Primary Lung Cancer Tissues Research Articles

Tertiary Lymphoid Structures in Brain Metastases of Lung Cancer: Prognostic Significance and Correlation With Clinical Outcomes.

The prognosis of patients with brain metastases (BMs) originating from lung cancer remains poor, despite advancements in treatment strategies. The role of tertiary lymphoid structures (TLSs) within the tumor immune microenvironment of BMs has not been extensively explored. This study utilized patient-derived clinical samples from 17 patients with histologically confirmed BMs of lung cancer, undergoing surgical resection. Immunohistochemistry was employed to analyze the presence and characteristics of TLS and tumor-infiltrating lymphocytes (TILs) within BM tissues, correlating these with clinical outcomes. TLSs, albeit in their immature form, were identified within BM tissues, distinguishing them from their mature counterparts in primary lung cancer tissues. A significant correlation between TLS density (but not TIL density) and improved postoperative survival was observed, underscoring the potential of TLS density as an independent prognostic marker. Furthermore, TLS density did not correlate with the Graded Prognostic Assessment (GPA) index, suggesting its unique prognostic value beyond conventional predictors. Our findings reveal the presence of TLSs in lung cancer-derived BMs and highlight their prognostic significance, independent of the GPA index. The identification of TLS within the unique central nervous system tumor microenvironment offers new insights into the immune landscape of BMs and suggests potential avenues for immunotherapeutic interventions targeting these structures to improve patient outcomes.

ALKBH5 promotes non-small cell lung cancer progression and susceptibility to anti-PD-L1 therapy by modulating interactions between tumor and macrophages

BackgroundUnderstanding the mechanisms that mediate the interaction between tumor and immune cells may provide therapeutic benefit to patients with cancer. The N6-methyladenosine (m6A) demethylase, ALKBH5 (alkB homolog 5), is overexpressed in non-small cell lung cancer. However, its role in the tumor microenvironment is unknown.MethodsDatasets and tissue samples were used to determine the relationship between ALKBH5 expression and immunotherapy efficacy. Bioinformatic analysis, colorimetric assay to determine m6A RNA methylation, dual luciferase reporter assay, RNA/m6A-modified RNA immunoprecipitation, RNA stability assay, and RNA sequencing were used to investigate the regulatory mechanism of ALKBH5 in non-small cell lung cancer. In vitro and in vivo assays were performed to determine the contribution of ALKBH5 to the development of non-small cell lung cancer.ResultsALKBH5 was upregulated in primary non-small cell lung cancer tissues. ALKBH5 was positively correlated with programmed death-ligand 1 expression and macrophage infiltration and was associated with immunotherapy response. JAK2 was identified as a target of ALKBH5-mediated m6A modification, which activates the JAK2/p-STAT3 pathway to promote non-small cell lung cancer progression. ALKBH5 was found to recruit programmed death-ligand 1-positive tumor-associated macrophages and promote M2 macrophage polarization by inducing the secretion of CCL2 and CXCL10. ALKBH5 and tumor-associated macrophage-secreted IL-6 showed a synergistic effect to activate the JAK2/p-STAT3 pathway in cancer cells.ConclusionsALKBH5 promotes non-small cell lung cancer progression by regulating cancer and tumor-associated macrophage behavior through the JAK2/p-STAT3 pathway and the expression of CCL2 and CXCL10, respectively. These findings suggest that targeting ALKBH5 is a promising strategy of enhancing the anti-tumor immune response in patients with NSCLC and that identifying ALKBH5 status could facilitate prediction of clinical response to anti-PD-L1 immunotherapy.

Consistency Analysis of Programmed Death Ligand 1 Expression in Non–Small Cell Lung Cancer Between Pleural Effusion and Matched Primary Lung Cancer Tissues by Immunohistochemical Double Staining

