Primary Human Lung Cancer Research Articles

Evaluating predictors of kinase activity of STK11 variants identified in primary human non-small cell lung cancers.

Critical evaluation of computational tools for predicting variant effects is important considering their increased use in disease diagnosis and driving molecular discoveries. In the sixth edition of the Critical Assessment of Genome Interpretation (CAGI) challenge, a dataset of 28 STK11 rare variants (27 missense, 1 single amino acid deletion), identified in primary non-small cell lung cancer biopsies, was experimentally assayed to characterize computational methods from four participating teams and five publicly available tools. Predictors demonstrated a high level of performance on key evaluation metrics, measuring correlation with the assay outputs and separating loss-of-function (LoF) variants from wildtype-like (WT-like) variants. The best participant model, 3Cnet, performed competitively with well-known tools. Unique to this challenge was that the functional data was generated with both biological and technical replicates, thus allowing the assessors to realistically establish maximum predictive performance based on experimental variability. Three out of the five publicly available tools and 3Cnet approached the performance of the assay replicates in separating LoF variants from WT-like variants. Surprisingly, REVEL, an often-used model, achieved a comparable correlation with the real-valued assay output as that seen for the experimental replicates. Performing variant interpretation by combining the new functional evidence with computational and population data evidence led to 16 new variants receiving a clinically actionable classification of likely pathogenic (LP) or likely benign (LB). Overall, the STK11 challenge highlights the utility of variant effect predictors in biomedical sciences and provides encouraging results for driving research in the field of computational genome interpretation.

Abstract 180: LINC00261 is functionally linked to tumor DNA mutational burden and tumor immune microenvironment composition in lung adenocarcinoma

Abstract Immunotherapy has emerged as a breakthrough in the improvement of survival outcomes for lung adenocarcinoma (LUAD), however effective response requires the combination of blocking inhibitory signals on the tumor surface and antigen presentation to the tumor surface for proper immune recognition. Several commercially available and robust methods exist for identification of tumors displaying immune-inhibitory surface receptors, such as PD-L1, however it is currently difficult to predict effectiveness of antigen presentation on the cell surface. To address this, we utilized clinical and next-generation sequencing data from The Cancer Genome Atlas (TCGA) to identify gene signatures that are correlated to tumor mutational burden (TMB) within cancers of epithelial origins as a surrogate for neoantigen signatures. We identified LINC00261 as a top gene correlated to TMB, whose expression activates DNA damage response pathways in vitro along with resistance to cisplatin. LINC00261 expression was also significantly correlated to MHC class I and II genes involved in endogenous neoantigen presentation expression within the TCGA-LUAD cohort. This relationship was confirmed in vitro through ectopic reintroduction of LINC00261 for key MHC class II presentation genes. Interferon gamma-induced MHC gene activation in vitro was also able to induce endogenous expression of LINC00261. Staining of primary human lung cancer sections suggested that loss of LINC00261 is associated with an immunosuppressive tumor microenvironment. Taken together, our results suggest there is a mechanistic relationship in the silencing of LINC00261 in LUAD and compromised DNA repair, accumulation of mutations, and reduced antitumor immune response. Citation Format: Jonathan Castillo, Tianchun Xue, Mayela Norwood, Samantha Joseph, Alan L. Epstein, William D. Wallace, Anthony W. Kim, Crystal N. Marconett. LINC00261 is functionally linked to tumor DNA mutational burden and tumor immune microenvironment composition in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 180.

