Non-small Cell Lung Cancer Lung Research Articles

Abstract 4803: Limiting heme availability as an effective strategy to target lung cancer growth and progression in vivo

Abstract Lung cancer is the leading cause of cancer-related death in the US. About 85% of cases are non-small cell lung cancer (NSCLC). Several chemotherapeutic and targeted therapeutic agents are approved for treating lung cancer, but the five-year survival rate remains low. Thus, alternative approaches for are required for treatment of lung cancer. Several studies demonstrate that enhanced mitochondrial respiration or oxidative phosphorylation (OXPHOS) is a key feature of NSCLC. Heme is a central metabolic molecule that serves as a prosthetic group in several proteins involved in oxygen transport, utilization, and storage. Previous studies from our lab show elevated levels of heme synthesis, uptake, and oxygen utilizing hemoproteins in NSCLC cells compared to normal cells. The results also demonstrate that inhibiting heme synthesis selectively inhibits proliferation of NSCLC cells. We hypothesize that elevated levels of heme are used as fuel for enhanced OXPHOS by NSCLC cells. Therefore, targeting heme flux and function can be an effective way of targeting NSCLC growth and progression. If elevated heme metabolism is crucial for the tumorigenic functions of NSCLC cells, limiting heme availability may be effective for suppressing lung tumor growth and progression. Therefore, we tried to lower heme availability by utilizing bacterial hemophores. We designed several heme-targeting peptides (HTAs) and tested their ability to inhibit heme uptake in NSCLC cell lines, subcutaneous xenografts, and lung orthotopic xenografts. NSCLC cells expressing luciferase were used to implant subcutaneous xenografts or lung orthotopic xenografts in NOD/SCID mice. The mice were treated with HTA to check if limiting heme availability would delay growth in NSCLC tumor xenografts. Also, mice with lung orthotopic xenografts treated with HTA were treated with a well-known chemotherapeutic agent, docetaxel, to determine if HTA pre-treatment improves survival in these mice. In vivo bioluminiscence imaging (BLI) was used to analyze growth and progression of orthotopic lung xenografts. Our BLI data show that HTA treatment causes a significant decrease in radiance (total photons/second) in subcutaneous xenografts and lung orthotopic xenografts. This is supported by Hematoxylin and Eosin (H & E) staining in paraffin-embedded tissue sections from lung orthotopic xenografts and tumor volumes of subcutaneous xenograft tumors resected from mice. Furthermore, in mice with lung orthotopic xenografts, HTA pre-treatment leads to a marked improvement in survival of mice further treated with docetaxel as compared to docetaxel treatment alone. Our results indicate that limiting heme availability can be an effective strategy to limit growth and progression of NSCLC and can be used to increase their vulnerability to chemotherapeutic agents like docetaxel. Citation Format: Poorva Ghosh, Sarada Preeta Kalainayakan, Sanchareeka Dey, Adnin Ashrafi, Li Zhang. Limiting heme availability as an effective strategy to target lung cancer growth and progression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4803.

Abstract 4447: Deep neural network for automatic histopathologic analysis of murine lung tumors

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, and genetically-engineered mouse models (GEMMs) of cancer provide important mechanistic and preclinical insights into this deadly disease. In particular, the “KP” model enables lung-specific inducible activation of oncogenic Kras G12D, and loss of Trp53, the two most common driver events of human non-small cell lung cancer (NSCLC). Importantly, the KP model is widely used and faithfully recapitulates molecular and histopathological features of the human disease, including progression from early hyperplasia and adenoma to invasive adenocarcinoma. However, the KP model results in multi-focal and heterogeneous tumor burden, and there is a need for improved tools to increase throughput and decrease subjectivity of tumor burden quantification and histopathological analyses. To this end, we trained a convolutional neural network (CNN) for semantic multi-class segmentation using the Aiforia(R) platform. The CNN was trained to classify and detect lung parenchyma, NSCLC tumors, and NSCLC tumor grades (grade 1-4). For supervised training, we used selected areas from 93 hematoxylin and eosin stained slides. For validation, we analyzed 34 slides completely independent of the CNN training. Tumor grades were manually annotated on the validation slides blinded to the CNN results. The overall F1 score of the CNN in grade classification was 98% and total area error 0.3%. The grade-specific F1-scores were 89%, 97%, 99%, and 98% for grades 1, 2, 3, and 4, respectively. Corresponding grade-specific total area errors were 0.4%, 0.2%, 0.4%, and 0.1%. Manual scoring independent of the training and CNN yielded similar tumor burden and grading results. In addition, the algorithm accurately recapitulates the increased tumor burden and grade seen in KP tumors harboring additional mutation of the tumor suppressor Keap1, and the delayed kinetics of KP tumors harboring a strong T cell antigen, in independent datasets. We have also extended this methodology to identification of tumors in a GEMM of small cell lung cancer, a distinct class of lung cancer with poor prognosis. In conclusion, we demonstrate that deep neural networks can be used for automated analysis and grading of preclinical models of lung cancer. We anticipate that this powerful technology will increase the throughput, sensitivity and reproducibility of hypothesis-driven studies of factors influencing tumor progression and immune response in mouse models of lung cancer. Citation Format: Peter Maxwell Kienitz Westcott, Tuomas Pitkänen, Sami Blom, Thomas Westerling, Tuomas Ropponen, Nathan Sacks, Katherine Wu, Roderick Bronson, Tuomas Tammela, Tyler Jacks. Deep neural network for automatic histopathologic analysis of murine lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4447.

