Kang-Yi Su

Pretreatment Epidermal Growth Factor Receptor (EGFR) T790M Mutation Predicts Shorter EGFR Tyrosine Kinase Inhibitor Response Duration in Patients With Non–Small-Cell Lung Cancer

PURPOSE Patients with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations have excellent response to EGFR tyrosine kinase inhibitors (TKIs), but T790M mutation accounts for most TKI drug resistance. This study used highly sensitive methods to detect T790M before and after TKI therapy and investigated the association of T790M and its mutation frequencies with clinical outcome. PATIENTS AND METHODS Direct sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and next-generation sequencing (NGS) were used to assess T790M in the following two cohorts of patients with NSCLC: TKI-naive patients (n = 107) and TKI-treated patients (n = 85). Results were correlated with TKI treatment response and survival. RESULTS MALDI-TOF MS was highly sensitive in detecting and quantifying the frequency of EGFR-activating mutations and T790M (detection limits, 0.4% to 2.2%). MALDI-TOF MS identified more T790M than direct sequencing in TKI-naive patients with NSCLC (27 of 107 patients, 25.2% v three of 107 patients, 2.8%, respectively; P < .001) and in TKI-treated patients (before TKI: 23 of 73 patients, 31.5% v two of 73 patients, 2.7%, respectively; P < .001; and after TKI: 10 of 12 patients, 83.3% v four of 12 patients, 33.3%, respectively; P = .0143). The EGFR mutations and their frequencies were confirmed by NGS. T790M was an independent predictor of decreased progression-free survival (PFS) in patients with NSCLC who received TKI treatment (P < .05, multivariate Cox regression). CONCLUSION T790M may not be a rare event before or after TKI therapy in patients with NSCLC with EGFR-activating mutations. The pretreatment T790M mutation was associated with shorter PFS with EGFR TKI therapy in patients with NSCLC.

Enterovirus-Induced miR-141 Contributes to Shutoff of Host Protein Translation by Targeting the Translation Initiation Factor eIF4E

Viruses rely on the host translation machinery to complete their life cycles. Picornaviruses use an internal ribosome entry site to initiate cap-independent protein translation and in parallel host cap-dependent translation is shut off. This process is thought to occur primarily via cleavage of host translation initiation factors eIF4GI and eIF4GII by viral proteases. Here we describe another mechanism whereby miR-141 induced upon enterovirus infection targets the cap-dependent translation initiation factor, eIF4E, for shutoff of host protein synthesis. Knockdown of miR-141 reduces viral propagation, and silencing of eIF4E can completely reverse the inhibitory effect of the miR-141 antagomiR on viral propagation. Ectopic expression of miR-141 promotes the switch from cap-dependent to cap-independent translation. Moreover, we identified a transcription factor, EGR1, which is partly responsible for miR-141 induction in response to enterovirus infection. Our results suggest that upregulation of miR-141 upon enterovirus infection can facilitate viral propagation by expediting the translational switch.

Slug Confers Resistance to the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

RATIONALE Non-small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown. OBJECTIVES To examine the role of EMT regulators in resistance to gefitinib. METHODS The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription-polymerase chain reaction and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from 44 patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS Slug expression, but not that of snail, twist, or zeb-1, was significantly increased in PC9/gef compared with PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib-induced apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim up-regulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naive cancer cells. CONCLUSIONS Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

Mice Deficient in Collapsin Response Mediator Protein-1 Exhibit Impaired Long-Term Potentiation and Impaired Spatial Learning and Memory

