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Transcriptome analysis of circulating tumor cells (CTCs), which migrate into blood vessels from primary tumor tissues, at the single-cell level offers critical insights into the biology of metastasis and contributes to drug discovery. However, transcriptome analysis of single CTCs has only been reported for a limited number of cancer types, such as multiple myeloma, breast, hepatocellular, and prostate cancer. Herein, we report the transcriptome analysis of gastric cancer single-CTCs. We utilized an antigen-independent strategy for CTC isolation from metastatic gastric cancer patients involving a size-dependent recovery of CTCs and a single cell isolation technique. The transcriptomic profile of single-CTCs revealed that a majority of gastric CTCs had undergone epithelial-mesenchymal transition (EMT), and indicated the contribution of platelet adhesion toward EMT progression and acquisition of chemoresistance. Taken together, this study serves to employ CTC characterization to elucidate the mechanisms of chemoresistance and metastasis in gastric cancer.
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Células Neoplásicas Circulantes , Neoplasias Gástricas , Transcriptoma/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Humanos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Análise de Célula Única , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Poly (ADP-ribose) glycohydrolase (PARG) is the main enzyme responsible for catabolism of poly (ADP-ribose) (PAR), synthesized by PARP. PARG dysfunction sensitizes certain cancer cells to alkylating agents and cisplatin by perturbing the DNA damage response. The gene mutations that sensitize cancer cells to PARG dysfunction-induced death remain to be identified. Here, we performed a comprehensive analysis of synthetic lethal genes using inducible PARG knockdown cells and identified dual specificity phosphatase 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction. DUSP22 is considered a tumor suppressor and its mutation has been frequently reported in lung, colon, and other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells reduced survival compared with single-knockdown counterparts. Dual depletion of PARG and DUSP22 increased the apoptotic sub-G1 fraction and upregulated PUMA in lung cancer A549, PC14, and SBC5 cells, and inhibited the PI3K/AKT/mTOR pathway in A549 cells, suggesting that dual depletion of PARG and DUSP22 induced apoptosis by upregulating PUMA and suppressing the PI3K/AKT/mTOR pathway. Consistently, the growth of tumors derived from double knockdown A549 cells was slower compared with those derived from control siRNA-transfected cells. Taken together, these results indicate that DUSP22 deficiency exerts a synthetic lethal effect when combined with PARG dysfunction, suggesting that DUSP22 dysfunction could be a useful biomarker for cancer therapy using PARG inhibitors. SIGNIFICANCE: This study identified DUSP22 as a novel synthetic lethal gene under the condition of PARG dysfunction and elucidated the mechanism of synthetic lethality in lung cancer cells.
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Glicosídeo Hidrolases/efeitos adversos , Neoplasias Pulmonares/genética , Animais , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , TransfecçãoRESUMO
About 5% of Triple negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) experience progressive disease (PD). Few reports are published on TNBCs with PD during NAC, whereas TNBCs that respond to NAC have been well-studied. We investigated kinase activity profiles of TNBCs to explore the biological differences underlying the lack of response to NAC. Among 740 TNBCs, 20 non-responders were identified. Seven non-responders and 10 TNBCs that did not receive NAC (control group) were evaluated. No correlation was observed between NAC response and age, menopausal status, tumor size and axillary lymph node status. Tyrosine kinase activity profiles of TNBC primary tissues from NAC non-responders and the controls were determined with a peptide microarray system. Kinase activity measurements showed that 35 peptides had significantly (p < 0.05) lower phosphorylation in non-responders. ZAP70, LCK, SYK and JAK2 were identified as differentially active upstream kinases. Pathway analysis suggested lower activity in immune-related pathways in non-responders. The number of tumor infiltrating lymphocytes (TILs) was significantly lower (p = 0.0053) in non-responders. Kinases related to the immune system are less activated in non-responders. TILs evaluation suggested that the immune system is hardly active in non-responders and is not activated by NAC treatment.
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We investigated the plasma levels of tumor-specific cell-free DNA (cfDNA) in 17 stage I-II (early) and IV (advanced) non-small cell lung cancer (NSCLC) patients who underwent radiotherapy. Digital polymerase chain reaction (PCR) and targeted sequencing showed that total and tumor-specific cfDNA levels increased in response to radiotherapy in both early- and advanced-stage NSCLC patients. We detected high copy numbers of epidermal growth factor receptor mutations (L858R and T790M) in the cfDNA samples from stage IV NSCLC patients who underwent stereotactic body radiation therapy to treat brain metastasis related to tyrosine kinase inhibitor (TKI) treatment failure. In conclusion, our study demonstrates that radiotherapy increases tumoral cfDNA levels in the plasma and shows potential to serve as an indicator for diagnosing drug-resistant tumor-related gene mutations in early-stage NSCLC patients or those undergoing molecular targeted therapy.