Abstract 955: Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC

Abstract Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple murine and human NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation and clonogenicity only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. Taken together, this data suggest direct regulation of EGFR protein expression by MSI2 at post transcriptional level. NSCLC lung tissue microarray immunohistochemistry analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. In vivo experiments of erlotinib treatments of wild type or MSI2 depleted tumors are ongoing and these results will be presented. Citation Format: Alexander Kudinov, Petr Makhov, Bulat Faezov, Igor Bychkov, Alexander Deneka, Emmanuelle Nicolas, Kathy Q Q. Caid, Rohan Brebione, Eleanor Avrilf, Anna S. Nikonova, Ilya G. Serebriiskii, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 955.

MiR-34b-3p represses cell proliferation, cell cycle progression and cell apoptosis in non-small-cell lung cancer (NSCLC) by targeting CDK4.

Lung cancer is the most common incident cancer, with a high mortality worldwide, and non‐small‐cell lung cancer (NSCLC) accounts for approximately 85% of cases. Numerous studies have shown that the aberrant expression of microRNAs (miRNAs) is associated with the development and progression of cancers. However, the clinical significance and biological roles of most miRNAs in NSCLC remain elusive. In this study, we identified a novel miRNA, miR‐34b‐3p, that suppressed NSCLC cell growth and investigated the underlying mechanism. miR‐34b‐3p was down‐regulated in both NSCLC tumour tissues and lung cancer cell lines (H1299 and A549). The overexpression of miR‐34b‐3p suppressed lung cancer cell (H1299 and A549) growth, including proliferation inhibition, cell cycle arrest and increased apoptosis. Furthermore, luciferase reporter assays confirmed that miR‐34b‐3p could bind to the cyclin‐dependent kinase 4 (CDK4) mRNA 3′‐untranslated region (3′‐UTR) to suppress the expression of CDK4 in NSCLC cells. H1299 and A549 cell proliferation inhibition is mediated by cell cycle arrest and apoptosis with CDK4 interference. Moreover, CDK4 overexpression effectively reversed miR‐34‐3p‐repressed NSCLC cell growth. In conclusion, our findings reveal that miR‐34b‐3p might function as a tumour suppressor in NSCLC by targeting CDK4 and that miR‐34b‐3p may, therefore, serve as a biomarker for the diagnosis and treatment of NSCLC.

