Non-small Cell Lung Carcinoma Tumors Research Articles

Development of a Fluorinated Analogue of Erlotinib for PET Imaging of EGFR Mutation\u2013Positive NSCLC

PurposePositron emission tomography (PET) using [11C]erlotinib identifies non-small cell lung carcinoma (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFRm). The short half-life of C-11, however, limits its clinical utility to centers with a nearby cyclotron. We therefore developed a F-18–labeled analogue of erlotinib for imaging EGFRm NSCLC.Procedures6-O-Fluoroethylerlotinib (6-O-FEE) was synthesized and its anti-proliferative activity was tested using human NSCLC cell lines. The F-18–labeled compound, 6-O-[18F]FEE, was obtained in a two-step synthesis, and PET acquisitions were carried out following its injection to NSCLC tumor–bearing mice.ResultsIn vitro, 6-O-FEE had maintained the selectivity and potency of erlotinib to EGFRm NSCLC. In vivo, 6-O-[18F]FEE accumulation in EGFRm tumors at 60 min after injection was 2- and 3.3-fold higher than in erlotinib-resistant or erlotinib-insensitive tumors, respectively.Conclusions6-O-[18F]FEE holds promise for imaging EGFRm NSCLC, warranting further investigation to fully explore its potential for stratifying NSCLC patients.

FBXO17 promotes cell proliferation through activation of Akt in lung adenocarcinoma cells

BackgroundThe ubiquitin-proteasome pathway, mediated in part, by ubiquitin E3 ligases, is critical in regulating cellular processes such as cell proliferation, apoptosis, and migration. FBXO17 was recently identified as an F-box protein that targets glycogen synthase kinase-3β to the E3 ubiquitin ligase protein complex for polyubiquitination and proteasomal degradation. Here, we identified that in several lung adenocarcinoma cell lines, FBXO17 cellular protein was detected at relatively high levels, as was expression in a subset of lung cancers. Hence, we investigated the effects of FBXO17 on cell proliferation.MethodsSingle cell RNA sequencing analysis was performed on a resection of a non-small cell lung carcinoma tumor to examine FBXO17 expression. Multiple lung cancer cell lines were immunoblotted, and The Cancer Genome Atlas was analyzed to determine if FBXO17 expression was amplified in a subset of lung cancers. A549 cells were transfected with empty vector or FBXO17-V5 plasmid and immunoblotted for Akt pathway mediators including PDK1, ERK1/2, ribosomal protein S6, and CREB. Cell proliferation and viability were analyzed by trypan blue exclusion, BrdU incorporation and an MTS-based fluorometric assay. Studies were also performed after transfecting with sifbxo17. Samples were used in an RNA microarray analysis to evaluate pathways affected by reduced FBXO17 gene expression.ResultsWe observed that overexpression of FBXO17 increased A549 cell proliferation coupled with Akt activation. Ectopically expressed FBXO17 also increased ERK1/2 kinase activation and increased phosphorylation of RPS6, a downstream target of mTOR. We also observed an increased number of cells in S-phase and increased metabolic activity of lung epithelial cells expressing FBXO17. FBXO17 knockdown reduced Akt Ser 473 phosphorylation approaching statistical significance with no effect on Thr 308. However, ERK1/2 phosphorylation, cellular metabolic activity, and overall cell numbers were reduced. When we analyzed RNA profiles of A549 cells with reduced FBXO17 expression, we observed downregulation of several genes associated with cell proliferation and metabolism.ConclusionsThese data support a role for FBXO17 abundance, when left unchecked, in regulating cell proliferation and survival through modulation of Akt and ERK kinase activation. The data raise a potential role for the F-box subunit in modulating tumorigenesis.

Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity.

