for chronic myeloid leukemia, for instance, lead to rapid tumor responses. Unfortunately, however, resistance often emerges and renders these agents ineffective after a variable amount of time. The FDA-approved dosing schedules for these drugs were not designed to optimally prevent the emergence of resistance. To this end, we have previously utilized evolutionary mathematical modeling of treatment responses to elucidate the dosing schedules best able to prevent or delay the onset of resistance. Here we expand on our approaches by taking into account dose-dependent mutation rates at which resistant cells emerge. The relationship between the serum drug concentration and the rate at which resistance mutations arise can lead to non-intuitive results about the best dose administration strategies to prevent or delay the emergence of resistance.

Methods

We used mathematical modeling, available clinical trial data, and different considerations of the relationship between mutation rate and drug concentration to predict the effectiveness of different dosing strategies.

Results

We designed several distinct measures to interrogate the effects of different treatment dosing strategies and found that a low-dose continuous strategy coupled with high-dose pulses leads to the maximal delay until clinically observable resistance. Furthermore, the response to treatment is robust against different assumptions of the mutation rate as a function of drug concentration.

Conclusions

For new and existing targeted drugs, our methodology can be employed to compare the effectiveness of different dose administration schedules and investigate the influence of changing mutation rates on outcomes.     

Meta-Analysis of EGFR Tyrosine Kinase Inhibitors Compared with Chemotherapy as Second-Line Treatment in Pretreated Advanced Non-Small Cell Lung Cancer

Background

Since efficacy and safety of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) versus chemotherapy in the treatment of patients with pretreated advanced non-small cell lung cancer (NSCLC) remain controversial, we performed a meta-analysis to compare them.

Methods

An internet search of several databases was performed, including PubMed, Embase, and the Cochrane database. Randomized trials that compared an EGFR-TKI with chemotherapy in the second-line setting were included. The outcomes were progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and grade 3–4 toxicities. The PFS, OS for the EGFR mutation-positive (EGFR M⁺) and EGFR mutation-negative (EGFR M⁻) subgroups were pooled. The pooled hazard ratios (HRs) and odds ratios (ORs) with their corresponding confidence intervals (CIs) were calculated on the STATA software.

Results

Our meta-analysis combined 3,825 patients from 10 randomized trials. Overall, EGFR-TKIs and second-line chemotherapy have equivalent efficacy in terms of PFS (HR, 1.03; 95%CI, 0.87–1.21; P = 0.73; I² = 78.7%, P_{heterogeneity}<0.001), OS (HR, 1.00; 95%CI, 0.92–1.08; P = 0.90; I² = 0.0%, P_{heterogeneity} = 0.88), and ORR (OR, 1.34; 95%CI, 0.86–2.08; P = 0.20; I² = 73.1%, P_{heterogeneity}<0.001). However, subgroup analysis based on EGFR mutation status showed that second-line chemotherapy significantly improved PFS (HR, 1.35; 95%CI, 1.09–1.66; P = 0.01; I² = 55.7%, P_{heterogeneity} = 0.046) for EGFR M⁻ patients, whereas OS was equal (HR, 0.96; 95%CI, 0.77–1.19; P = 0.69; I² = 0.0%, P_{heterogeneity} = 0.43); EGFR-TKIs significantly improved PFS (HR, 0.28; 95%CI, 0.15–0.53; P<0.001; I² = 4.1%, P_{heterogeneity} = 0.35) for EGFR M⁺ patients, whereas OS was equal (HR, 0.86; 95%CI, 0.44–1.68; P = 0.65; I² = 0.0%, P_{heterogeneity} = 0.77). Compared with chemotherapy, EGFR-TKIs led to more grade 3–4 rash, but less fatigue/asthenia disorder, leukopenia and thrombocytopenia.

Conclusions

Our analysis suggests that chemotherapy in the second-line setting can prolong PFS in EGFR M⁻ patients, whereas it has no impact on OS. EGFR-TKIs seem superior over chemotherapy as second-line therapy for EGFR M⁺ patients. Our findings support obtaining information on EGFR mutational status before initiation of second-line treatment.     

The Role of Angiopoietins as Potential Therapeutic Targets in Renal Cell Carcinoma

Angiopoietin 2 (Ang2) is a secreted glycoprotein upregulated at sites of angiogenesis and has been implicated in cancer neovascularization. Recent studies have suggested efficacy of combined Ang and vascular endothelial growth factor receptor (VEGFR) inhibition for patients with metastatic renal cell carcinoma (mRCC). We measured Ang2 expression in human tissue and plasma, and tested the effect of dual Ang1/2 (trebananib; AMG386) or Ang2 alone (L1-7) inhibition with VEGFR inhibition on murine RCC growth and blood flow. Ang2 levels were higher in human tumors than normal tissues with RCC ranking highest for Ang2 expression across all tumor types tested. Plasma Ang2 was significantly higher in patients with mRCC compared to controls or patients with stage I disease. Plasma Ang2 decreased with sunitinib treatment and increased at time of disease progression. In the RCC mouse, dual Ang1/2 and Ang2 inhibition improved the activity of sunitinib. Combined Ang1/2 and VEGFR inhibition prevented the resumption of blood flow associated with sunitinib resistance. Thus, Ang2 inhibition, independent of Ang1 inhibition, improves the activity of sunitinib and plasma Ang2 increases in the setting of progression on sunitinib possibly contributing to resistance. Further, arterial spin-labeled perfusion magnetic resonance imaging might be a non-invasive marker of the antiangiogenic activity of Ang inhibitors.     