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[This corrects the article DOI: 10.18632/oncotarget.25053.].
RESUMO
Poly(ADP-ribose) polymerases (PARPs) family proteins catalyze poly(ADP-ribosylation) (PARylation) by conjugating ADP-ribose residues repeatedly on amino acid residues using nicotinamide adenine dinucleotide as a substrate. The inhibitors of PARP widely block DNA repair processes and are currently examined in clinical trials of cancer therapy. Poly(ADP-ribose) glycohydrolase (PARG) is the main nuclear enzyme, which digests poly(ADP-ribose) into ADP-ribose. PARG inhibitor could also be considered as a chemotherapeutic agent for cancer, because of its involvement in DNA repair. Various PARG inhibitors with IC50 value of micromolar to submicromolar range have been reported. However, for most of these chemicals, the specificity of inhibition has not been fully evaluated. PARG functional inhibition models in various organisms have been developed. Here, inducible PARG knockdown system was developed in HeLa cells and the cell line will be useful for identifying the synthetic lethal genes or affecting genes for PARG inhibitor treatment and also for functional elucidation of PARP superfamily molecules.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/química , Modelos Biológicos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Células HeLa , Humanos , Mutação , Fenótipo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNARESUMO
Various types of circulating tumor cell (CTC) detection systems have recently been developed that show a high CTC detection rate. However, it is a big challenge to find a system that can provide better prognostic value than CellSearch in head-to-head comparison. We have developed a novel semi-automated CTC enumeration system (fluidic cell microarray chip system, FCMC) that captures CTC independently of tumor-specific markers or physical properties. Here, we compared the CTC detection sensitivity and the prognostic value of FCMC with CellSearch in breast cancer patients. FCMC was validated in preclinical studies using spike-in samples and in blood samples from 20 healthy donors and 22 breast cancer patients in this study. Using spike-in samples, a statistically higher detection rate (p=0.010) of MDA-MB-231 cells and an equivalent detection rate (p=0.497) of MCF-7 cells were obtained with FCMC in comparison with CellSearch. The number of CTC detected in samples from patients that was above a threshold value as determined from healthy donors was evaluated. The CTC number detected using FCMC was significantly higher than that using CellSearch (p=0.00037). CTC numbers obtained using either FCMC or CellSearch had prognostic value, as assessed by progression free survival. The hazard ratio between CTC+ and CTC- was 4.229 in CellSearch (95% CI, 1.31 to 13.66; p=0.01591); in contrast, it was 11.31 in FCMC (95% CI, 2.245 to 57.0; p=0.000244). CTC detected using FCMC, like the CTC detected using CellSearch, have the potential to be a strong prognostic factor for cancer patients.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/terapia , Estudos de Casos e Controles , Contagem de Células , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Resultado do TratamentoRESUMO
Methods for the enumeration and molecular characterization of circulating tumor cells (CTC) have been actively investigated. However, such methods are still technically challenging. We have developed a novel epithelial cell adhesion molecule independent CTC enumeration system integrated with a sorting system using a microfluidics chip. We compared the number of CTC detected using our system with those detected using the CellSearch system in 46 patients with various cancers. We also evaluated epidermal growth factor receptor (EGFR) and PIK3CA mutations of captured CTC in a study of 4 lung cancer and 4 breast cancer patients. The percentage of samples with detected CTC was significantly higher with our system (65.2%) than with CellSearch (28.3%). The number of detected CTC per patient using our system was statistically higher than that using CellSearch (median 5, 0; P = 0.000172, Wilcoxon test). In the mutation analysis study, the number of detected CTC per patient was low (median for lung, 4.5; median for breast, 5.5); however, it was easy to detect EGFR and PIK3CA mutations in the CTC of 2 lung and 1 breast cancer patient, respectively, using a commercially available kit. Our system is more sensitive than CellSearch in CTC enumeration of various cancers and is also capable of detecting EGFR and PIK3CA mutations in the CTC of lung and breast cancer patients, respectively.
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Células Neoplásicas Circulantes , Adulto , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/secundário , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Estudos de Casos e Controles , Contagem de Células , Linhagem Celular Tumoral , Separação Celular , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Receptores ErbB/genética , Feminino , Citometria de Fluxo , Humanos , Dispositivos Lab-On-A-Chip , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Deleção de SequênciaRESUMO
Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%-90.0%) and excellent linear performance (R2 = 0.8189-0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.