Abstract A016: LINC00261 is functionally linked to tumor DNA mutational burden and tumor immune microenvironment composition in lung adenocarcinoma

Abstract Immunotherapy has emerged as a breakthrough in the improvement of survival outcomes for lung adenocarcinoma (LUAD), however effective response requires the combination of blocking inhibitory signals on the tumor surface and antigen presentation to the tumor surface for proper immune recognition. Several commercially available and robust methods exist for identification of tumors displaying immune-inhibitory surface receptors, such as PD-L1, however it is currently difficult to predict effectiveness of antigen presentation on the cell surface. To address this, we utilized clinical and next-generation sequencing data from The Cancer Genome Atlas (TCGA) to identify gene signatures that are correlated to tumor mutational burden (TMB) within cancers of epithelial origins as a surrogate for neoantigen signatures. We identified LINC00261 as a top gene correlated to TMB, whose expression activates DNA damage response pathways in vitro along with resistance to cisplatin. LINC00261 expression was also significantly correlated to MHC class I and II genes involved in endogenous neoantigen presentation expression within the TCGA-LUAD cohort. This relationship was confirmed in vitro through ectopic reintroduction of LINC00261 for key MHC class II presentation genes. Interferon gamma-induced MHC gene activation in vitro was also able to induce endogenous expression of LINC00261. The staining of primary human lung cancer sections suggested that loss of LINC00261 is associated with an immunosuppressive tumor microenvironment. Taken together, our results suggest there is a mechanistic relationship in the silencing of LINC00261 in LUAD and compromised DNA repair, accumulation of mutations, and reduced antitumor immune response. Citation Format: Jonathan Castillo, Tianchun Xue, Mayela Norwood, Samantha Joseph, Alan L. Epstein, W. Dean Wallace, Anthony W. Kim, Crystal N. Marconett. LINC00261 is functionally linked to tumor DNA mutational burden and tumor immune microenvironment composition in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A016.

Pair bonding and disruption impact lung transcriptome in monogamous Peromyscus californicus

Social interactions affect physiological and pathological processes, yet their direct impact in peripheral tissues remains elusive. Recently we showed that disruption of pair bonds in monogamous Peromyscus californicus promotes lung tumorigenesis, pointing to a direct effect of bonding status in the periphery (Naderi et al., 2021). Here we show that lung transcriptomes of tumor-free Peromyscus are altered in a manner that depends on pair bonding and superseding the impact of genetic relevance between siblings. Pathways affected involve response to hypoxia and heart development. These effects are consistent with the profile of the serum proteome of bonded and bond-disrupted Peromyscus and were extended to lung cancer cells cultured in vitro, with sera from animals that differ in bonding experiences. In this setting, the species’ origin of serum (deer mouse vs FBS) is the most potent discriminator of RNA expression profiles, followed by bonding status. By analyzing the transcriptomes of lung cancer cells exposed to deer mouse sera, an expression signature was developed that discriminates cells according to the history of social interactions and possesses prognostic significance when applied to primary human lung cancers. The results suggest that present and past social experiences modulate the expression profile of peripheral tissues such as the lungs, in a manner that impacts physiological processes and may affect disease outcomes. Furthermore, they show that besides the direct effects of the hormones that regulate bonding behavior, physiological changes influencing oxygen metabolism may contribute to the adverse effects of bond disruption.

Multidirectional characterization of cellular composition and spatial architecture in human multiple primary lung cancers

Multiple primary lung cancers (MPLCs) pose diagnostic and therapeutic challenges in clinic. Here, we orchestrated the cellular and spatial architecture of MPLCs by combining single-cell RNA-sequencing and spatial transcriptomics. Notably, we identified a previously undescribed sub-population of epithelial cells termed as CLDN2+ alveolar type II (AT2) which was specifically enriched in MPLCs. This subtype was observed to possess a relatively stationary state, play a critical role in cellular communication, aggregate spatially in tumor tissues, and dominate the malignant histopathological patterns. The CLDN2 protein expression can help distinguish MPLCs from intrapulmonary metastasis and solitary lung cancer. Moreover, a cell surface receptor−TNFRSF18/GITR was highly expressed in T cells of MPLCs, suggesting TNFRSF18 as one potential immunotherapeutic target in MPLCs. Meanwhile, high inter-lesion heterogeneity was observed in MPLCs. These findings will provide insights into diagnostic biomarkers and therapeutic targets and advance our understanding of the cellular and spatial architecture of MPLCs.