Clinical significance of serum miR-25 in non-small-cell lung cancer

ABSTRACTBackground: MicroRNAs (miRNAs) are becoming recognized as novel diagnostic and prognostic biomarkers in several malignancies, including non-small-cell lung cancer (NSCLC). miR-25 is overexpressed in small cell lung cancer (SCLC) and NSCLC tissues, and high miR-25 expression is associated with poorer overall survival of women with lung ADC. We hypothesised links between serum miR-25 levels and clinicopathological characteristics, diagnosis and prognosis of NSCLC patients.Methods: Serum miR-25 was determined by real-time quantitative polymerase chain reaction in 128 NSCLC patients and 128 healthy controls, and links between miR-25 level and cliniopathological characteristics including diagnosis and prognosis were explored.Results: Median (IQR) serum miR-25 levels were significantly increased in NSCLC compared to healthy controls at 0.86 relative units (0.14–1.78) versus 0.23 (0.08–0.96) (P < 0.001). Using a cut-off of 0.67 units, miR-25 had a sensitivity of 76.4%, specificity of 84.6%, accuracy of 72.6%, positive predictive value of 92.8% and negative predictive value of 68.5% for the diagnosis of NSCLC. High serum miR-25 level was significantly associated with gender (P = 0.042), tumour stage (P = 0.014) and lymph node metastasis (P < 0.001). In multivariate analyses, miR-25 was an independent prognostic factor for overall survival and relapse-free survival.Conclusions: Serum levels of miR-25 could improve NSCLC screening, and be a useful diagnostic and prognostic marker of NSCLC.

Lung Cancer Part II

In the United States, lung cancer is the second most common cancer, surpassed only by prostate cancer in men and breast cancer in women. But lung cancer is the leading cause of cancer deaths, accounting for 29% and 26% of all cancer-related deaths in men and women, respectively. The four major pathologic cell types of lung cancer are small cell carcinoma, adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Because they have overlapping clinical behaviors and responses to treatment, adenocarcinoma, squamous cell carcinoma, and large cell carcinoma are generally grouped together in the category of non–small cell lung cancer (NSCLC). This review discusses treatment of both NSCLC and small cell lung cancer (SCLC). This review 2 figures, 19 tables, and 90 references. Keywords: lung cancer, mediastinoscopy, chemoradiotherapy, TNM staging system, pulmonary parenchyma, segmentectomy

LncRNA BANCR suppresses cell viability and invasion and promotes apoptosis in non-small-cell lung cancer cells in vitro and in vivo.

Background: As a leading cause of deaths worldwide, lung cancer is a collection of diseases with diverse etiologies which includes non-small-cell lung cancer (NSCLC). Increasing evidence reported that aberrant expression of BRAF activated non-coding RNA (BANCR) was involved in the tumorigenesis and progression of various malignancies.Purpose and methods: However, its role in NSCLC has not been completely clarified. In the present study, we identified the role of BANCR in the regulation of NSCLC cell viability, invasion, and apoptosis. Down-regulation of BANCR expression was significantly observed in different NSCLC cell lines (A549, H1299, H1650, H1975, SPC-A1, and PC-9), tumor tissue from NSCLC mouse model and 30 human NSCLC tissues compared with adjacent normal tissues.Result: Overexpression of BANCR in these six NSCLC cell lines attenuated the cell viability and invasion. An increased apoptotic level caused by BANCR overexpression was also detected and displayed a conversed influence on Bcl-2 and Bax expression in mRNA and protein level. Furthermore, we identified the effect of BANCR overexpression on tumor growth in NSCLC mouse model. The restoration of BANCR expression inhibits NSCLC.Conclusion: Taken together, our findings shed an insight on the novel molecular mechanisms of lung NSCLC oncogenesis and provide the information for new therapeutic approaches on the disease.

Pyrroline-5-Carboxylate Reductase 1 Accelerates the Migration and Invasion of Nonsmall Cell Lung Cancer In Vitro.

Background: Pyrroline-5-carboxylate reductase 1 (PYCR1) is involved in tumor progression, for instance, breast cancer and prostate cancer. However, its role in tumor metastasis, especially in nonsmall cell lung cancer (NSCLC), is still elusive. Materials and Methods: The messenger RNA (mRNA) expression of PYCR1 between NSCLC and normal lung specimens was compared using Oncomine database. The endogenous PYCR1 expressions in NSCLC cell lines 95C and H1299 were knocked down by lentiviral-mediated delivery of short hairpin RNA (shRNA). Then the effects of PYCR1 on the migration and invasion of NSCLC cells were studied by wound healing assay and transwell assay. Results: PYCR1 mRNA expression was significantly higher in NSCLC specimens than that in normal lung tissues. Depletion of PYCR1 in NSCLC cell significantly repressed the cell migration and invasion. Moreover, depletion of PYCR1 influenced the expression of epithelial-mesenchymal transition molecules E-cadherin, Vimentin, N-cadherin, and Snail1. Conclusions: Our data suggested that PYCR1 plays a positive role in NSCLC metastasis in vitro and might be a promising target for treating NSCLC.