Collapsing response mediator protein-1 (CRMP-1) was initially identified in brain and has been implicated in plexin-dependent neuronal function. The high amino acid sequence identity among the five CRMPs has hindered determination of the functions of each individual CRMP. We generated viable and fertile CRMP-1 knock-out (CRMP-1−/−) mice with no evidence of gross abnormality in the major organs. CRMP-1−/− mice exhibited intense microtubule-associated protein 2 (MAP2) staining in the proximal portion of the dendrites, but reduced and disorganized MAP2 staining in the distal dendrites of hippocampal CA1 pyramidal cells. Immunoreactivity to GAP-43 (growth-associated protein-43) and PSD95 (postsynaptic density-95) (a postsynaptic membrane adherent cytoskeletal protein) was also decreased in the CA1 region of the knock-out mice. These changes were consistent with the mutant mice showing a reduction in long-term potentiation (LTP) in the CA1 region and impaired performance in hippocampal-dependent spatial learning and memory tests. CRMP-1−/− mice showed a normal synapsin I labeling pattern in CA1 and normal paired-pulse facilitation. These findings provide the first evidence suggesting that CRMP-1 may be involved in proper neurite outgrowth in the adult hippocampus and that loss of CRMP-1 may affect LTP maintenance and spatial learning and memory.

RNA is favourable for analysing EGFR mutations in malignant pleural effusion of lung cancer

Malignant pleural effusion (MPE) is a useful specimen allowing for the evaluation of EGFR status in nonsmall cell lung cancer (NSCLC). However, direct sequencing of genomic DNA from MPE samples was found not to be sensitive for EGFR mutation detection. To test whether EGFR analysis from RNA is less prone to interference from nontumour cells that have no or lower EGFR expression, we compared three methods (sequencing from cell-derived RNA versus sequencing and mass-spectrometric analysis from genomic DNA), in parallel, for EGFR mutation detection from MPE samples in 150 lung adenocarcinoma patients receiving first-line tyrosine kinase inhibitors (TKIs). Among these MPE samples, EGFR mutations were much more frequently identified by sequencing using RNA than by sequencing and mass-spectrometric analysis from genomic DNA (for all mutations, 67.3 versus 44.7 and 46.7%; for L858R or exon 19 deletions, 61.3 versus 41.3 and 46.7%, respectively). The better mutation detection yield of sequencing from RNA was coupled with the superior prediction of clinical efficacy of first-line TKIs. In patients with acquired resistance, EGFR sequencing from RNA provided satisfactory detection of T790M (54.2%). These results demonstrated that EGFR sequencing using RNA as template greatly improves sensitivity for EGFR mutation detection from samples of MPE, highlighting RNA as the favourable source for analysing EGFR mutations from heterogeneous MPE specimens in NSCLC.

Rutin, a Flavonoid and Principal Component of Saussurea Involucrata, Attenuates Physical Fatigue in a Forced Swimming Mouse Model

This study investigated the antifatigue effects of rutin, a flavonoid extracted from the ethyl acetate extract of S. involucrata. Mice were subjected to a weight-loaded forced swim test (WFST) on alternate days for 3 wk. Rutin was administered orally to the mice for 7 days in dosages of 15, 30, and 60 mg/kg body weight, and several biomarkers of physical fatigue were evaluated: swimming time, change in body weight, lipid peroxidation, lactic acid (LA), glycogen, and the activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). On Day 7, the rutin-treated mice had a 3-fold longer exhaustive swimming time than the control mice, as well as significantly reduced blood LA concentrations. The 15, 30, and 60 mg/kg body weight rutin-supplemented groups displayed 11.2%, 22.5%, and 37.7% reduced malondialdehyde (MDA) concentrations, respectively, in brain and muscle tissues compared with the control exercised group. Our results indicated that the administration of rutin protected the mice against the depletion of SOD and GPx activities significantly. Following 7 days of rutin treatment, we sacrificed the mice and analyzed their soleus muscle and brain for peroxisome proliferator-activated receptor-α coactivator (PGC-1α) and sirtuin 1 (SIRT1) mRNA expression. We observed that rutin treatment increased PGC-1α and SIRT1 mRNA and protein expression. The changes in these markers of mitochondrial biogenesis were associated with increased maximal endurance capacity. The application of 2D gel electrophoresis to analyze the rutin-responsive protein profiles in the WFST mouse brain further revealed the upregulation of the CB1 cannabinoid receptor-interacting protein 1, myelin basic protein, Rho GDP dissociation inhibitor (GDI) alpha, and TPI, indicating that rutin might inhibit anxiety through the upregulation of the expression of anxiety-associated proteins. Western blot analysis of MAPK expression further confirmed the antianxiety effects of rutin. Our study results thus indicate that rutin treatment ameliorates the various impairments associated with physical fatigue.