An integrated single-cell transcriptomic dataset for non-small cell lung cancer

As single-cell RNA sequencing (scRNA-seq) has emerged as a great tool for studying cellular heterogeneity within the past decade, the number of available scRNA-seq datasets also rapidly increased. However, reuse of such data is often problematic due to a small cohort size, limited cell types, and insufficient information on cell type classification. Here, we present a large integrated scRNA-seq dataset containing 224,611 cells from human primary non-small cell lung cancer (NSCLC) tumors. Using publicly available resources, we pre-processed and integrated seven independent scRNA-seq datasets using an anchor-based approach, with five datasets utilized as reference and the remaining two, as validation. We created two levels of annotation based on cell type-specific markers conserved across the datasets. To demonstrate usability of the integrated dataset, we created annotation predictions for the two validation datasets using our integrated reference. Additionally, we conducted a trajectory analysis on subsets of T cells and lung cancer cells. This integrated data may serve as a resource for studying NSCLC transcriptome at the single cell level.

Phototherapeutic Induction of Immunogenic Cell Death and CD8+ T Cell-Granzyme B Mediated Cytolysis in Human Lung Cancer Cells and Organoids.

Simple SummaryImmunogenic cancer cell death and photodynamic therapy play an important emerging role in cancer treatment. Understanding mechanisms involved in tumor killing via immune cells in response to photodynamic therapy is important for developing new anticancer approaches. In this study, we report that methylene blue photodynamic therapy decreases the proliferation of lung cancer cells and patient derived non-small cell lung cancer organoids via immunogenic cells death and granzyme B mediated cytolysis.Augmenting T cell mediated tumor killing via immunogenic cancer cell death (ICD) is the cornerstone of emerging immunotherapeutic approaches. We investigated the potential of methylene blue photodynamic therapy (MB-PDT) to induce ICD in human lung cancer. Non-Small Cell Lung Cancer (NSCLC) cell lines and primary human lung cancer organoids were evaluated in co-culture killing assays with MB-PDT and light emitting diodes (LEDs). ICD was characterised using immunoblotting, immunofluorescence, flow cytometry and confocal microscopy. Phototherapy with MB treatment and low energy LEDs decreased the proliferation of NSCLC cell lines inducing early necrosis associated with reduced expression of the anti-apoptotic protein, Bcl2 and increased expression of ICD markers, calreticulin (CRT), intercellular cell-adhesion molecule-1 (ICAM-1) and major histocompatibility complex I (MHC-I) in NSCLC cells. MB-PDT also potentiated CD8+ T cell-mediated cytolysis of lung cancer via granzyme B in lung cancer cells and primary human lung cancer organoids.

Multidirectional Characterization of Cellular Composition and Spatial Architecture in Human Multiple Primary Lung Cancers

Multidirectional Characterization of Cellular Composition and Spatial Architecture in Human Multiple Primary Lung Cancers

Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers.

Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.

Autophagic flux in cancer cells at the invasive front in the tumor-stroma border.

Cancer cells at the invasive front directly interact with stromal tissue that provides a microenvironment with mechanical, nutrient, and oxygen supply characteristics distinct from those of intratumoral tissues. It has long been known that cancer cells at the invasive front and cancer cells inside the tumor body exhibit highly differentiated functions and behaviors. However, it is unknown whether cancer cells at different locations exhibit a variety of autophagic flux, an important catabolic process to maintain cellular homeostasis in response to environmental changes. Here, using transmission electron microscopy (TEM), we found that invading cancer cells at the invasive front, which show mesenchymal transcriptomic traits, exhibit higher autophagic flux than cancer cells inside the tumor body in human primary non-small cell lung cancer (NSCLC) tissues. This autophagic feature was further confirmed by a live cell autophagic flux monitoring system combined with a 3D organotypic invasion coculture system. Additionally, the increased autophagic flux endows cancer cells with invasive behavior and positively correlates with the advanced tumor stages and the reduced survival period of lung cancer patients. These findings expand the understanding of autophagic dynamics during cancer invasion.