Stathmin-1 Is a Useful Diagnostic Marker for High-Grade Lung Neuroendocrine Tumors

Stathmin-1 regulates microtubule dynamics and is associated with malignant phenotypes in non-small cell lung cancer (NSCLC). This study evaluated its diagnostic value for differentiating between NSCLC and high-grade lung neuroendocrine tumor (HGNET). Stathmin-1 protein expression was assessed by immunohistochemistry in 414 NSCLC (305 adenocarcinoma [AD], 102 squamous cell carcinoma [SCC], 7 large-cell carcinoma), 5 typical carcinoid (low-grade lung neuroendocrine tumor), and 34 HGNET (17 small-cell carcinoma [SCLC] and 17 large-cell neuroendocrine carcinoma [LCNEC]) surgical specimens and 57 NSCLC (29 AD and 28 SCC) and 42 HGNET (17 LCNEC and 25 SCLC) biopsy specimens. We also analyzed stathmin-1 mRNA levels in 81 NSCLCs and 26 HGNETs with the use of reverse transcription-polymerase chain reaction. Among NSCLC samples, we saw high stathmin-1 protein expression in only three ADs, one SCC, and one large-cell carcinoma surgical samples, all five of which showed neuroendocrine characteristics in pathologic re-review; and low or intermediate expression in all five typical carcinoid surgical samples and all 57 NSCLC biopsy samples. In contrast, all HGNET surgical (n= 34) and biopsy (n= 42) samples showed highstathmin-1 expression. In reverse transcription-polymerase chain reaction, stathmin-1 expression was significantly higher in HGNET tissues than in NSCLC tissues (p < 0.001). Stathmin-1 expression can help in differentiating NSCLC from HGNET.

Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer.

(1) The TBX2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) are markedly down-regulated in human non-small cell lung cancer (NSCLC) and exert tumor suppressor effects in lung malignancy. Yet, mechanisms underlying suppressed expression of the TBX2 subfamily in NSCLC are elusive. Here, we interrogated probable epigenetic mechanisms in suppressed expression of the TBX2 subfamily in human NSCLC. (2) TBX2 subfamily gene expression and methylation levels in NSCLC and normal lung tissues were surveyed using publicly available RNA-sequence and genome-wide methylation datasets. Methylation β-values of the four genes were statistically compared between NSCLCs and normal lung tissues, correlated with gene expression levels, and interrogated with clinicopathological variables. Expression and methylation levels of TBXs were quantified in NSCLC cells using real-time PCR and methylation-specific PCR assays, respectively. Effects of the DNA methyltransferase inhibitor 5-azacytidine (Aza) on TBX2 subfamily expression were assessed in NSCLC cells. Impact of TBX2 subfamily expression on Aza-treated cells was evaluated by RNA interference. (3) All four TBXs were significantly hypermethylated in NSCLCs relative to normal lung tissues (p < 0.05). Methylation β-values of the genes, with exception of TBX2, were significantly inversely correlated with corresponding mRNA expression levels (p < 0.05). We found no statistically significant differences in hypermethylation levels of the TBX2 subfamily by clinicopathological features including stage and tobacco history. Expression levels of the TBX genes were overall suppressed in NSCLC cells relative to normal alveolar cells. Members of the subfamily were significantly hypermethylated in all tested NSCLC cell lines relative to normal alveolar cells. Treatment with Aza induced the expression of the TBX2 subfamily concomitant with NSCLC cell growth inhibition. Further, simultaneous knockdown of the four TBX genes markedly reduced anti-growth effects of Aza in NSCLC cells. (4) Our study sheds light on new epigenetic profiles in the molecular pathogenesis of human NSCLC.

Association of EGFR 1 Gene Alteration and their Association with Lung Adenocarcinoma Patients