Tumor-associated myeloid cells maintain immunosuppressive microenvironments within tumors. Identification of myeloid-specific receptors to modulate tumor-associated macrophage and myeloid-derived suppressor cell (MDSC) functions remains challenging. The leukocyte immunoglobulin-like receptor B (LILRB) family members are negative regulators of myeloid cell activation. We investigated how LILRB targeting could modulate tumor-associated myeloid cell function. LILRB2 antagonism inhibited receptor-mediated activation of SHP1/2 and enhanced proinflammatory responses. LILRB2 antagonism also inhibited AKT and STAT6 activation in the presence of M-CSF and IL-4. Transcriptome analysis revealed that LILRB2 antagonism altered genes involved in cell cytoskeleton remodeling, lipid/cholesterol metabolism, and endosomal sorting pathways, as well as changed differentiation gene networks associated with inflammatory myeloid cells as opposed to their alternatively activated phenotype. LILRB2 blockade effectively suppressed granulocytic MDSC and Treg infiltration and significantly promoted in vivo antitumor effects of T cell immune checkpoint inhibitors. Furthermore, LILRB2 blockade polarized tumor-infiltrating myeloid cells from non-small cell lung carcinoma tumor tissues toward an inflammatory phenotype. Our studies suggest that LILRB2 can potentially act as a myeloid immune checkpoint by reprogramming tumor-associated myeloid cells and provoking antitumor immunity.

EGF-induced nuclear localization of SHCBP1 activates \u03b2-catenin signaling and promotes cancer progression

Aberrant activation of EGFR represents a common event in non-small cell lung carcinoma (NSCLC) and activates various downstream signaling pathways. While EGFR activation of β-catenin signaling was previously reported, the mediating mechanism remains unclear. Our current study found that EGFR activation in NSCLC cells releases SHC-binging protein 1 (SHCBP1) from SHC adaptor protein 1 (SHC1), which subsequently translocates into the nucleus and directly promotes the transactivating activity of β-catenin, consequently resulting in development of NSCLC cell stemness and malignant progression. Furthermore, SHCBP1 promotes β-catenin activity through enhancing the CBP/β-catenin interaction, and most interestingly, a candidate drug that blocks the CBP/β-catenin binding effectively abrogates the aforementioned biological effects of SHCBP1. Clinically, SHCBP1 level in NSCLC tumors was found to inversely correlate with patient survival. Together, our study establishes a novel convergence between EGFR and β-catenin pathways and highlights a potential significance of SHCBP1 as a prognostic biomarker and a therapeutic target.

Intra-tumoural heterogeneity characterization through texture and colour analysis for differentiation of non-small cell lung carcinoma subtypes

Radiomics has shown potential in disease diagnosis, but its feasibility for non-small cell lung carcinoma (NSCLC) subtype classification is unclear. This study aims to explore the diagnosis value of texture and colour features from positron emission tomography computed tomography (PET-CT) images in differentiation of NSCLC subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). Two patient cohorts were retrospectively collected into a dataset of 341 18F-labeled 2-deoxy-2fluoro-d-glucose ([18F] FDG) PET-CT images of NSCLC tumours (125 ADC, 174 SqCC, and 42 cases with unknown subtype). Quantification of texture and colour features was performed using freehand regions of interest. The relation between extracted features and commonly used parameters such as age, gender, tumour size, and standard uptake value (SUVmax) was explored. To classify NSCLC subtypes, support vector machine algorithm was applied on these features and the classification performance was evaluated by receiver operating characteristic curve analysis. There was a significant difference between ADC and SqCC subtypes in texture and colour features (P < 0.05); this showed that imaging features were significantly correlated to both SUVmax and tumour diameter (P < 0.05). When evaluating classification performance, features combining texture and colour showed an AUC of 0.89 (95% CI, 0.78–1.00), colour features showed an AUC of 0.85 (95% CI, 0.71–0.99), and texture features showed an AUC of 0.68 (95% CI, 0.48–0.88). DeLong’s test showed that AUC was higher for features combining texture and colour than that for texture features only (P = 0.010), but not significantly different from that for colour features only (P = 0.328). HSV colour features showed a similar performance to RGB colour features (P = 0.473). The colour features are promising in the refinement of NSCLC subtype differentiation, and features combining texture and colour of PET-CT images could result in better classification performance.