Abstract 2434: Transcriptional crosstalk between YAP, TEAD and TP63 is associated with early lung carcinogenesis

Abstract Introduction: The Hippo pathway effector YAP contributes to carcinogenesis by directing stem cell activity, cell proliferation and cell survival signals. In the lung, aberrant nuclear YAP activity promotes bronchial epithelial growth, which in animal models can drive pathology that resembles lung premalignant lesions (PMLs). However, the molecular mechanisms by which YAP contributes to aberrant lung epithelial pathology are poorly understood. In this project, we investigated the DNA binding pattern and gene expression program regulated by YAP and associated TEAD and TP63 transcriptional factors in primary normal human bronchial epithelial cells (HBECs) and lung cancer cell lines. Methods: RNA-seq experiments were conducted using HBEC, H2170 and SW900 cells transfected with siRNA targeting YAP, TEADs or TP63, and expression profiles were compared to corresponded controls to derive gene signatures. In parallel, ChIP-seq experiments were carried out using antibodies specific for the respective proteins to identify genome binding peaks. Direct target genes were inferred with Binding and Expression Target Analysis (BETA) using gene signature and peak profile of each factor in each cell line respectively. The metagene score for target genes in longitudinally collected endobronchial biopsies from patients with PMLs was calculated using GSVA and the association with PML phenotypes was tested with a mixed-effects model adjusting for batch and patient as random effect (GSE109743). Results: Chromatin binding analyses revealed that YAP co-occupies genomic regions with TEAD and TP63 in normal bronchial epithelial cells aligning with H3K27ac chromatin modifications. Integrating genomic binding and gene expression profiling, we identified large overlaps in target genes directly regulated by YAP, TEAD and TP63 in HBECs, demonstrating cooperation between these factors in the control of bronchial epithelial transcription. YAP-TEAD-TP63 induced targets in HBECs are enriched for genes involved in cell proliferation and extracellular matrix alteration, while repressed targets are enriched for genes involved in cell fate specification. Notably, cooperatively regulated genes are strongly associated with gene expression profile identified in progressive PML, and in particular with genes linked to early immune responses that are observed with PML pathology progression. Similar analyses of YAP, TEAD and TP63 in lung cancer cells revealed an altered transcriptional program for YAP with a divergence of YAP-TEAD away from TP63, suggesting contextual changes in YAP activity with cancer progression. Conclusion: We found that YAP, TP63 and TEADs co-regulate lung epithelial gene expression related to PML pathology progression. These findings provide insight into potential YAP function in the early stages of lung carcinogenesis and offer potential new avenues to develop lung cancer interception strategies. Citation Format: Boting Ning, Andrew Tilston-Lunel, Julia Hicks-Berthet, Sarah A. Mazzilli, Jennifer E. Beane, Xaralabos Varelas, Marc E. Lenburg. Transcriptional crosstalk between YAP, TEAD and TP63 is associated with early lung carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2434.

Novel Primary Human Cancer Stem-Like Cell Populations from Non-Small Cell Lung Cancer: Inhibition of Cell Survival by Targeting NF-κB and MYC Signaling.