Background:The epidermal growth factor receptor 1 (EGFR1) plays a significant role in cell proliferation and development. Its regulation in humans is very critical and incompletely understood in Non small cell lung cancer (NSCLC).Methods:100 newly diagnosed NSCLC (lung adenocarcinoma) patients and 100 healthy controls were included and allele specific (AS) polymerase chain reaction (PCR) was used to genotype and expression was analyzed by quantitative real time PCR. Overall survival of patients was analyzed by Kaplan-Meier method and for prognostic significance ROC curve was plotted.Results:A statistically significant difference (p<0.0001) in CC, AA and CA genotypes distribution among patients and healthy controls was observed. Compared to the CC genotype as reference, OR was 30.40 (95%CI 1.75- 524.9, p=0.0002) and 3.97 (95%CI 1.49-10.52, p=0.003) for the homozygous AA and heterozygous CA genotypes respectively. Kaplan-Meier survival analysis was also performed to analyze the relationship of EGFR1 (-191C/A) genotypes with progression free median survival of NSCLC patients and the difference was found to be significantly (p=0.0002) associated with different genotypes. In the ROC curve with respect to TNM stage at optimal cut-off value of 9.88 fold increase in EGFR1 mRNA expression, sensitivity and specificity were 92.9%, 83.3% respectively (AUC=0.95, p<0.0001). ROC curve w.r.t. distant metastases at optimal cut-off value of 13.5 fold change EGFR1 mRNA expression, sensitivity and specificity were 68.2%, 71.4% respectively (AUC=0.81, p<0.0001). In ROC curve w.r.t to presence/ absence of pleural effusion at optimal cut-off value of 14.8 fold change EGFR1 mRNA expression sensitivity and specificity were 66.7%, 68.2% respectively (AUC=0.71, p=0.009).Conclusions:Study concluded EGFR1 promoter polymorphism could be a risk factor associated with disease and may be used as prognostic marker for patients’ survival and predictor for disease worseness.

CHAF1B promotes proliferation and reduces apoptosis in 95‑D lung cancer cells and predicts a poor prognosis in non‑small cell lung cancer.

Chromatin assembly factor 1 subunit B (CHAF1B) participates in DNA synthesis and repair. High CHAF1B expression has been associated with a poor prognosis in several types of cancers. However, no study has evaluated the clinical significance and biological function of CHAF1B in non‑small cell lung cancer (NSCLC). In the present study, we aimed to investigate CHAF1B expression and its role in NSCLC. In the present study, it was revealed that CHAF1B was highly expressed in NSCLC lung tissues and 95‑D cells. Kaplan‑Meier survival analysis indicated that high CHAF1B expression in tumour tissue was associated with poor clinical outcomes in NSCLC patients. Multivariate Cox analyses revealed that lymph node metastasis, tumour‑node‑metastasis (TNM) stage and CHAF1B expression were independent prognostic factors in NSCLC patients. Moreover, CHAF1B knockdown in 95‑D cells markedly inhibited tumour proliferation, reduced colony formation, induced cell cycle arrest and promoted apoptosis. In vivo studies demonstrated that CHAF1B knockdown inhibited the growth of transplanted tumours. Furthermore, our results revealed that the mechanism by which CHAF1B induced apoptosis was mediated by the activation of the p53‑dependent apoptotic signalling pathway (BAK/Bcl‑2/caspase‑3) in 95‑D cells. These data indicated that CHAF1B plays an important role in tumourigenesis and may be a therapeutic molecular target to counter NSCLC progression.

Proteome analysis of non-small cell lung cancer cell line secretomes and patient sputum reveals biofluid biomarker candidates for cisplatin response prediction

Molecular markers are urgently needed to select non-small cell lung cancer (NSCLC) patients most likely to benefit from platinum-based chemotherapies. Of particular interest are proteins that can be found in biofluids like sputum for non-invasive detection. Therefore, we profiled the secretomes of 6 NSCLC cell lines with varying IC50-values for cisplatin, using label-free GeLC-MS/MS-based proteomics. Out of a total dataset of 2610 proteins, 304 proteins showed significant differences in expression levels between cisplatin sensitive and insensitive cell lines. Functional data mining revealed that the secretion of typically extracellular factors was associated with a higher sensitivity towards cisplatin, while cisplatin insensitivity correlated with increased secretion of theoretically intra-cellular proteins. Stringent statistical analysis and quantitative filtering yielded 58 biomarker candidates, 34 of which could be detected in clinical biofluids of lung cancer patients such as sputum using label-free LC-MS/MS-based proteomics. To assess performance of these biofluid biomarker candidates, we correlated protein expression with patient survival using a publically available clinical gene expression data set (GSE14814). We thus identified 3 top candidates with potential predictive value in determining cisplatin response (UGGT1, COL6A1 and MAP4) for future development as non-invasive biomarkers to guide treatment decisions. SignificancePlatinum-based chemotherapies are still the standard of care for NSCLC and other lung cancer types in the clinic today. However, due to chemoresistance, many patients suffer from the toxic side effects of these treatments without gaining any benefit in terms of survival. To date, no molecular biomarkers are available to predict clinical outcome of platinum-based chemotherapy. Because proteins present the functional read-out of genetic, epigenetic and translational events in the cell, a protein test is likely to be particularly suitable for response prediction. Of high relevance are proteins that are shed or secreted from cells, for example at primary tumor sites, and can be found in easily accessible biofluids like sputum for non-invasive detection. Here, we report the proteome profiling of the conditioned media (secretomes) of a panel of NSCLC cell lines in relation to cisplatin IC50 values, as a pre-clinical model, and of patient sputum as a clinical, lung cancer relevant biofluid. Using this approach in conjunction with exploration of the predictive potential in a transcriptome lung cancer patient dataset, we reveal biofluid biomarker candidates that, with further validation, may be used for non-invasive cisplatin response prediction in the future.