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Eletroforese/métodos , Células Neoplásicas Circulantes , Análise de Célula Única , Linhagem Celular Tumoral , HumanosAssuntos
Neoplasias da Mama/patologia , Carcinoma Papilar/secundário , Dispneia/etiologia , Hipertensão Pulmonar/etiologia , Neoplasias Pulmonares/secundário , Células Neoplásicas Circulantes/patologia , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Papilar/diagnóstico , Evolução Fatal , Feminino , Humanos , Neoplasias Pulmonares/diagnósticoRESUMO
Small cell lung cancer (SCLC) accounts for 15% of all lung cancer cases and is a highly lethal disease. For the last several decades, the standard treatment for SCLC has been deadlocked, and new therapeutic strategies are urgently needed. HER2 is a member of the HER family and has been reported to be overexpressed in 30% of SCLC cases with poor prognosis. However, the clinical relevance of HER2-targeted therapy for SCLC remains unclear. Here, we first identify that cytotoxic drugs induce significant HER2 overexpression through microRNA-125a (miR-125a) and miR-125b downregulation, which in turn act as a novel therapeutic target for trastuzumab-mediated cellular cytotoxicity in SCLC. In this study, we showed that treatment of the HER2-positive SCLC cells, SBC-3 and SBC-5, with cytotoxic drugs induced a significant upregulation of HER2. Cisplatin (CDDP) treatment of SCLC cells resulted in a significant downregulation of miR-125a and miR-125b. We confirmed that miR-125a and miR-125b bound to the 3'-untranslated regions of HER2 mRNA, and that downregulation of miR-125a and miR-125b resulted in upregulation of HER2 in SCLC cells, suggesting a relationship between cytotoxic drug exposure and miR-125/HER2 dysregulation. Furthermore, using a calcein assay, we demonstrated a significantly enhanced cytotoxic effect of CDDP and trastuzumab that was mediated via antibody-dependent cellular cytotoxicity. Finally, we clearly demonstrated the enhanced antitumor effect of these agents in an orthotopic lung cancer model in vivo. Our results offer a novel therapeutic strategy for HER2-positive SCLCs by using trastuzumab combined with cytotoxic drugs.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , MicroRNAs/genética , Receptor ErbB-2/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Trastuzumab/farmacologia , Regiões 3' não Traduzidas/genética , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Masculino , Camundongos SCID , MicroRNAs/metabolismo , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Trastuzumab/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This report describes a patient with intravascular large B-cell lymphoma (IVLBCL) with central nervous system involvement at the time of diagnosis who achieved complete remission for over 5 years in response to therapy. The patient, a 71 year-old woman, was previously healthy with the exception of taking verapamil for paroxysmal supraventricular tachycardia. She had presented with pyrexia and gradually progressive anemia. Brain magnetic resonance imaging revealed an infarct-like lesion in the pons, although no paralysis was observed. She was diagnosed with IVLBCL on the basis of random skin biopsy. After eight cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy, abnormal laboratory data had normalized, and no pontine lesion was evident on magnetic resonance imaging without receiving any intrathecal chemotherapy. IVLBCL is associated with poor prognosis, particularly in patients with central nervous system involvement. Early initiation of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy and drug interactions between anticancer agents and verapamil as a p-glycoprotein inhibitor were considered the possible reasons for favorable outcome in the present case.
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Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O6-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.