In clinical practice, programmed death ligand 1 (PD-L1) detection is prone to nonspecific staining due to the complex cellular composition of pleural effusion smears. In this study, diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) immunohistochemistry double staining was performed to investigate PD-L1 expression in tumor cells from malignant pleural effusion (MPE). MPE was considered as a metastasis in non–small cell lung cancer patients; thus, the heterogeneity between metastatic and primary lung cancer was revealed as well. Ninety paired specimens of MPE cell blocks and matched primary lung cancer tissues from non–small cell lung cancer patients were subjected to PD-L1 and thyroid transcription factor-1(TTF-1)/p63 immunohistochemistry double staining. Two experienced pathologists independently evaluated PD-L1 expression using 3 cutoffs (1%, 10%, and 50%). PD-L1 expression in MPE was strongly correlated with that in matched primary lung cancer tissues (R = 0.813; P < .001). Using a 4-tier scale (cutoffs: 1%, 10%, and 50%), the concordance was 71.1% (Cohen’s κ = .534). Using a 2-tier scale, the concordance was 75.6% (1%, Cohen’s κ = 0.53), 78.9% (10%, Cohen’s κ = 0.574), and 95.6% (50%, Cohen’s κ = 0.754). The rates of PD-L1 positivity in MPE (56.7%) were higher than that in lung tissues (32.2%). All 27 discordant cases had higher scores in MPE. The double-staining method provided superior identification of PD-L1-positive tumor cells on a background with nonspecific staining. In conclusion, PD-L1 expression was moderately concordant between metastatic MPE cell blocks and matched primary lung carcinoma tissues, with variability related to tumor heterogeneity. MPE should be considered to detect PD-L1 when histological specimens are unattainable, especially when PD-L1 expression is >50%. PD-L1 positivity rates were higher in MPE. Double staining can improve PD-L1 detection by reducing false-negative/positive results.

Distinct fibroblast subpopulations associated with bone, brain or intrapulmonary metastasis in advanced non-small-cell lung cancer.

Bone or brain metastases may develop in 20-40% of individuals with late-stage non-small-cell lung cancer (NSCLC), resulting in a median overall survival of only 4-6 months. However, the primary lung cancer tissue's distinctions between bone, brain and intrapulmonary metastases of NSCLC at the single-cell level have not been underexplored. We conducted a comprehensive analysis of 14 tissue biopsy samples obtained from treatment-naïve advanced NSCLC patients with bone (n=4), brain (n=6) or intrapulmonary (n=4) metastasis using single-cell sequencing originating from the lungs. Following quality control and the removal of doublets, a total of 80084 cells were successfully captured. The most significant inter-group differences were observed in the fraction and function of fibroblasts. We identified three distinct cancer-associated fibroblast (CAF) subpopulations: myofibroblastic CAF (myCAF), inflammatory CAF (iCAF) and antigen-presenting CAF (apCAF). Notably, apCAF was prevalent in NSCLC with bone metastasis, while iCAF dominated in NSCLC with brain metastasis. Intercellular signalling network analysis revealed that apCAF may play a role in bone metastasis by activating signalling pathways associated with cancer stemness, such as SPP1-CD44 and SPP1-PTGER4. Conversely, iCAF was found to promote brain metastasis by activating invasion and metastasis-related molecules, such as MET hepatocyte growth factor. Furthermore, the interaction between CAFs and tumour cells influenced T-cell exhaustion and signalling pathways within the tumour microenvironment. This study unveils the direct interplay between tumour cells and CAFs in NSCLC with bone or brain metastasis and identifies potential therapeutic targets for inhibiting metastasis by disrupting these critical cell-cell interactions.

Circulating hsa_circ_0072309, acting via the miR-100/ACKR3 pathway, maybe a potential biomarker for the diagnosis, prognosis, and treatment of brain metastasis from non-small-cell lung cancer.