Aberrant Expression of miR-362 Promotes Lung Cancer Metastasis through Downregulation of Sema3A.

miR-362 is a recently discovered member of the microRNA family, and it modulates a variety of physical activities and plays an important role in the occurrence and development of many tumors. However, the biological functions of hsa-miR-362-5p in non-small-cell lung carcinoma (NSCLC) are unknown. Transwell assay and colony formation were used to determine the migration, invasion, and proliferation of NSCLC cells in vitro. A subcutaneous tumor model in nude mice was established to detect NSCLC tumor growth in vivo. The direct binding of miR-362 to the 3′UTR of Semaphorin 3A (Sema3A) was confirmed by luciferase reporter assay. In this study, we found that the level of miR-362 was higher in NSCLC tissues than in adjacent normal tissues and that the level of miR-362 expression was also elevated in five NSCLC cell lines (A549, 95-D, H1299, H292, and H460) relative to a human normal lung epithelial cell line (BEAS2B). Furthermore, miR-362 promoted NSCLC cell invasion, migration, and colony formation in vitro and tumor formation in vivo. Next, we identified the miR-362 target gene Sema3A, which is significantly correlated with metastasis. Sema3A expression was increased in normal tissues relative to NSCLC tissues. This result is consistent with the fact that miR-362 expression is negatively correlated with Sema3A expression in clinical tissue samples and indicated that miR-362 can regulate Sema3A expression in NSCLC cells and consequently affect NSCLC invasion, migration, and colony formation. Taken together, these findings on the newly identified miR-362/Sema3A axis elucidate the molecular mechanism of NSCLC invasion and migration and could lead to a potential therapeutic target in NSCLC treatment.

Abstract 2595: Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer

Abstract The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR), one of the most studied targets. Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective, however TKI resistance is common due to the acquisition of the T790M mutation in the EGFR kinase domain. There are currently very few studies suggesting the role of VEGFR in mediating erlotinib resistance. We suggest that over-expression of vascular endothelial growth factor receptors (VEGFRs) in tumors may create an autocrine loop that may mediate TKI resistance. The purpose of this study is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling. H2170 NSCLC parental cells were obtained from ATCC. The resistant cells were obtained by treating parental cells with increasing concentrations of erlotinib. DNA sequencing was performed to exclude the presence of the T790M mutation. Quantitative real-time PCR was carried out for target genes coding for VEGF, VEGFR-2, and neuropilin-1 (NP-1). Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis were performed with antibodies against VEGFR-2 and NP-1. The viability of resistant cells was assessed after being treated with increasing concentrations of a VEGF inhibitor (VEGF neutralizing antibody) in medium with or without erlotinib using a cell proliferation assay (MTT). Thirty paraffin embedded tumor biopsies were immunostained (IHC) with an antibody against VEGFR-2. On the transcriptional level, real-time PCR revealed a 13.1, 3.4 and a 2.9 fold increase in gene expression for VEGF, VEGFR-2 and NP-1 respectively in the resistant cells compared to the parental cells. When looking at membrane bound receptors, FACS analysis showed a 2.1 and a 3.8 fold increase in the expression of VEGFR and NP-1 in resistant cells, respectively. IF studies correlated with the FACS results, showing significantly higher expression of both VEGFR-2 and NP-1 in the resistant cells. MTT assays demonstrated that the VEGF inhibitor was able to decrease cell death from 10 to 80% (0.1-10ug/ml) as evidenced by decreasing viability of resistant cells treated with increasing concentrations of VEGF inhibitor compared to the untreated control. Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (7mo vs 25mo, p=0.05) in NSCLC tumors in patients diagnosed with late stage NSCLC. Our results suggest that by up-regulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKI's on the EGFR pathway, leading to resistance. Furthermore, IHC demonstrated the presence of high expression of VEGFR-2 in tumors, suggesting its potential as a prognostic biomarker for lung cancer. Citation Format: Chike Osude, Leo Lin, Brad Foster, Neelu Puri. Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2595.