R331W Missense Mutation of Oncogene YAP1 Is a Germline Risk Allele for Lung Adenocarcinoma With Medical Actionability

PURPOSE Adenocarcinoma is the most dominant type of lung cancer in never-smoker patients. The risk alleles from genome-wide association studies have small odds ratios and unclear biologic roles. Here we have taken an approach featuring suitable medical actionability to identify alleles with low population frequency but high disease-causing potential. PATIENTS AND METHODS Whole-genome sequencing was performed for a family with an unusually high density of lung adenocarcinoma with available DNA from the affected mother, four affected daughters, and one nonaffected son. Candidate risk alleles were confirmed by matrix-assisted laser desorption ionization time of flight mass spectroscopy. Validation was conducted in an external cohort of 1,135 participants without cancer and 1,312 patients with lung adenocarcinoma. Family follow-ups were performed by genotyping the relatives of the original proband and the relatives of the identified risk-allele carriers. Low-dose computed tomography scans of the chest were evaluated for lung abnormalities. RESULTS YAP1 R331W missense mutation from the original family was identified and validated in the external controls and the cohort with lung adenocarcinoma. The YAP1 mutant-allele carrier frequency was 1.1% in patients with lung adenocarcinoma compared with 0.18% in controls (P = .0095), yielding an odds ratio (adjusted for age, sex, and smoking status) of 5.9. Among the relatives, YAP1-mutant carriers have overwhelmingly higher frequencies of developing lung adenocarcinoma or ground-glass opacity lung lesions than those who do not carry the mutation (10:0 v 1:7; P < .001). YAP1 mutation was shown to increase the colony formation ability and invasion potential of lung cancer cells. CONCLUSION These results implicated YAP1 R331W as an allele predisposed for lung adenocarcinoma with high familial penetrance. Low-dose computed tomography scans may be recommended to this subpopulation, which is at high risk for lung cancer, for personalized prevention and health management.

Clustered Genomic Alterations in Chromosome 7p Dictate Outcomes and Targeted Treatment Responses of Lung Adenocarcinoma With EGFR-Activating Mutations

PURPOSE Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been proven more effective for patients with lung adenocarcinoma with EGFR-activating mutation rather than wild type, the former group still includes approximately 30% nonresponders. The molecular basis of this substantial response heterogeneity is unknown. Our purpose was to seek molecular aberrations contributing to disease progression at the genome-wide level and identify the prognostic signature unique to patients with EGFR-activating mutation. PATIENTS AND METHODS We first investigated the molecular differences between tumors with EGFR-activating mutation and wild-type tumors by conducting high-density array comparative genomic hybridization on a collection of 138 adenocarcinoma tissues. We then used an independent group of 114 patients to validate the clinical relevance of copy-number alterations (CNAs) in predicting overall and disease-free survival. Finally, focusing on 23 patients with EGFR mutation receiving EGFR-TKI treatment, we investigated the association between CNAs and response to EGFR-TKIs. RESULTS We identified chromosome regions with differential CNAs between tumors with EGFR-activating mutation and wild-type tumors and found the aberration sites to cluster highly on chromosome 7p. A cluster of six representative chromosome 7p genes predicted overall and disease-free survival for patients with EGFR-activating mutation but not for those with wild type. Importantly, simultaneous presence of more genes with increased CNAs in this cluster correlated with less favorable response to EGFR-TKIs in patients with EGFR-activating mutation. CONCLUSION Our results shed light on why responses to EGFR-TKIs are heterogeneous among patients with EGFR-activating mutation. They may lead to better patient management in this population.