One of the main causes of lung cancer-related death is brain metastasis (BM). Finding early indicators of BM derived from lung cancer is crucial. Therefore, this study was designed to determine if serum hsa_circ_0072309 may be employed as a potential biomarker for BM induced by non-small-cell lung cancer (NSCLC) and to understand its possible underlying mechanism. Primary lung cancer and healthy neighboring tissues were obtained from all patients, while BM tissues were taken from BM+ patients. Serum specimens were collected from all patients and healthy volunteers. Hsa_circ_001653, miR-100, and ACKR3 RNA expressions were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and atypical chemokine receptor 3 (ACKR3) protein expression by western blotting (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). In order to examine the effect of serum hsa_circ_0072309 and its relevant mechanism on BM development, an NSCLC-associated BM model in mice was established. According to the results, miR-100 expression was down-regulated in primary lung cancer tissues compared to healthy lung tissues in all NSCLC patients, and circ_0072309 and ACKR3 expression were up-regulated. In BM tissues compared with primary lung tumors of BM+ patients, in serum samples from all patients compared to healthy volunteers, and in lung tumors of BM+ patients compared to those from BM- patients. Patients' serum exhibits the same level of hsa_circ_0072309/miR-100/ACKR3 expression as in BM samples. Advanced tumor-node-metastasis (TNM) stage, higher BM, shorter post-operative overall survival (OS), and progression-free survival (PFS) are all substantially associated with increased serum circ_0072309 levels in BM+ patients. In animal models, serum owning hsa_circ_0072309 from BM+ patients facilitates BM formation by regulating the miR-100/ACKR3 pathway. The current preliminary research reveals serum hsa_circ_0072309 as a possible biomarker and target for early diagnosis, prognosis, and therapy of NSCLC-derived BM and suggests a substantial role for the hsa_circ_0072309/miR-100/ACKR3 axis in the formation of BM from NSCLC.

Abstract 3808: Spatially resolved transcriptomics of cellular architecture in EGFR-mutated lung cancer

Abstract Introduction: Single-cell RNA sequencing of dissociated tumors enables the profiling of cellular states in fine detail, but erases the cellular organization of the analyzed tissue. Spatially resolved transcriptomics (SRT) using 10X Genomics’ Visium platform combines histological staining and RNA sequencing by capturing each transcript across spatially barcoded microarrays. SRT yields gene-expression matrices resolved within 55-micron array spots, and creates opportunities to explore how lung biology is affected by lung adenocarcinoma and how tumor cells and the proximal microenvironment are modulated by targeted therapy. Methods: SRT reactions (n=8) were performed on surgical specimens from human lung adenocarcinomas driven by kinase domain mutations of EGFR (exon 19 deletion, L858R point mutation, exon 20 insertion). SRT reactions analyzed primary lung cancer (n=5) or paired tumor-adjacent lung tissues (n=3). Results: 28458 array spots were recorded across all samples, and array spots captured a median of 5800 total transcripts and a median of 2416 unique transcripts. Single marker gene expression and unsupervised clustering across array spots corresponded with histological annotations of lung structures and adenocarcinoma. Integration of single-cell RNA sequencing data from lung adenocarcinoma specimens permitted mapping and quantification of 15 major cell types, including cancer cells, T- and B- lymphocytes, macrophages, dendritic cells, fibroblasts, and endothelial cells. Compared with paired tumor-adjacent lung tissues, adenocarcinoma tissues contained fibroblast-enriched desmoplasia and B-cell enriched tertiary lymphoid structures. In a clinical case with resistance to Osimertinib, the standard tyrosine kinase inhibitor, gene signature analysis of cancer-containing array spots revealed enrichment of gap-junction, fatty acid metabolism, and kynurenine pathway signatures compared to treatment naïve array spots. Conclusion: This pilot study demonstrates the feasibility of using SRT in lung adenocarcinoma. Paired with a single-cell atlas of lung adenocarcinoma during targeted therapy, this approach enables hypothesis-generation to investigate the alterations of tumor and tumor microenvironmental architecture in relation to targeted therapy. Citation Format: Daniel L. Kerr, Wei Wu, Whitney Tamaki, Anatoly Urisman, Yu-Ting Chou, Philippe Gui, David M. Jablons, Trever G. Bivona, Collin M. Blakely. Spatially resolved transcriptomics of cellular architecture in EGFR-mutated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3808.

Abstract 3070: Non-small cell lung cancer patient-derived organoids as high-throughput chemotherapy testing platforms