Abstract 1174: Characterization of the immunologic intra-tumor heterogeneity in early stages of non-small cell lung carcinoma using multiplex immunofluorescence and image analysis approaches

Abstract Introduction. Recurrence of non-small cell lung carcinoma (NSCLC) is associated with genetic and epigenetic intra-tumor heterogeneity (ITH). The interaction between malignant cells, stromal cells, and tumor-associated immune-cells (TAICs), such as T-cell lymphocytes (TCLs) and tumor-associated macrophages (TAMs), is important for progression of NSCLC and the characterization of the immunologic ITH might be relevant to predict recurrence in surgically treated patients at early stages of NSCLC. The aim of this study was to characterize the immunologic ITH of primary NSCLC tumors at early stages using image analysis and multiplex immunofluorescence (mIF) approaches. Material and methods. Eight cases of stage IA and 8 cases of stage IB surgically resected NSCLC (11 adenocarcinomas, ADCs; and 5 squamous-cell carcinomas, SCCs) with a history of early recurrence were selected for this preliminary analysis. Formalin-fixed, paraffin-embedded (FFPE) blocks were obtained and consecutive sections were stained with two panels of mIF for immune profiling, panel 1: pan-cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B (GB), CD45RO, and FOXP3. Three not adjacent, intra-tumor regions (3mm2 each) per case were randomly selected after gridding the whole tumor section. A total of 41 intra-tumor regions were scanned by Vectra multispectral-microscope (PerkinElmer) and analyzed using InForm-software (PerkinElmer). TAICs were quantified in the epithelial and stromal compartments from each intra-tumor region. Results. The median density of TCLs and TAMs were 1527 cells/mm2 and 635 cells/mm2, respectively, without significant differences between histologic subtypes. TCLs were predominantly concentered in the stromal compartment (median, 2222 cells/mm2) when compared with epithelial compartment (median, 332 cells/mm2). The percentage and density of TCLs and TAMs varied 4 and 8 times, respectively, between cases and regions. Non-cytotoxic T-cells and inactive cytotoxic T-cells were the most prevalent phenotypes. Higher density of TAMs and antigen-experienced TCLs were observed in stage IB than stage IA in the primary tumor of patients with NSCLC. Conclusion. The characterization of the immunologic ITH of NSCLC is able by mIF and image analysis with FFPE tumor tissue. There is a variability of TAICs densities between regions from the same tumor and different subpopulations were observed. TAMs and exhausted T-cells were more prominent in stage IB (tumor &amp;gt;3cm) suggesting these cells may play an important role in recurrence. Ongoing studies with a larger cohort and comparison with non-recurrent surgically treated patients are warranted. Supported in part by CPRIT RP160668 grant Citation Format: Alejandro Francisco Cruz, Edwin R. Parra, Mei Jiang, Junya Fujimoto, Chi-Wan Chow, Jaime Rodriguez-Canales, Carmen Behrens, Neda Kalhor, Annikka Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, J. Jack Lee, Cesar Moran, P. Andrew Futreal, Jianjun Zhang, Ignacio I. Wistuba. Characterization of the immunologic intra-tumor heterogeneity in early stages of non-small cell lung carcinoma using multiplex immunofluorescence and image analysis approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1174.

Phytochemical naphtho[1,2-b] furan-4,5‑dione induced topoisomerase II-mediated DNA damage response in human non-small-cell lung cancer

BackgroundPhytochemical naphtho[1,2-b] furan-4,5‑dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC). PurposeTo examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism. Study designThe anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay. ResultsNFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo. ConclusionThese findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.