There is growing evidence that cancer stem cells (CSCs), a small subpopulation of self-renewal cancer cells, are responsible for tumor growth, treatment resistance, and cancer relapse and are thus of enormous clinical interest. Here, we aimed to isolate new CSC-like cells derived from human primary non-small cell lung cancer (NSCLC) specimens and to analyze the influence of different inhibitors of NF-κB and MYC signaling on cell survival. CSC-like cells were established from three squamous cell carcinomas (SCC) and three adenocarcinomas (AC) of the lung and were shown to express common CSC markers such as Prominin-1, CD44-antigen, and Nestin. Further, cells gave rise to spherical cancer organoids. Inhibition of MYC and NF-κB signaling using KJ-Pyr-9, dexamethasone, and pyrrolidinedithiocarbamate resulted in significant reductions in cell survival for SCC- and AC-derived cells. However, inhibition of the protein–protein interaction of MYC/NMYC proto-oncogenes with Myc-associated factor X (MAX) using KJ-Pyr-9 revealed the most promising survival-decreasing effects. Next to the establishment of six novel in vitro models for studying NSCLC-derived CSC-like populations, the presented investigations might provide new insights into potential novel therapies targeting NF-κB/MYC to improve clinical outcomes in NSCLC patients. Nevertheless, the full picture of downstream signaling still remains elusive.

Histone Monoubiquitination in Chromatin Remodelling: Focus on the Histone H2B Interactome and Cancer.

Simple SummaryPost-translational modifications (PTM) of histone tails represent epigenomic regulation of the chromatin landscape, influencing gene expression and the response to DNA damage. This review focusses on cancer-associated roles of ubiquitin as a histone PTM, specifically in conjunction with an E3 ubiquitin ligase cascade that results in the addition of a single ubiquitin (monoubiquitination) to histone H2B at lysine 120 (H2Bub1). H2Bub1 has roles in chromatin accessibility important for transcriptional elongation, the DNA damage response, cellular proliferation and developmental transitions, including in stem cell plasticity. It has been implicated in inflammation and tumour progression, with examples of its loss associated with a worse prognosis for patients with some cancers. Many factors involved in the H2Bub1 interactome are well known cancer-associated proteins, including p53, BRCA1 and components of the SWI/SNF remodelling complex. Increased knowledge of H2Bub1 and its interactome offers new opportunities for therapeutic targeting of malignancy.Chromatin remodelling is a major mechanism by which cells control fundamental processes including gene expression, the DNA damage response (DDR) and ensuring the genomic plasticity required by stem cells to enable differentiation. The post-translational modification of histone H2B resulting in addition of a single ubiquitin, in humans at lysine 120 (K120; H2Bub1) and in yeast at K123, has key roles in transcriptional elongation associated with the RNA polymerase II-associated factor 1 complex (PAF1C) and in the DDR. H2Bub1 itself has been described as having tumour suppressive roles and a number of cancer-related proteins and/or complexes are recognised as part of the H2Bub1 interactome. These include the RING finger E3 ubiquitin ligases RNF20, RNF40 and BRCA1, the guardian of the genome p53, the PAF1C member CDC73, subunits of the switch/sucrose non-fermenting (SWI/SNF) chromatin remodelling complex and histone methyltransferase complexes DOT1L and COMPASS, as well as multiple deubiquitinases including USP22 and USP44. While globally depleted in many primary human malignancies, including breast, lung and colorectal cancer, H2Bub1 is selectively enriched at the coding region of certain highly expressed genes, including at p53 target genes in response to DNA damage, functioning to exercise transcriptional control of these loci. This review draws together extensive literature to cement a significant role for H2Bub1 in a range of human malignancies and discusses the interplay between key cancer-related proteins and H2Bub1-associated chromatin remodelling.

Microenvironmental Th9 and Th17 lymphocytes induce metastatic spreading in lung cancer.