Nuclear TAZ activity distinctly associates with subtypes of non-small cell lung cancer

The transcription co-factor TAZ plays critical roles in the regulation of human carcinogenesis. However, the pathological role for TAZ in lung cancer has remained incompletely understood. TAZ expression was examined by immunohistochemistry for 163 NSCLC tissues. TAZ expression was also examined by western blotting for 20 frozen paired NSCLC and adjacent normal lung tissues. We report that TAZ is overexpressed in non-small cell lung cancer (NSCLC) tissues and correlates with shorter patient survival. Intriguingly, we find that TAZ is overexpressed primarily in lung squamous cell carcinomas (LUSC) but not lung adenocarcinomas (LUAD) compared to normal lung tissues, and that the expression levels of TAZ are significantly higher in LUSC than LUAD. The nuclear localization of TAZ correlates worse clinical outcomes in LUSC, but not LUAD, further suggesting a prognostic value for activated TAZ in LUSC. A meta-analysis of the public datasets from TCGA, Broad institute, and Oncomine shows that the TAZ gene (WWTR1) copy numbers are significantly increased in LUSC and correlate with the increase of TAZ mRNA expression, suggesting that TAZ is overexpressed in LUSC at least partly through gene amplifications. Collectively, our results suggest that TAZ expression distinctly associates with subtypes of NSCLC and may be useful for developing novel therapeutics treating LUSC.

Expression of PD-1 and PD-L1 in the peripheral blood of advanced non-small-cell lung cancer patients and its implications

Objective: To investigate the expression of programmed death 1(PD-1) and programmed death ligand 1 (PD-L1) on T lymphocyte and monocyte from peripheral blood of advanced non-small-cell lung cancer (NSCLC) patients and its potential role in immune escape of NSCLC. Methods: Forty-eight patients with advanced NSCLC (Lung Cancer Group) were included from the Department of Respiratory Diseases in The Second Affiliated Hospital of Soochow University from June 2014 to June 2015. Thirty-six healthy volunteers who received health examination at the same time, matching in sex, age were also enrolled as controls. The expression of PD-1 on peripheral blood CD4(+)T cells and CD8(+)T cells and PD-L1 on monocytes were detected by flow cytometry. Patients who received chemotherapy alone for 2-4 cycles and received sequential sampling were assessed with Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1). Seven cases of patients with significant response to treatment were selected as partial response (PR) group and ten patients with poor response to treatment were treated as progression disease(PD) group. The differences in the expression of PD-1 on the surface of CD4(+)T cells, CD8(+)T cells, and PD-L1 on the surface of monocyte before and after treatment were analyzed. Results: Compared with healthy control group, PD-1 expression level on both CD4(+) T and CD8(+) T cells from peripheral blood in lung cancer group were significantly increased [(25.9±7.4)% vs (20.6±6.2)%, (19.9±9.8)% vs (14.0±5.6)%, both P<0.05]. A higher level of PD-L1 expression on monocyte in lung cancer group was also found compared with the control group [(33.1±15.1)% vs (13.6±5.3)%, P<0.001]. The expression level of PD-1 on CD4(+)T and CD8(+)T cells and PD-L1 on monocytes in lung cancer group with good response to treatment was relatively lower than the baseline level of before treatment [(22.8±8.5)% vs (25.9±7.8)%, (17.1±8.4)% vs (20.4±8.6)%, (18.1±6.9)% vs (31.3±13.2)%, all P<0.05], but in lung cancer group with poor response to treatment, it was higher than the baseline level of before treatment [(33.5±6.5)% vs (23.9±4.2)%, (25.2±9.1)% vs (19.1±8.8)%, (43.1±18.3)% vs (29.7±10.6)%, all P<0.05]. Conclusion: Abnormal expression of PD-1 and PD-L1 exists in T cells and monocytes respectively, prompting PD-1/PD-L1 pathway may inhibit T cell proliferation during the interaction of T cell and monocyte, which may lead to non-small cell lung cancer immune escape.