Evaluation of a Dual ALK/ROS1 Fluorescent In Situ Hybridization Test in Non–Small-cell Lung Cancer

BackgroundSeveral therapeutics targets have emerged to treat patients with non–small-cell lung carcinoma (NSCLC), with numerous biomarkers available to test for treatment choices. Minimum tumor wastage is necessary to permit the analysis of every potentially relevant target. Searching for targetable ALK and ROS1 rearrangements is now mandatory in NSCLC. In the present study, we evaluated the performance of a dual ALK/ROS1 fluorescent in situ hybridization (FISH) probe that concurrently analyzed the 2 oncogenes on a same FISH slide. Materials and MethodsWe used the FlexISH ALK/ROS1 DistinguISH Probe (Zytovision, Bremerhaven, Germany) to analyze a set of 28 formalin-fixed paraffin-embedded NSCLC tumor samples enriched in tumors with ALK- and ROS1-rearranged status. ResultsThe dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests (15 ALK and 3 ROS1 rearrangements) without any false-positive results. Dual- and single-probe FISH test results were also concordant regarding the unusual ALK FISH patterns. These included 1 sample that had negative FISH results with diffuse single 5′-ALK signals and positive ALK immunohistochemistry findings in a patient with a response to crizotinib, 2 paired samples with high percentages of ALK FISH-rearranged nuclei despite negative ALK immunohistochemistry findings, and ALK FISH-positive samples from 2 patients lacking a response to crizotinib therapy despite concordant ALK FISH and immunohistochemistry-positive results. ConclusionThe dual ALK/ROS1 FISH probe test is a valuable tool to search concurrently for both ALK and ROS1 rearrangements on a same FISH slide and could help to spare tumor tissue for other biomarkers tests.

Wide Expression and Significance of Alternative Immune Checkpoint Molecules, B7x and HHLA2, in PD-L1-Negative Human Lung Cancers.

Purpose: Immunotherapy targeting the PD-1/PD-L1 pathway has changed the treatment landscape of non-small cell lung carcinoma (NSCLC). We demonstrated that HHLA2, a newly identified immune inhibitory molecule, was widely expressed in NSCLC. We now compared the expression and function of PD-L1 with alternative immune checkpoints, B7x and HHLA2.Experimental Design: Expression was examined in tissue microarrays consisting of 392 resected NSCLC tumors. Effects of PD-L1, B7x, and HHLA2 on human T-cell proliferation and cytokine production were investigated.Results: PD-L1 expression was identified in 25% and 31% of tumors in the discovery and validation cohorts and was associated with higher stage and lymph node involvement. The multivariate analysis showed that stage, TIL status, and lymph node involvement were independently associated with PD-L1 expression. B7x was expressed in 69% and 68%, whereas HHLA2 was positive in 61% and 64% of tumors in the two sets. The coexpression of PD-L1 with B7x or HHLA2 was infrequent, 6% and 3%. The majority (78%) of PD-L1-negative cases expressed B7x, HHLA2, or both. The triple-positive group had more TIL infiltration than the triple-negative group. B7x-Ig and HHLA2-Ig inhibited TCR-mediated proliferation of CD4 and CD8 T cells more robustly than PD-L1-Ig. All three significantly suppressed cytokine productions by T cells.Conclusions: The majority of PD-L1-negative lung cancers express alternative immune checkpoints. The roles of the B7x and HHLA2 pathway in mediating immune evasion in PD-L1-negative tumors deserve to be explored to provide the rationale for an effective immunotherapy strategy in these tumors. Clin Cancer Res; 24(8); 1954-64. ©2018 AACR.

Tests immunohistochimiques PD-L1 dans les cancers du poumon non à petites cellules : recommandations par le groupe PATTERN de pathologistes thoraciques