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Dacarbazina/análogos & derivados , Difosfonatos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Imidazóis/farmacologia , O(6)-Metilguanina-DNA Metiltransferase/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dacarbazina/farmacologia , Sinergismo Farmacológico , Feminino , Glioma/genética , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido Zoledrônico , Proteínas ras/metabolismoRESUMO
BACKGROUND: Lenvatinib (E7080), an oral multi-kinase inhibitor, has inhibitory action on tumor cell proliferation and tumor angiogenesis in preclinical models. We evaluated correlations between pharmacodynamic (PD) biomarkers with patient clinical outcomes in a lenvatinib phase 1 dose-escalation study. METHODS: Plasma angiogenic proteins were evaluated as potential PD biomarkers of response to lenvatinib in a dose-escalation phase 1 study. Lenvatinib was administered to 27 patients by twice-daily dosing in 3-week cycles; 2 weeks of treatment followed by 1 week of rest until discontinuation. Blood samples for plasma proteins were collected on days 1 (baseline), 8, and 15 of cycle 1, and days 1, 8, and 15 of cycle 2. Selected clinical outcomes, including tumor shrinkage and adverse events (AEs), were used for correlative analyses of pharmacokinetic parameters and PD biomarkers. RESULTS: Tumor shrinkage and changes in PD biomarkers (increased vascular endothelial growth factor [VEGF] and stromal cell-derived factor 1 alpha [SDF1α] levels and decreased soluble VEGF receptor 2 [sVEGFR2] levels) significantly correlated with increasing lenvatinib exposure. Observed changes in levels of VEGF, SDF1α, and sVEGFR2 were maintained on day 15 of cycle 1, but returned to baseline during the 1-week rest period, and similar changes were induced by reinstitution of treatment in cycle 2. The worst grades of hypertension, proteinuria, and fatigue were associated with changes in VEGF and HGF at day 8 of cycle 1. Maximum tumor shrinkage was correlated with increased SDF1α levels. Decreased sVEGFR2 level was also correlated with tumor shrinkage and frequency of hypertension, proteinuria, and fatigue. Tumor shrinkage significantly correlated with the worst grade of proteinuria, but not with hypertension or fatigue. CONCLUSION: PD biomarker changes observed in plasma angiogenic proteins are correlated with lenvatinib-induced tumor shrinkage and AEs. Our findings warrant further assessment of plasma proteins associated with angiogenesis as potential biomarkers of lenvatinib activity. TRIAL REGISTRATION: ClinicalTrial.gov: NCT00280397 (January 20, 2006).
Assuntos
Proteínas Angiogênicas/sangue , Biomarcadores Farmacológicos/sangue , Neoplasias/tratamento farmacológico , Compostos de Fenilureia/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Quinolinas/administração & dosagem , Proteínas Sanguíneas/metabolismo , Progressão da Doença , Esquema de Medicação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/patologia , Compostos de Fenilureia/efeitos adversos , Compostos de Fenilureia/farmacocinética , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Quinolinas/efeitos adversos , Quinolinas/farmacocinética , Resultado do TratamentoRESUMO
BACKGROUND: We previously reported the role of IL-6 in a murine model of cancer cachexia and currently documented a patient in whom tocilizumab, anti-IL-6 receptor antibody, dramatically improved cachexia induced by IL-6 over-expressing lung cancer. Despite this potential to alleviate cancer cachexia, tocilizumab has not been approved for this clinical use. Therefore, preceding our planned clinical trial of tocilizumab, we designed the two studies described here to evaluate the levels of IL-6 in patients with lung cancer and the effect of tocilizumab in a murine model of human cancer cachexia. METHODS: First, we measured serum IL-6 levels in patients with lung cancer and analyzed its association with cachexia and survival. Next, we examined the effect of a rodent analog of tocilizumab (MR16-1) in the experimental cachexia model. RESULTS: Serum IL-6 levels were higher in patients with cachexia than those without cachexia. In patients with chemotherapy-resistant lung cancer, a high IL-6 serum level correlated strongly with survival, and the cut-off level for affecting their prognosis was 21 pg/mL. Meanwhile, transplantation of IL-6-expressing Lewis Lung Carcinoma cells caused cachexia in mice, which then received either MR16-1 or 0.9% saline. Tumor growth was similar in both groups; however, the MR16-1 group lost less weight, maintained better food and water intake and had milder cachectic features in blood. MR16-1 also prolonged the survival of LLC-IL6 transplanted mice (36.6 vs. 28.5 days, p = 0.016). CONCLUSION: Our clinical and experimental studies revealed that serum IL-6 is a surrogate marker for evaluating cachexia and the prognosis of patients with chemotherapy resistant metastatic lung cancer and that tocilizumab has the potential of improving prognosis and ameliorating the cachexia that so devastates their quality of life. This outcome greatly encourages our clinical trials to evaluate the safety and efficacy of tocilizumab treatment for patients with increased serum IL-6.
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Anticorpos Monoclonais Humanizados/administração & dosagem , Caquexia/tratamento farmacológico , Carcinoma Pulmonar de Lewis/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Interleucina-6/sangue , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/sangue , Caquexia/sangue , Caquexia/patologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Receptores de Interleucina-6/sangueRESUMO
BACKGROUND: Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis; however, the technical difficulties in isolating and analyzing rare CTCs have limited progress to date. In this preclinical study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology. METHODS: We developed a cell capture platform using On-chip Sort (On-Chip Biotechnologies), a novel bench-top cell sorter equipped with a disposable microfluidic chip. Spike-in experiments comprising a series of lung cancer cell lines with varying epithelial cell adhesion molecule (EpCAM) expression levels were conducted to assess the capture and purification efficiency of the platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by sample fixation and labeling. The enriched and labeled samples were then sorted by On-chip Sort based on cytokeratin, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification followed by mutation analysis using deep targeted sequencing, and copy number analysis using quantitative polymerase chain reaction (qPCR). RESULTS: Spike-in experiments revealed an excellent overall mean capture rate of 70.9%. A 100% success rate in the detection of EGFR, KRAS and BRAF mutations from captured cells was achieved using pyrosequencing and deep sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR in captured tumor cells. CONCLUSIONS: Using a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.