Dynamic Plasma EGFR Mutation Status as a Predictor of EGFR-TKI Efficacy in Patients with EGFR-Mutant Lung Adenocarcinoma

Background: Epidermal growth factor receptor (EGFR) mutation status in lung cancer can effectively predict EGFR-tyrosine kinase inhibitor (TKI) efficacy. We evaluated the role of dynamic plasma cell-free DNA EGFR mutation status in outcome prediction. Methods: Advanced lung adenocarcinoma patients were enrolled and prospectively observed for outcomes of EGFR-TKI treatment. Peptide nucleic acid–zip nucleic acid polymerase chain reaction clamp method was developed to assess EGFR mutations in matched tumor and serial plasma cell-free DNA specimens. Results: A total of 72 patients were enrolled in this study, of which 62 patients (86.1%) had EGFR-mutant tumors (34 patients with exon 19 deletions, and 28 patients with L858R). Pretreatment plasma used for EGFR mutation testing showed a sensitivity of 59.7% and a specificity of 100%. Detection sensitivity was significantly higher in stage IV-M1b patients compared with stage IIIb and IV-M1a patients (78.0% versus 23.8%, p < 0.001). All patients who presented with EGFR-mutant tumors received first-line EGFR-TKI therapy. The objective response rate and disease control rate were 74.2% and 82.3%, respectively. Median progression-free survival and overall survival were 8.8 months (95% CI: 6.6–11.0) and 20.5 months (95% CI 15.1–26.0), respectively. Failure to clear plasma EGFR mutations after EGFR-TKI treatment was an independent predictor of lower disease control rate (odds ratio 5.26 [95% CI: 1.13–24.44]; p = 0.034), shorter progression-free survival (hazard ratio: 1.97 [95% CI: 1.33–2.91]; p = 0.001), and shorter overall survival (hazard ratio: 1.82 [95% CI: 1.04–3.18], p = 0.036). Conclusion: Changes in plasma EGFR mutation status can be successfully assessed using the peptide nucleic acid–zip nucleic acid polymerase chain reaction clamp method and can serve as an independent outcome predictor.

3,4-Dihydroxytoluene, a metabolite of rutin, inhibits inflammatory responses in lipopolysaccharide-activated macrophages by reducing the activation of NF-κB signaling

BackgroundSaussurea involucrata (Kar. et Kir.) (S. involucrate), is a rare traditional Chinese medicinal herb. Rutin and hispidulin as well as their metabolites are flavonoids of the flavonol type that abound in S. involucrata, which has been reported to inhibit nonoxidative advanced glycation end products which was involved in physiological inflammation. This study aims to investigate the role of 3,4-dihydroxytoluene (DHT), a metabolite of rutin, in inflammatory inhibition and its involved mechanism.MethodsThis study utilized lipopolysaccharide (LPS) stimulated murine macrophage cell line RAW 264.7 as inflammatory model. The inhibitory effects of DHT were evaluated by the expression level of several inflammation markers such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 after LPS treatment. In addition, underlying mechanisms, the activation of mitogen-activated protein kinases (MAPKs) and NF-κB, were also investigated.ResultsOur results showed that DHT significantly suppressed the LPS-induced production of nitric oxide (NO), iNOS, and COX-2 in a dose-dependent manner without cytotoxicity. DHT also reduced the generation of proinflammatory cytokines majorly in tumor necrosis factor (TNF)-α and minor in interleukin (IL)-1β and IL-6. In addition, LPS-stimulated I-κBα phosphorylation and degradation followed by translocation of the nuclear factor κB (NF-kB)-p65 from the cytoplasm to the nucleus were attenuated after DHT treatment.ConclusionsCombined, the results suggest that DHT might exert anti-inflammatory effects in vitro in LPS stimulated RAW 264.7 macrophages and is potential in adjuvant treatment in inflammation disease.