Abstract Background: Lung cancer is associated with phenotypic, genotypic, and transcriptomic heterogeneity among patients that poses a threat to the development of precision medicine. Despite being considered as potential drug screening platforms, patient-derived xenograft (PDX) models are limited by low rates and prolonged durations of tumor engraftment. To overcome these major limitations, patient-derived organoid (PDO) models have been developed for standard of care drug testing and the outcome of which is driven by differential transcriptome trajectories. Experimental methods: Single cell suspension from ten surgically resected patient primary non-small cell lung cancer (NSCLC) tissues (adenocarcinoma and squamous cell carcinoma) was mixed with matrigel (growth factor reduced) and cultured in organoid growth medium. Organoids were characterized for structural (Hematoxylin &amp; Eosin staining) and pathological marker (immunohistochemistry for cytokeratin (CK) 5/6, CK7, Napsin A, Thyroid transcription factor-1 (TTF-1) and p40) similarities with primary tumor. Upon passaging, organoids were seeded in triplicates, allowed to acclimate for 24h and subjected to standard of care chemotherapy testing versus vehicle control. PDOs and corresponding primary tumors were subjected to bulk RNA sequencing to understand the transcriptome driven outcome in chemotherapy sensitive versus resistant organoids. Results: Organoid growth was monitored by light microscopy. PDOs exhibited different growth kinetics indicating the inter-tumor heterogeneity. Time for PDOs to reach 100-200 μm ranged from 6 days to 14 days and exhibited structural features and pathological marker expression similar to their corresponding primary tumors reflecting respective histological subtypes - adenocarcinoma or squamous cell carcinoma. Chemotherapy testing (1 μg/ml Carboplatin plus 0.5 μg/ml Paclitaxel) showed differential responses (day 6 of treatment). Based on the scoring, PDOs were categorized as partially sensitive, very sensitive or resistant (based on organoid growth or regression in presence of chemotherapeutic drugs). Results of RNA sequencing may reveal distinct transcriptomic signatures and molecular pathways reflecting in differential chemoresponses. Conclusions and future perspectives: PDOs capitulate structural and pathological marker expression of patient primary tumors and serve as versatile, high-throughput drug testing platforms to treat NSCLC patients. Further, identification of key transcriptomic signatures and molecular pathways resulting in chemoresistance will be critical in formulating personal precision medicine to treat NSCLC patients. Citation Format: Yariswamy Manjunath, Kanve Nagaraj Suvilesh, Iuliia Innokenteva, Wesley Warren, Eric T. Kimchi, Kevin F. Staveley-O’Carroll, Guangfu Li, Jonathan B. Mitchem, Jussuf T. Kaifi. Non-small cell lung cancer patient-derived organoids as high-throughput chemotherapy testing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3070.

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.

MPDZ as a novel epigenetic silenced tumor suppressor inhibits growth and progression of lung cancer through the Hippo-YAP pathway.

MPDZ also named MUPP1 is involved in signal transduction mediated by the formation of protein complexes. However, the expression regulation, clinical significance, potential function, and mechanism of this gene in lung cancer remain unclear. Methylation status of MPDZ was measured by methylation-specific PCR and bisulfite genomic sequencing. Kaplan-Meier and Cox regression analyses were performed to identify the prognostic value of MPDZ. The tumor suppressing effects of MPDZ were determined in vitro and in vivo. The target molecules and signaling pathway that mediated the function of MPDZ were also identified. MPDZ methylation was identified in 61.2% of primary lung cancer tissues and most lung cancer cell lines but not in normal lung tissues. MPDZ expression was significantly downregulated in lung cancer tissues and negatively associated with DNA hypermethylation, and attenuated MPDZ expression predicted a poor outcome. Furthermore, MPDZ overexpression prominently dampened cell growth, migration, and invasion of tumor cells. Conversely, MPDZ knockdown promoted cell proliferation, migration, and invasion in vitro and in vivo. Moreover, MPDZ deficiency promotes tumor metastasis and reduces the survival of MPDZ knockout mice. Importantly, MPDZ promotes tumor suppressor ability that depends on the Hippo pathway-mediated repression of YAP. MPDZ activates the phosphorylation of YAP (Ser127) and inhibits YAP expression through stabilizing MST1 and interaction with LATS1. We first identified and validated that MPDZ methylation and expression could be a good diagnostic marker and independent prognostic factor for lung cancer. MPDZ functions as a tumor suppressor by inhibiting cell proliferation, migration, and invasion through regulating the Hippo-YAP signaling pathway.