MiR-126-3p suppresses the growth, migration and invasion of NSCLC via targeting CCR1.

MicroRNA (miRNA) plays vital roles in the development of different cancers. In the current work, we explored the function of miR-126-3p in the growth and metastasis of non-small-cell lung cancer (NSCLC) cell in vitro and in vivo. The expressions of miR-126-3p in NSCLC cell lines were assessed using the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, wound healing and transwell invasion were applied to reveal the role of miR-126b-3p on NSCLC cell growth, migration and invasion. The expressions of epithelial-mesenchymal transition (EMT) associated markers (E-cadherin and N-cadherin) were assessed by immunofluorescence staining. The Xenograft model and lung metastasis model were applied to explore the impact of miR-126-3p on the growth and aggressiveness of NSCLC cell in vivo. MiR-126-3p was significantly down-regulated in NSCLC cell lines and tissues. The up-regulation of miR-126-3p inhibited the growth, colony formation, migration and invasion of NSCLC cell. Furthermore, the xenograft model indicated that miR-126-3p suppressed NSCLC cell growth and lung metastasis by targeting chemokine (C-C motif) receptor 1 (CCR1). In addition, we demonstrated that the over-expression of CCR1 rescued the inhibitory effects of miR-126-3p on NSCLC cells growth, migration and invasion. Finally, knocked-down of CCR1 was able to mimic the inhibitory effects of miR-126-3p on the progression of NSCLC cell. These findings indicate that miR-126-3p plays an important role in the growth, migration and invasion of NSCLC by targeting CCR1.

Is routine dissection of lymph node station 4L clinically significant in left lung NSCLC?

I read with special interest the article entitled “Clinical significance of 4L lymph node dissection in left lung cancer” published in Journal of Clinical Oncology in October 2018 (1). The authors did a retrospective analysis of their patients who underwent lung parenchyma resection for non-small cell lung cancer (NSCLC) of the left lung during the 5-year period 2005–2009. The main conclusions of their study were that dissection of the 4L lymph node station revealed a quite high rate of involvement by the tumor (20.9%), while 4L lymph node dissection was found to be an independent prognostic factor for improved disease-free survival (DFS) and overall survival (OS) in left lung NSCLC. In case the above-mentioned results will be reproduced in future studies, dissection of the 4L lymph node station will be definitely considered an essential step in the surgical management of left lung NSCLC which will affect the clinical practice and the guidelines for lymph node dissection of left lung NSCLC.

Anti-tumoral activity of the human-specific duplicated form of α7-nicotinic receptor subunit in tobacco-induced lung cancer progression

Objectives: Tobacco smoking is strongly correlated with the onset and progression of non-small cell lung cancer (NSCLC). By activating α7 nicotinic acetylcholine receptors (α7-nAChRs) in these tumors nicotine and its tobacco-derived nitrosamine, NNK, contribute to these oncogenic processes. Here, we investigated whether the human-specific duplicated form of the α7-nAChR subunit (dupα7) behaves as an endogenous negative regulator of α7-nAChR-mediated tumorigenic activity induced by tobacco in NSCLC cells, similarly to its influence on other α7-nAChR-controlled functions in non-tumor cells.Methods: Two human NSCLC cell lines, lung adenocarcinoma (A549) and squamous cell carcinoma of the lung (SK-MES-1), both wild-type or with stable overexpression of dupα7 (A549dupα7 or SK-MES-1dupα7), were used to investigate in vitro anti-tumor activity of dupα7 on nicotine- or NNK-induced tumor progression. For this purpose, migration, proliferation or epithelial-mesenchymal transition (EMT) were examined. The anti-tumor effect of dupα7 on nicotine-promoted tumor growth, proliferation or angiogenesis was also assessed in vivo in an athymic mouse model implanted with A549dupα7 or A549 xenografts.Results: Overexpression of dupα7 in both cell lines almost completely suppresses the in vitro tumor-promoting effects induced by nicotine (1 μM) or NNK (100 nM) in wild-type cells. Furthermore, in mice receiving nicotine, A549dupα7 xenografts show: (i) a significant reduction of tumor growth, and (ii) decreased expression of cell markers for proliferation (Ki67) or angiogenesis (VEGF) compared to A549 xenografts.Conclusion: Our study demonstrates, for the first time, the in vitro and in vivo anti-tumor capacity of dupα7 to block the α7-nAChR-mediated tumorigenic effects of tobacco in NSCLC, suggesting that up-regulation of dupα7 expression in these tumors could offer a potential new therapeutic target in smoking-related cancers.