Assuntos
Neoplasias Pulmonares/sangue , Células Neoplásicas Circulantes , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Receptores ErbB/genética , Citometria de Fluxo , Genes ras , Humanos , Separação Imunomagnética , Neoplasias Pulmonares/genética , Mutação , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Epidermal growth factor receptor (EGFR) and PI3K/mTOR pathway are drug targets for non-small cell lung cancer (NSCLC). Herein, we investigated anti-tumor effects of the combination of BGT226, a novel PI3K/mTOR dual inhibitor, and gefitinib on NSCLC cell lines which are high sensitive to gefitinib. The combination of BGT226 and gefitinib exhibited supra-additive growth inhibitory effects in PC-9 and HCC827 cells. Apoptotic induction and the inhibition of PI3K/mTOR signaling were enhanced by the combination. Significant tumor growth suppression was observed in xenograft model by the combination. These results suggest that the combination is effective in EGFR inhibitor-sensitive NSCLC therapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Imidazóis/farmacologia , Neoplasias Pulmonares/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/farmacologia , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Gefitinibe , Humanos , Imidazóis/administração & dosagem , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Quinazolinas/administração & dosagem , Quinolinas/administração & dosagem , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as "gefitinib-resistant persisters" (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1-all genes involved in stemness-were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Quinazolinas/farmacologia , Receptor IGF Tipo 1/metabolismo , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Separação Celular , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Gefitinibe , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos Endogâmicos NOD , Mutação/genética , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Peptídeos/metabolismo , Quinazolinas/uso terapêutico , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Small-cell lung cancer in young patients is very rare and has not been adequately described. In addition, malignancies associated with genetic rearrangements of nuclear protein of the testis (NUT) have been reported in young patients. PATIENTS AND METHODS: We reviewed the clinical records of patients younger than 40 years of age who had been diagnosed as having SCLC and had been treated for this condition. We also examined NUT rearrangements using immunohistochemistry (IHC) staining and fluorescence in situ hybridization (FISH) analysis. RESULTS: We evaluated the diagnoses and treatment outcomes of 8 young patients among 747 SCLC patients. Based on further analyses using IHC staining and FISH, NUT rearrangements were found in 2 of these cases. The range of the overall survival period was 3.6 to 49.7 months. The 2 patients with NUT rearrangements survived for less than 12 months. CONCLUSION: NUT rearrangements were identified in 2 patients who had been previously diagnosed as having SCLC. Further attention regarding the diagnosis of SCLC in young patients is needed.
Assuntos
Neoplasias Pulmonares/diagnóstico , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Adolescente , Adulto , Fatores Etários , Feminino , Rearranjo Gênico , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Neoplasias , Estudos Retrospectivos , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Taxa de Sobrevida , Adulto JovemRESUMO
Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that targets the extracellular domain of epidermal growth factor receptor (EGFR). Oncogenic KRAS mutations in tumors have been shown to be a negative predictor of the response of colorectal cancer (CRC) to cetuximab treatment. Cetuximab exerts its therapeutic effects through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC). However, the influence of KRAS mutations on cetuximab-mediated ADCC is not fully understood. Here, we investigated cetuximab-mediated ADCC in two pairs of isogenic CRC cells with or without a KRAS mutation. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and NK92, a natural killer (NK) cell line that exogenously expresses FcγRIIIa (CD16a), were used as effector cells. In an ADCC assay, perforin-dependent target cell lysis was not affected by the KRAS mutation status. On the other hand, perforin-independent ADCC was observed only in CRC cells with wild-type KRAS, but not in cells with mutant KRAS. Neutralizing experiments revealed that the Fas-Fas ligand (FasL) interaction was responsible for the induction of apoptosis and perforin-independent ADCC. Furthermore, the presence of effector cells clearly enhanced the growth-inhibitory effect of cetuximab only in CRC cells with wild-type KRAS, but not in those with mutant KRAS. These findings suggest that ADCC is an important mode of action of cetuximab and that KRAS mutation impairs the therapeutic effect exerted by cetuximab-mediated ADCC.