DNA methylome profiling of circulating tumor cells in lung cancer at single base-pair resolution

DNA methylation plays a pivotal role in regulating cellular processes, and altered DNA methylation pattern is a general hallmark of cancer. However, DNA methylome in circulating tumor cells (CTCs) is still a mystery due to the lack of proper analytical techniques. We introduced an efficient workflow, LCM–µWGBS, which can efficiently profile the DNA methylation of microdissected CTC samples. LCM–µWGBS combines the laser capture microdissection (LCM)-based CTC capture method and whole-genome bisulfite sequencing in very small CTC population (µWGBS) to gain insight into the DNA methylation landscape of CTCs. We herein profiled the DNA methylome of CTCs from lung cancer patients. Deriving from a comprehensive analysis of CTC methylome, a unique “CTC DNA methylation signature” that is distinct from primary lung cancer tissues was identified. Further analysis showed that promoter hypermethylation of epithelial genes is a hallmark of stable epithelial–mesenchymal transition process. Moreover, it has been suggested that CTCs are endowed with a stemness-related feature during dissemination and metastasis. This work constitutes a unique DNA methylation analysis of CTCs at single base-pair resolution, which might facilitate to propose noninvasive CTC DNA methylation biomarkers contributing to clinical diagnosis.

High expression of hexokinase 2 promotes lung cancer proliferation and metastasis

IntroductionHigh metabolic plasticity, as one of the hallmarks of multiple cancers, usually promotes cancer growth and metastasis. Therefore, metabolic enzymes could be selective targets in suppressing the growth and invasion of cancer cells. It is generally believed that hexokinase 2 (HK2) catalysing glucose into glucose-6-phosphate is the first step in glucose metabolism. It is unclear, however, if HK2 is involved in lung cancer (LC) proliferation and metastasis.Material and methodsTumours and adjacent tissue from LC patients were used to study the expression of HK2 by RT-PCR and western blot analysis. Several different LC cell lines were used to explore the oncogene function of HK2 via MTS and BrdU assay in vitro, as well as xenografts model and Lung metastasis model in vivo.ResultsHK2 was upregulated in primary LC tissue and metastatic foci. The expression of HK2 can be modulated by multiple mutations of oncogenes or tumour suppressors, including Kras, p53, and Keap1. Functionally, suppression of HK2 expression by shRNA transfection in LC cell lines hindered lactate production and therefore suppressed cell migration and invasion. The depletion of HK2 also sensitised the LC cells to cisplatin-induced apoptosis, suggesting that HK2 also mediated the chemoresistance in LC. Finally, our in vivo data indicated that stable knockdown of HK2 inhibited xenografted tumour growth, sensitized the tumour response to cisplatin treatment, and decreased the incidence of lung metastasis in nude mice after tail vein injection.ConclusionsWe demonstrated that HK2 is required for LC cell growth and metastasis in vitro and in vivo, and for its relationship between multiple mutations in LC. Our results revealed that HK2 promotes LC cell proliferation and metastasis in various mutation backgrounds of LC cells, indicating that HK2 is a high-potential therapy target for LC treatment. Our data provide strong evidence that HK2 plays an important role in promoting LC disease progression, and it may be a promising target for lung cancer therapy.

MPDZ As a Novel Epigenetic Silenced Tumor Suppressor Inhibits Growth and Progression of Lung Cancer Through the Hippo-YAP Pathway

MPDZ is involved in signal transduction mediated by the formation of protein complexes. However, the expression regulation, clinical significance, potential function and mechanism of this gene in lung cancer remain unclear. Methylation status of MPDZ was measured by methylation-specific PCR and bisulfite genomic sequencing. Kaplan-Meier and Cox regression analyses were performed to identify the prognostic value of MPDZ. The tumor suppressing effects of MPDZ were determined in vitro and in vivo. The target molecules and signaling pathway that mediated the function of MPDZ were also identified. MPDZ methylation was identified in 61.2% of primary lung cancer tissues and most lung cancer cell lines but not in normal lung tissues. MPDZ expression was significantly downregulated in lung cancer tissues and negatively associated with DNA hypermethylation, and attenuated MPDZ expression predicted a poor outcome. Furthermore, MPDZ overexpression prominently dampened cell growth, migration and invasion of tumor cells. Conversely, MPDZ knockdown promoted cell proliferation, migration and invasion in vitro and in vivo. Importantly, MPDZ promotes tumor suppressor ability depends on the Hippo pathway-mediated repression of YAP. MPDZ activates the phosphorylation of YAP (Ser127) and inhibits YAP expression through interaction with MST1/LATS/YAP complex. Moreover, MPDZ deficiency promotes tumor metastasis and reduces the survival of MPDZ knock-out mice. We first identified and validated that MPDZ methylation and expression could be a good diagnostic marker and independent prognostic factor for lung cancer. MPDZ functions as a tumor suppressor by inhibiting cell proliferation, migration and invasion through regulating the Hippo-YAP signaling pathway. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (No. 81573179 and 81573114). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This study was approved by the Clinical Research Ethics Committee of the Third Military Medical University, and informed consent was obtained from all patients. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Third Military Medical University, China.