Lung cancer is the leading cause of cancer death in France with low response rates to conventional chemotherapy. Nevertheless, new therapies have emerged recently, among which PD1 immune checkpoint inhibitors (ICI), such as nivolumab (OPDIVO®, Bristol-Myers Squibb) and pembrolizumab (KEYTRUDA®, Merck & Co), or PD-L1 ICI, such as atezolizumab (TECENTRIQ®, Genentech), durvalumab (IMFINZI®, Astra-Zeneca), and avelumab (BAVENCIO®, EMD Serono). The prescription of pembrolizumab for advanced stage non-small cell lung carcinoma (NSCLC) patients requires the demonstration of PD-L1 expression by tumor cells by immunohistochemistry (IHC) (minimum of 50% of positive tumor cells is required for first-line setting, and of 1% for second-line and beyond) and PD-L1 assay is now considered as a companion diagnostic tool for this drug. Numerous standardized PD-L1 assays performed on dedicated platforms have been validated in clinical trials, each antibody being associated to one specific PD1 or PD-L1 inhibitor. However, not all pathologists have access to the dedicated platforms and the high cost of these assays is still a limitation to their implementation; in addition, the small size of the NSCLC tumor samples does not allow to perform at the same time multiple assays for multiple drugs. The use of laboratory-developed tests seems feasible but their validation must guarantee the same sensitivities and specificities as standardized tests. In this context, the French group of thoracic pathologists PATTERN has teamed up with thoracic oncologists to provide recommendations on the indication, the critical technical steps and the interpretation of the PD-L1 IHC test to help pathologists to implement quickly and in the best conditions this new theranostic test.

Estrogen receptor-alpha isoforms are the main estrogen receptors expressed in non-small cell lung carcinoma

The expression profile of estrogen receptors (ER) in Non-Small Cell Lung Carcinoma (NSCLC) remains contradictory. Here we investigated protein and transcriptome expression of ERα wild type and variants. Tissue Micro-Arrays of 200 cases of NSCLC (paired tumor/non-tumor) were assayed by immunohistochemistry using a panel of ERα antibodies targeting different epitopes (HC20, 6F11, 1D5, ERα36 and ERα17p). ERβ epitopes were also examined for comparison. In parallel we conducted a probe-set mapping (Affymetrix HGU133 plus 2 chip) meta-analysis of 12 NSCLC tumor public transcriptomic studies (1418 cases) and 39 NSCLC cell lines. Finally, we have investigated early transcriptional effects of 17β-estradiol, 17β-estradiol-BSA, tamoxifen and their combination in two NSCLC cell lines (A549, H520). ERα transcript and protein detection in NSCLC specimens and cell lines suggests that extranuclear ERα variants, like ERα36, prevail, while wild-type ERα66 is minimally expressed. In non-tumor lung, the wild-type ERα66 is quasi-absent. The combined evaluation of ERα isoform staining intensity and subcellular localization with sex, can discriminate NSCLC subtypes and normal lung. Overall ERα transcription decreases in NSCLC. ERα expression is sex-related in non-tumor tissue, but in NSCLC it is exclusively correlating with tumor histologic subtype. ERα isoform protein expression is higher than ERβ. ERα isoforms are functional and display specific early transcriptional effects following steroid treatment. In conclusion, our data show a wide extranuclear ERα-variant expression in normal lung and NSCLC that is not reported by routine pathology ER evaluation criteria, limited in the nuclear wild type receptor.

Correlation Between Programmed Death Receptor-1 Expression in Tumor-Infiltrating Lymphocytes and Programmed Death Ligand-1 Expression in Non-Small Cell Lung Carcinoma.