Immune microenvironment plays a critical role in lung cancer control versus progression and metastasis. In this investigation, we explored the effect of tumor-infiltrating lymphocyte subpopulations on lung cancer biology by studying in vitro cocultures, in vivo mouse models, and human lung cancer tissue. Lymphocyte conditioned media (CM) induced epithelial-mesenchymal transition (EMT) and migration in both primary human lung cancer cells and cell lines. Correspondingly, major accumulation of Th9 and Th17 cells was detected in human lung cancer tissue and correlated with poor survival. Coculturing lung cancer cells with Th9/Th17 cells or exposing them to the respective CM induced EMT in cancer cells and modulated the expression profile of genes implicated in EMT and metastasis. These features were reproduced by the signatory cytokines IL-9 and IL-17, with gene regulatory profiles evoked by these cytokines partly overlapping and partly complementary. Coinjection of Th9/Th17 cells with tumor cells in WT, Rag1-/-, Il9r-/-, and Il17ra-/- mice altered tumor growth and metastasis. Accordingly, inhibition of IL-9 or IL-17 cytokines by neutralizing antibodies decreased EMT and slowed lung cancer progression and metastasis. In conclusion, Th9 and Th17 lymphocytes induce lung cancer cell EMT, thereby promoting migration and metastatic spreading and offering potentially novel therapeutic strategies.

Retraction: Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines.

Retraction: Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines.

MEN1 deficiency leads to neuroendocrine differentiation of lung cancer and disrupts the DNA damage response

The MEN1 gene, a tumor suppressor gene that encodes the protein menin, is mutated at high frequencies in neuroendocrine (NE) tumors; however, the biological importance of this gene in NE-type lung cancer in vivo remains unclear. Here, we established an ATII-specific KrasG12D/+/Men1−/− driven genetically engineered mouse model and show that deficiency of menin results in the accumulation of DNA damage and antagonizes oncogenic Kras-induced senescence and the epithelial-to-mesenchymal transition during lung tumorigenesis. The loss of menin expression in certain human primary lung cancers correlates with elevated NE profiles and reduced overall survival.

RETRACTED ARTICLE: N6-methyladenosine induced miR-143-3p promotes the brain metastasis of lung cancer via regulation of VASH1

BackgroundBrain metastasis (BM) is one of the principal causes of mortality for lung cancer patients. While the molecular events that govern BM of lung cancer remain frustrating cloudy.MethodsThe miRNA expression profiles are checked in the paired human BM and primary lung cancer tissues. The effect of miR-143-3p on BM of lung cancer cells and its related mechanisms are investigated.ResultsmiR-143-3p is upregulated in the paired BM tissues as compared with that in primary cancer tissues. It can increase the invasion capability of in vitro blood brain barrier (BBB) model and angiogenesis of lung cancer by targeting the three binding sites of 3’UTR of vasohibin-1 (VASH1) to inhibit its expression. Mechanistically, VASH1 can increase the ubiquitylation of VEGFA to trigger the proteasome mediated degradation, further, it can endow the tubulin depolymerization through detyrosination to increase the cell motility. m6A methyltransferase Mettl3 can increase the splicing of precursor miR-143-3p to facilitate its biogenesis. Moreover, miR-143-3p/VASH1 axis acts as adverse prognosis factors for in vivo progression and overall survival (OS) rate of lung cancer.ConclusionsOur work implicates a causal role of the miR-143-3p/VASH1 axis in BM of lung cancers and suggests their critical roles in lung cancer pathogenesis.

Prognostic value of insulin-like growth factor 2 mRNA-binding protein 3 and vascular endothelial growth factor-A in patients with primary non-small-cell lung cancer.