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.

CAMK2A Supported Tumor Initiating Cells of Lung Adenocarcinoma by Upregulating SOX2 Through EZH2 Phosphorylation

Tumor initiating cells (TIC) of lung cancer are mainly induced by stress-related plasticity. Calcium/Calmodulin dependent protein kinase II alpha (CAMK2A) is a key calcium signaling molecule activated by exogenous and endogenous stimuli with effects on multiple cell functions but little is known about its role on TIC. In human lung adenocarcinomas (AD), CAMK2A was aberrantly activated in a proportion of cases and was an independent risk factor predicting shorter survivals. Functionally, CAMK2A enhanced TIC phenotypes in vitro and in vivo. CAMK2A regulated SOX2 expression by reducing H3K27me3 and EZH2 occupancy at SOX2 regulatory regions, leading to its epigenetic de-repression with functional consequences. Further, CAMK2A caused kinase-dependent phosphorylation of EZH2 at T487 with suppression of EZH2 activity. Together, the data demonstrated the CAMK2A-EZH2-SOX2 axis on TIC regulation. This study provided phenotypic and mechanistic evidence for the TIC supportive role of CAMK2A, implicating a novel predictive and therapeutic target for lung cancer. Funding: This study was supported by the Research Grants Council General Research Fund (GRF) (GRF 17150916), and the Small Project Funding of the University of Hong Kong (201309176108). Declaration of Interest: The authors disclose no potential conflicts of interest. Ethical Approval: Primary lung cancer tissues from ethnic Chinese were collected in Queen Mary Hospital with informed consent after approval by HKU/HAHKWC Ethics Board. All animal experiments were approved and performed according to guidelines by the Animal Ethics Committee, the University of Hong Kong.

Carboxypeptidase A4 promotes migration and invasion of lung cancer cells, and is closely associated with lymph node metastasis

AbstractObjectiveThe incidence and mortality rate of lung cancer ranks first among all malignant tumors. Lymph node metastasis is the most common mode of metastasis in lung cancer, and it is also an important indicator for treatment strategies and prognosis. Several studies have shown that the carboxypeptidase family plays important roles in cancer metastasis. In the present study, we investigated the effect of carboxypeptidase A4 (CPA4) on human lung cancer cells, and the clinical significance of its levels in lung cancer tissues and corresponding lymph node metastasis tissues.MethodsReal‐time quantitative polymerase chain reaction and western blotting were used to detect the expression levels of CPA4 in cancer cells after transfecting them with CPA4 interference sequence (siRNA‐CPA4) and the negative control sequence. Transwell assay with/without Matrigel was used to assess the effect of CPA4 on cell invasion and migration. CPA4 expression was detected in eight normal lung tissues, 46 lung cancer tissues, and the corresponding lymph node metastasis tissues using immunohistochemistry.ResultsReverse transcription polymerase chain reaction and western blot showed that siRNA‐CPA4 significantly decreased CPA4 mRNA levels in H226 and SKMES‐1 cells compared with the control cells. Knockdown of CPA4 in lung cancer cells significantly inhibited cell invasion by 62.3% and 77.3% in H226 and SKMES‐1 cell, respectively. Furthermore, siRNA‐CPA4 significantly suppressed cell migration in H226 and SKMES‐1 cells by 48.1% and 42.4%, respectively. Immunohistochemistry results showed that the positive expression rate of CPA4 in lung normal tissues, cancer tissues, and lymph node metastatic tissues was 12.5% (1/8), 50.0% (23/46), and 76.1% (35/46), respectively. Importantly, the levels of CPA4 in the lymph node metastatic tissues were significantly higher than those in corresponding primary lung cancer tissues (t = 5.176, P &lt; 0.001). The clinical correlation analysis showed that high levels of CPA4 were closely associated with the tumor pathology type, differentiation, and stage; however, there was no significant correlation with the age, sex, and depth of invasion.ConclusionsCPA4 promoted the invasion and migration of human lung cancer cells, H226, and SKMES‐1, in vitro. The levels of CPA4 were gradually elevated in primary lung cancer and corresponding lymph node metastasis tissues, and CPA4 could be a potential candidate therapeutic target for lung cancer.