The interaction between programmed death ligand-1 (PD-L1) and programmed death receptor-1 (PD-1) on activated T cells sends an inhibitory signal that dampens the immune response. Tumors can express PD-L1 and evade the immune system. In advanced non-small cell lung carcinoma, expression of PD-1 in tumor-infiltrating lymphocytes (TILs) correlates with PD-L1 expression in tumor cells (TCs). However, this relationship has not been thoroughly explored in early disease. To investigate the correlation of PD-1 and PD-L1 in non-small cell lung carcinoma tumor samples, with emphasis on stage I disease. Whole tissue sections from non-small cell lung carcinoma tumors were retrospectively evaluated by immunohistochemistry for PD-1 and PD-L1 expression. The scoring was based on the percentage of cells positive for PD-1 in TILs and PD-L1 in TCs and tumor-infiltrating immune cells (ICs). Expression of PD-1 in TILs was observed in 147 of 161 non-small cell lung carcinoma cases (91%). The majority of cases negative for PD-1 also lacked PD-L1 in TCs. The 68 cases with highest PD-1 expression in TILs included 33 (49%) with expression of PD-L1 in TCs and ICs. Strong correlations were observed in patients with elevated PD-1 expression in TILs and PD-L1 in TCs ( P = .01) and ICs ( P = .003). Expression of PD-1 also correlated with increased PD-L1 in TCs and ICs when the 2 were grouped together ( P < .001). Finally, stage I patients with negative PD-1 and PD-L1 expression showed trends toward increased disease-specific survival. Expression of PD-1 in TILs correlates with PD-L1 expression in both TCs and ICs. Furthermore, negative expression of PD-1 and PD-L1 suggest trends toward disease-specific survival, even in early disease stages.

Metastasis-associated protein 2 promotes the metastasis of non-small cell lung carcinoma by regulating the ERK/AKT and VEGF signaling pathways.

Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer-associated mortality in the world and accounts for ~85% of human lung cancers. Metastasis-associated protein 2 (MTA2) is a component of the histone deacetylase complex and serves a role in tumor progression; however, the mechanism through which MTA2 is involved in the progression of NSCLC remains unclear. The aim of the present study was to investigate the expression and function of MTA2 and the MTA2-mediated signaling pathway in NSCLC cells. Expression of MTA2 and its target genes was analyzed in MTA2-overexpressing and anti-MTA2 antibody (AbMTA2)-treated NSCLC cells, as well as growth, migration, invasion and apoptotic-resistance. The inhibitory effects on tumor formation were analyzed using AbMTA2-treated NSCLC cells and in a mouse model. Histological assessment was conducted to analyze the expressions levels of extracellular signal-regulated kinase (ERK), RAC-α serine/threonine protein kinase (AKT) and vascular endothelial growth factor (VEGF) in experimental tumors. Results of the present study demonstrated that MTA2 was overexpressed in NSCLC cells. The growth, migration and invasion of NSCLC cells were markedly inhibited by AbMTA2. In addition, it was observed that the ERK/AKT and VEGF signaling pathways were both upregulated in MTA2-overexpressing NSCLC cells, and downregulated following silencing of MTA2 activation. ERK and AKT phosphorylation levels were downregulated in NSCLC cells and tumors following MTA2 silencing. The in vivo study demonstrated that tumor growth was markedly inhibited following siRNA-MTA2 treatment. In conclusion, the results of the present study suggested that MTA2 silencing may significantly inhibit the growth and aggressiveness of NSCLC cells. Results from the present study indicated that the mechanism underlying the MTA2-mediated invasive potential of NSCLC cells involved the ERK/AKT and VEGF signaling pathways, which may be a potential therapeutic target for the treatment of NSCLC.

Overexpression of NR4A1 is associated with tumor recurrence and poor survival in non-small-cell lung carcinoma.

The expression level and clinical significance of NR4A1 are presently unknown in the non-small-cell lung carcinoma (NSCLC). This study aimed to explore the expression, prognostic value, and function of NR4A1 in NSCLC. Methods: Clinicopathological parameters of 167 NSCLC patients who received radical surgery from January 2007 and December 2012 were retrospectively reviewed. The NR4A1 expression in NSCLC tumors and the adjacent matched para-carcinoma specimens were examined, and the association between NR4A1 expression and clinical variables was explored. Cell viability assay, and transwell migration and invasion assays were used to access the function of NR4A1 in NSCLC. Kaplan-Meier analysis and Cox regression were performed to investigate the prognostic significance of NR4A1 for NSCLC. Results: NR4A1 was overexpressed in NSCLC tissues compared with the para-carcinoma specimens. Consistently, Oncomine analysis showed that NR4A1 was overexpressed in NSCLC tissues compared with normal tissues in published datasets (P < 0.001). The elevated NR4A1 expression was associated with carcinoma recurrence (P < 0.05). The 5-year median overall survival (OS) and progression free survival (PFS) were significantly poorer in the NR4A1-overexpression group. Multivariate Cox analysis showed that NR4A1 overexpression was an independent factor for OS (HR, 95%CI: P < 0.05) and PFS (HR, 95%CI: P < 0.05) in NSCLC. Moreover, knockdown of NR4A1 significantly reduced NSCLC cell proliferation, migration, and invasion. Conclusions: NR4A1 exhibits a tumor-promoting effect on NSCLC, and might serve as a promising prognostic biomarker and a therapeutic target for NSCLC.