MiRNA-19 promotes non-small-cell lung cancer cell proliferation via inhibiting CBX7 expression.

BackgroundmiR-19 is a critical carcinogenic miRNA that participates in important biological processes of human malignancies. CBX7 plays a key role in lung cancer development and progression. In the present study, for the first time, we investigated the correlation between miR-19 and CBX7 in non-small-cell lung cancer (NSCLC).MethodsmiR-19 expression in NSCLC tissues and lung cancer cell lines was detected using quantitative reverse transcriptase PCR (qRT-PCR). Luciferase reporter assay, qRT-PCR, Western blot, and immunohistochemical assay were conducted to identify the target reaction of miR-19 and CBX7. Moreover, the influence of miR-19 on lung cancer cell proliferation, migration, and invasion was studied including cell counting kit-8 assay, scratch assay, transwell assay, flow cytometry assay, and staining assays.ResultsmiR-19 was overexpressed in NSCLC tissues and lung cancer cell lines. Luciferase reporter assay demonstrated that miR-19 could inhibit CBX7 expression via binding to the 3′-UTR of CBX7. Furthermore, miR-19 remarkably decreased CBX7 protein and mRNA expression. Additionally, overexpression of miR-19 could significantly enhance lung cancer cell proliferation and migration.ConclusionmiR-19 functions as a tumor accelerator promoting lung cancer cell proliferation through targeting CBX7 and inhibiting its expression.

Comparison of predictive powers of functional and anatomic dosimetric parameters for radiation-induced lung toxicity in locally advanced non-small cell lung cancer

PurposeTo investigate the predictive value of the perfusion (Q) single-photon emission computed tomography (SPECT)-weighted dose–function histogram (DFH) obtained mid-treatment (mid-Tx) with radiotherapy (RT) for radiation-induced lung toxicity (RILT) in patients with non-small cell lung cancer (NSCLC). Materials and methodsThe study population consisted of NSCLC patients who were undergoing RT treatment and enrolled in prospective imaging studies. Q-SPECT was performed prior to and during RT (at ∼40–45 Gy). A baseline dose–volume histogram (DVH) and mid-Tx DVH based on simulation CT as well as a baseline DFH and mid-Tx DFH based on Q-SPECT were calculated. Only patients with stage III NSCLC and visible functional lung (FL) changes on the mid-Tx scan were eligible for this enriched analysis. RILT was graded according to a reported scale. ResultsForty-two stage III NSCLC patients met the criteria for inclusion. The accumulative incidence of grade ≥2 RILT was 31% in this high-risk population. Significant differences in functional metrics such as functional lung volume FV5–FV20 at increments of 5 Gy and functional MLD (FMLD) were observed between patients with and without grade ≥2 RILT (p < 0.05). Similar results were also obtained for anatomical metrics from V5–V20 and MLD (p < 0.05). The areas under the receiver operating characteristic curves (AUCs) ranged from 0.724to 0.812 for baseline DVH parameters, from 0.745 to 0.830 for mid-Tx DVH parameters, from 0.764 to 0.878 for baseline DFH parameters, and from 0.767 to 0.891 for mid-Tx DFH parameters. Further principal components analysis showed that the AUCs were 0.814/0.817 and 0.790/0.857 for baseline/mid-Tx DVH and baseline/mid-Tx DFH, respectively. ConclusionsMid-Tx DFH parameters based on Q-SPECT were significantly elevated in patients with grade ≥2 RILT in this study population. Among the metrics compared, mid-Tx DFH seemed to have better predictive accuracy, but this difference did not reach statistical difference.