Abstract A221: Epigenetic therapy restores polyfunctionality of malignant pleural effusion T-cells in patients with non-small cell lung cancer without downregulation of PD-1

Abstract Cytotoxic T-cells in patients with advanced tumors are often dysfunctional due to persistent cancer antigen exposure. These dysfunctional T-cells, known as exhausted T-cells, are characterized by an increased expression of inhibitory receptors and the gradual loss of effector functions, which contribute to immune evasion of cancer cells. Immune checkpoint blockade therapy works by removing the inhibitory signals, reversing the T-cell exhausted state, thereby leading to immune-mediated tumor regression. While immunotherapy has become a front-line treatment for many lung cancer patients for its promising clinical outcome, the durability of this reversal of T-cell exhaustion still remains unsatisfactory, which contributes to eventual treatment failure. Emerging evidence in animal models suggests that exhausted T-cells have a distinct epigenetic landscape that remains unaltered following immune checkpoint blockade therapy. Thus, we hypothesized that epigenetic therapy may reprogram tumor-reactive T-cells to enhance their effector functions. We isolated CD3+ T-cells from malignant pleural effusion or primary tumor tissues of 20 lung cancer patients and examined their immunophenotypes, including surface markers (CD4 and CD8), proliferative index (Ki67), as well as inhibitory receptors (PD-1, TIM3, and CTLA4). T-cells were subject to in vitro daily treatment of decitabine, a DNA methyltransferase inhibitor, for 72 hours. Polyfunctional T-cells that co-express effector functions (i.e., TNF-α, IFN-γ, IL-2 and CD107a) were analyzed by multichromatic flow cytometry. We discovered that T-cells in the malignant pleural effusion or primary lung cancer tissues have higher CD4/CD8 ratios as compared with peripheral blood mononuclear cells from healthy volunteers. The majority of T-cells from lung cancer patients exhibited the exhausted phenotype and expressed at least one inhibitory receptor at higher levels. This indicates that T-cells from malignant pleural effusion could potentially reflect dysfunctional immune states in lung cancer patients when tumor tissues are not available. Moreover, treatment of decitabine at clinically relevant concentrations significantly increased the polyfunctionality of PD-1 positive CD4+ and CD8+ T-cells from selected patients. Interestingly, the reinvigoration of polyfunctional T-cell responses did not associate with the downregulation of PD-1. On the other hand, the effects of decitabine on PD-1 negative CD4+ and CD8+ T-cells were only modest. This suggests that malignant pleural effusion T-cells, which are often PD-1 positive, may respond to epigenetic reprogramming via PD-1-independent mechanisms. Our data shed light on the potential uses of epigenetic therapy in lung cancer treatment by immunomodulation. Citation Format: Hsing-Chen Tsai, Yi-Chieh Wu, Shu-Yung Lin, I-Yu Chen, Jih-Hsiang Lee, Kuang-Hua Cheng, Ping-Huai Wang, Huan-Jang Ko, Wen-Chien Huang, Yi-Jhen Huang, Kai-Lin Wei, Chong-Jen Yu, Yen-Ling Chiu. Epigenetic therapy restores polyfunctionality of malignant pleural effusion T-cells in patients with non-small cell lung cancer without downregulation of PD-1 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A221.

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.