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC50] at 3.5–7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

MiR-768-3p is involved in the proliferation, invasion and migration of non-small cell lung carcinomas.

Altered microRNA expression has been found to be a common feature of several cancers, including lung carcinomas. However, the possible roles of miR-768-3p in the pathological changes of lung carcinomas are still unknown. The aim of the present study was to investigate the expression and possible effects of miR-768-3p in human non-small cell lung carcinomas (NSCLC). Eighty-three NSCLC patients attending the clinic of Kunming Hospital were invited to participate in the study. Their tumor samples were obtained for qRT-PCR analysis. Human NSCLC cell lines, A549 and HCC4006, were employed and transfected with either miR-768-3p mimics or miR-768-3p antagomir. Following transfection, the invitro and invivo proliferation, apoptosis fractions, migration and invasion of NSCLC cells were evaluated. The data revealed that: i)upregulated miR-768-3p in tumors were associated with the clinicopathological features of NSCLC patients; ii)inhibiting miR-768-3p function by miR-768-3p antagomir induced distinctly apoptosis and Fas/FasL expressional alteration of NSCLC cells; iii)miR-768-3p antagomir transduction also decreased the viability, migration and invasion, as well as MMP-2 and MMP-9 activities in A549 and HCC4006 cells; and iv)miR-768-3p antagomir transfection also inhibited the growth and proliferation of NSCLC xenografts in nude mice. The present results suggested that abnormal elevated miR-768-3p in NSCLC tumors and cell lines played important roles in NSCLC carcinogenic progression, and the targeting of miR-768-3p might be a potential therapeutic strategy for the treatment of NSCLC.

BOK displays cell death-independent tumor suppressor activity in non-small-cell lung carcinoma.

As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFβ-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients.

MiR-1254 suppresses HO-1 expression through seed region-dependent silencing and non-seed interaction with TFAP2A transcript to attenuate NSCLC growth.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, which direct post-transcriptional gene silencing (PTGS) and function in a vast range of biological events including cancer development. Most miRNAs pair to the target sites through seed region near the 5’ end, leading to mRNA cleavage and/or translation repression. Here, we demonstrated a miRNA-induced dual regulation of heme oxygenase-1 (HO-1) via seed region and non-seed region, consequently inhibited tumor growth of NSCLC. We identified miR-1254 as a negative regulator inhibiting HO-1 translation by directly targeting HO-1 3’UTR via its seed region, and suppressing HO-1 transcription via non-seed region-dependent inhibition of transcriptional factor AP-2 alpha (TFAP2A), a transcriptional activator of HO-1. MiR-1254 induced cell apoptosis and cell cycle arrest in human non-small cell lung carcinoma (NSCLC) cells by inhibiting the expression of HO-1, consequently suppressed NSCLC cell growth. Consistently with the in vitro studies, mouse xenograft studies validated that miR-1254 suppressed NSCLC tumor growth in vivo. Moreover, we found that HO-1 expression was inversely correlated with miR-1254 level in human NSCLC tumor samples and cell lines. Overall, these findings identify the dual inhibition of HO-1 by miR-1254 as a novel functional mechanism of miRNA, which results in a more effective inhibition of oncogenic mRNA, and leads to a tumor suppressive effect.