Ascorbic Acid and a Cytostatic Inhibitor of Glycolysis Synergistically Induce Apoptosis in Non-Small Cell Lung Cancer Cells

Ascorbic acid (AA) exhibits significant anticancer activity at pharmacologic doses achievable by parenteral administration that have minimal effects on normal cells. Thus, AA has potential uses as a chemotherapeutic agent alone or in combination with other therapeutics that specifically target cancer-cell metabolism. We compared the effects of AA and combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small cell lung cancer (NSCLC) cell lines to the effects on an immortalized lung epithelial cell line. AA concentrations of 0.5 to 5 mM caused a complete loss of viability in all NSCLC lines compared to a <10% loss of viability in the lung epithelial cell line. Combinations of AA and 3-PO synergistically enhanced cell death in all NSCLC cell lines at concentrations well below the IC₅₀ concentrations for each compound alone. A synergistic interaction was not observed in combination treatments of lung epithelial cells and combination treatments that caused a complete loss of viability in NSCLC cells had modest effects on normal lung cell viability and reactive oxygen species (ROS) levels. Combination treatments induced dramatically higher ROS levels compared to treatment with AA and 3-PO alone in NSCLC cells and combination-induced cell death was inhibited by addition of catalase to the medium. Analyses of DNA fragmentation, poly (ADP-ribose) polymerase cleavage, annexin V-binding, and caspase activity demonstrated that AA-induced cell death is caused via the activation of apoptosis and that the combination treatments caused a synergistic induction of apoptosis. These results demonstrate the effectiveness of AA against NSCLC cells and that combinations of AA with 3-PO synergistically induce apoptosis via a ROS-dependent mechanism. These results support further evaluation of pharmacologic concentrations of AA as an adjuvant treatment for NSCLC and that combination of AA with glycolysis inhibitors may be a promising therapy for the treatment of NSCLC.     

Alteration in Mir-21/PTEN Expression Modulates Gefitinib Resistance in Non-Small Cell Lung Cancer

Resistance to TKI treatment is a major obstacle in effective treatment of NSCLC. Besides EGFR mutation status, the mechanisms involved are largely unknown. Some evidence supports a role for microRNA 21 in modulating drug sensitivity of chemotherapy but its role in NSCLC TKI resistance still remains unexplored. This study aimed to investigate whether NSCLC miR-21 mediated resistance to TKIs also results from Pten targeting. Here, we show miR-21 promotes cancer by negatively regulating Pten expression in human NSCLC tissues: high miR-21 expression levels were associated with shorter DFS in 47 NSCLC patients; high miR-21/low Pten expression levels indicated a poor TKI clinical response and shorter overall survival in another 46 NSCLC patients undergoing TKI treatment. In vitro assays showed that miR-21 was up-regulated concomitantly to down-regulation of Pten in pc-9/GR cells in comparison with pc-9 cells. Moreover, over-expression of miR-21 significantly decreased gefitinib sensitivity by down-regulating Pten expression and activating Akt and ERK pathways in pc-9 cells, while miR-21 knockdown dramatically restored gefitinib sensitivity of pc-9/GR cells by up-regulation of Pten expression and inactivation of AKT and ERK pathways, in vivo and in vitro. We propose alteration of miR-21/Pten expression as a novel mechanism for TKI resistance in NSCLC cancer. Our findings provide a new basis for using miR 21/Pten-based therapeutic strategies to reverse gefitinib resistance in NSCLC.     

Detection and Clinical Significance of Intratumoral EGFR Mutational Heterogeneity in Chinese Patients with Advanced Non-Small Cell Lung Cancer

Purpose

This study evaluated occurrence and potential clinical significance of intratumoral EGFR mutational heterogeneity in Chinese patients with non-small cell lung cancer (NSCLC).

Materials and Methods

Eighty-five stage IIIa-IV NSCLC patients who had undergone palliative surgical resection were included in this study. Of these, 45 patients carried EGFR mutations (group-M) and 40 patients were wild-type (group-W). Each tumor sample was microdissected to yield 28–34 tumor foci and Intratumoral EGFR mutation were determined using Denaturing High Performance Liquid Chromatography (DHPLC) and Amplification Refractory Mutation System (ARMS). EGFR copy numbers were measured using fluorescence in situ hybridization (FISH).

Results

Microdissection yielded 1,431 tumor foci from EGFR mutant patients (group-M) and 1,238 foci from wild-type patients (group-W). The EGFR mutant frequencies in group-M were 80.6% (1,154/1,431) and 87.1% (1,247/1,431) using DHPLC and ARMS, respectively. A combination of EGFR-mutated and wild-type cells was detected in 32.9% (28/85) of samples by DHPLC and 28.2% (24/85) by ARMS, supporting the occurrence of intratumoral heterogeneity. Thirty-one patients (36.5%) were identified as EGFR FISH-positive. Patients harboring intratumoral mutational heterogeneity possessed lower EGFR copy numbers than those tumors contained mutant cells alone (16.7% vs. 71.0%, P<0.05). Among 26 patients who had received EGFR-TKIs, the mean EGFR mutation content was higher in patients showing partial response (86.1%) or stable disease (48.7%) compared with patients experiencing progressive disease (6.0%) (P = 0.001). There also showed relationship between progression-free survival (PFS) and different content of EGFR mutation groups (pure wild type EGFR, EGFR mutation with heterogeneity and pure mutated EGFR) (P = 0.001).

Conclusion

Approximately 30% of patients presented intratumoral EGFR mutational heterogeneity, accompanying with relatively low EGFR copy number. EGFR mutant content was correlated with the response and prognosis of EGFR-TKIs.