Expression and correlation of MALAT1 and SOX9 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths in the world. MALAT1 and SOX9 have important roles in tumour formation and development in several types of cancers. However, little is known about the function and co-relationship of these 2 factors in NSCLC in vivo. To explore the role of MALAT1 and SOX9 expression relationship, their clinical pathological characteristics and OS on NSCLC patients. Paired of primary lung cancer tissues and the matched tumour adjacent tissues were collected in 121 NSCLC patients. MALAT1 and SOX9 mRNA expression was measured by SYBR green q RT-PCR assay. SOX-9 protein expression was measured by streptavidin-peroxidase (SP) staining method. MALAT1and SOX9 expression was higher in NSCLC tissues than the adjacent tissues, and they have positive correlation. Moreover, SOX9 protein expression was higher in NSCLC tissues, especially in MALAT1 mRNA higher expressed NSCLC tissues. MALAT1 and SOX9 mRNA expression were associated with age (x2 =11.474, P = .009), tumour size (x2 =26.839, P = .000), TNM stage (x2 =8.010, P = .046) and LEL. (x2 =53.908, P = .000). NSCLC patients with higher MALAT1 and SOX9 mRNA expression had poorer OS rates. MALAT1 and SOX9 could be used as prognostic co-biomarker in NSCLC.

Abnormal expression of angiopoietin-2 associated with invasion, metastasis and prognosis of lung cancer

Objective: To investigate the relationship between abnormal angiopoietin-2 (Ang-2) expression and invasion/metastasis of lung cancer. Methods: Totally 122 cases of postoperative primary lung cancer tissues and their paracancerous tissues from Jan. 2009 to Dec. 2010 were collected from Affiliated Hospital of Nantong University and Ang-2 expression was analyzed by immunohistochemistry. At the cellular level, the protein and mRNA levels of Ang-2 in lung epithelial cell line Beas-2B and four lung cancer cell lines (SPCA-1, NCI-1650, A549 and NCI-H1975) were observed. The most effective Ang-2-shRNA for Ang-2 transcription was screened and transfected into A 549 lung cancer cells. The Ang-2 expression, Ang-2 gene transcription, cell proliferation, invasion/metastasis, and epithelial-mesenchymal transition (EMT) abilities of lung cancer cells were analyzed by Western blotting, fluorescent quantitative reverse transcriptase PCR, Cell Counting Kit-8 assay, and Transwell cell models for exploring the relationship between Ang-2 expression and invasion/metastasis of lung cancer. Results: The higher Ang-2 expression levels in lung cancerous tissues were closely related to tumor diameter (P=0.008), differentiating degree (P=0.033), TNM stage (P=0.025) and 5-year survival rate (P<0.001). According to the Kaplan-Meier survival curves, the 5-year survival rate of patients with higher expression levels of Ang-2 (16.1%) was significantly poorer than that of patients with lower Ang-2 (80.0%, P<0.001). Significant difference of 5-year survival rate was found in patients with different Ang-2 levels at TNM stage Ⅰ(P<0.001), but not at stage Ⅱ, Ⅲ and Ⅳ. Among Beas-2B and four lung cancer cell lines, the protein and mRNA levels of Ang-2 in A549 cells were the highest. After Ang-2-shRNA-1 plasmid successfully transfected into A549 cells, cell proliferation rate was significantly lower than that in the shRNA-negative or blank group at a time-dependent manner. The significant decrease of the invasion, migration and EMT abilities were also found in A549 cells after transfection of Ang-2 shRNA. Conclusion: Abnormal expression of Ang-2 is closely related to invasion, migration and prognosis of lung cancer, and interfering the activation of Ang-2 would be a novel molecular-targeted therapy for lung cancer.

NatD promotes lung cancer progression by preventing histone H4 serine phosphorylation to activate Slug expression

N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation (Nt-acetylation) of histone H4 known to be involved in cell growth. Here we report that NatD promotes the migratory and invasive capabilities of lung cancer cells in vitro and in vivo. Depletion of NatD suppresses the epithelial-to-mesenchymal transition (EMT) of lung cancer cells by directly repressing the expression of transcription factor Slug, a key regulator of EMT. We found that Nt-acetylation of histone H4 antagonizes histone H4 serine 1 phosphorylation (H4S1ph), and that downregulation of Nt-acetylation of histone H4 facilitates CK2α binding to histone H4 in lung cancer cells, resulting in increased H4S1ph and epigenetic reprogramming to suppress Slug transcription to inhibit EMT. Importantly, NatD is commonly upregulated in primary human lung cancer tissues where its expression level correlates with Slug expression, enhanced invasiveness, and poor clinical outcomes. These findings indicate that NatD is a crucial epigenetic modulator of cell invasion during lung cancer progression.