Insulin-like growth factor 2 mRNA-binding protein 3 (IMP3) and vascular endothelial growth factor-A (VEGF-A) may play important roles in the process of tumor progression and tumor angiogenesis. The aim of the present study was to examine the co-expression of IMP3 and VEGF-A in primary human non-small cell lung cancer (NSCLC), to investigate the association between these two expression levels and determine the clinicopathological implications, including changes to microvessel density (MVD), and to assess the prognostic value of co-expression. Using immunohistochemical staining, the expression of IMP3, VEGF-A and CD34 expression was detected in 128 primary NSCLC tissue samples. According to the expression of IMP3 and VEGF-A, the cases were divided into four groups. Next, the clinicopathological features, MVD and survival time were investigated across the different groups. The immunohistochemical analyses demonstrated that there was a significant correlation between IMP3 and VEGF-A expression in NSCLC (r=0.181; P=0.041). Co-expression of IMP3 and VEGF-A was significantly associated with larger primary tumor size (P=0.016), poorer differentiation (P=0.014), more advanced Tumor-Node-Metastasis stage (P=0.012), increased MVD (P=0.004) and positive lymph node metastasis (P=0.002). Survival analysis demonstrated that cases with IMP3 and VEGF-A double-positive staining were significantly associated with lower survival rates compared with cases with double-negative staining (P=0.039). In the early NSCLC (I–IIa) subgroup, the mean survival time of the double-positive staining group was significantly shorter compared with that of the double-negative staining group (P=0.015). Co-expression of IMP3 and VEGF-A was associated with angiogenesis and a poorer prognosis in NSCLC, and may therefore play a critical role in NSCLC progression.

MEAK-7 Forms an Alternative mTOR Complex with DNA-PKcs in Human Cancer

SummaryMTOR associated protein, eak-7 homolog (mEAK-7), activates mechanistic target of rapamycin (mTOR) signaling in human cells through an alternative mTOR complex to regulate S6K2 and 4E-BP1. However, the role of mEAK-7 in human cancer has not yet been identified. We demonstrate that mEAK-7 and mTOR signaling are strongly elevated in tumor and metastatic lymph nodes of patients with non-small-cell lung carcinoma compared with those of patients with normal lung or lymph tissue. Cancer stem cells, CD44+/CD90+ cells, yield elevated mEAK-7 and activated mTOR signaling. mEAK-7 is required for clonogenic potential and spheroid formation. mEAK-7 associates with DNA-dependent protein kinase catalytic subunit isoform 1 (DNA-PKcs), and this interaction is increased in response to X-ray irradiation to regulate S6K2 signaling. DNA-PKcs pharmacologic inhibition or genetic knockout reduced S6K2, mEAK-7, and mTOR binding with DNA-PKcs, resulting in loss of S6K2 activity and mTOR signaling. Therefore, mEAK-7 forms an alternative mTOR complex with DNA-PKcs to regulate S6K2 in human cancer cells.

Myeloid cell-derived LL-37 promotes lung cancer growth by activating Wnt/β-catenin signaling.

Rationale: Antimicrobial peptides, such as cathelicidin LL-37/hCAP-18, are important effectors of the innate immune system with direct antibacterial activity. In addition, LL-37 is involved in the regulation of tumor cell growth. However, the molecular mechanisms underlying the functions of LL-37 in promoting lung cancer are not fully understood.Methods: The expression of LL-37 in the tissues and sera of patients with non-small cell lung cancer was determined through immunohistological, immunofluorescence analysis, and enzyme-linked immunosorbent assay. The animal model of wild-type and Cramp knockout mice was employed to evaluate the tumorigenic effect of LL-37 in non-small cell lung cancer. The mechanism of LL-37 involving in the promotion of lung tumor growth was evaluated via microarray analyses, recombinant protein treatment approaches in vitro, tumor immunohistochemical assays, and intervention studies in vivo.Results: LL-37 produced by myeloid cells was frequently upregulated in primary human lung cancer tissues. Moreover, its expression level correlated with poor clinical outcome. LL-37 activated Wnt/β-catenin signaling by inducing the phosphorylation of protein kinase B and subsequent phosphorylation of glycogen synthase kinase 3β mediated by the toll-like receptor-4 expressed in lung tumor cells. LL-37 treatment of tumor cells also decreased the levels of Axin2. In contrast, it elevated those of an RNA-binding protein (tristetraprolin), which may be involved in the mechanism through which LL-37 induces activation of Wnt/β-catenin.Conclusion: LL-37 may be a critical molecular link between tumor-supportive immune cells and tumors, facilitating the progression of lung cancer.