Human Lung Cancer Tissues Research Articles

ZNF652 exerts a tumor suppressor role in lung cancer by transcriptionally downregulating cyclin D3.

Dysfunction of zinc finger protein 652 (ZNF652) is associated with various malignant tumors. However, the role of ZNF652 in lung cancer (LC) is poorly understood. Here, we identified that ZNF652 was downregulated in human LC tissues and cell lines. Low ZNF652 expression was associated with poor survival in LC patients. Overexpression of ZNF652 inhibited cell viability, proliferation, migration, and invasion of LC cells, whereas ZNF652 knockdown promoted these malignant phenotypes. Using RNA-seq analysis revealed that ZNF652 overexpression resulted in obvious alterations of various biological processes, especially cell cycle and cellular senescence. Subsequently, we confirmed that ZNF652 overexpression arrested the cell cycle at the G1 phase, increased ROS-mediated DNA damage, induced LC cell senescence, and enhanced cisplatin-induced apoptosis in LC cells. Mechanistically, ZNF652 directly bound to the promoter of cyclin D3 (CCND3), inhibited its transcription, thereby arresting the cell cycle at the G1 phase. Ectopic expression of cyclin D3 rescued the decreased cell viability and cell cycle arrest induced by ZNF652. In vivo studies further showed that ZNF652 overexpression suppressed the tumorigenic potential of LC. Collectively, our findings reveal that ZNF652 exerts a tumor suppressor role in lung cancer by inducing cell cycle arrest and cellular senescence via transcriptionally downregulating cyclin D3. Thus, ZNF652 may be a prognostic predictive factor for LC patients.

Species Diversity and Network Diversity in the Human Lung Cancer Tissue Microbiomes.

This study explores the relationship between microbial diversity and disease status in human lung cancer tissue microbiomes, using a sample size of 1,212. Analysis divided the data into primary tumor (PT) and normal tissue (NT) categories. Differences in microbial diversity between PT and NT were significant in 57% of comparisons, although dataset dependence was a factor in the diversity levels. Shared species analysis (SSA) indicated no significant differences between PT and NT in over 90% of comparisons. Network diversity assessments revealed significant differences between NT and PT regarding species relative abundances and network link abundances for q=0-3. Additionally, at q=0, significant variations were found between NT and LUSC in network link probabilities, illustrating the diversity in species interactions. Our findings suggest a stable overall microbiome diversity and composition in lung cancer patients' lung tissues despite patients with diagnosed lung tumors, indicating modified microbial interactions within the tumor. These results highlight an association between altered microbiome interaction patterns and lung tumors, offering new insights into the ecological dynamics of lung cancer microbiomes.

O-GlcNAcylation promotes malignancy and cisplatin resistance of lung cancer by stabilising NRF2.

The transcription factor NRF2 plays a significant role in regulating genes that protect cells from oxidative damage. O-GlcNAc modification, a type of posttranslational modification, is crucial for cellular response to stress. Although the involvement of both NRF2 and O-GlcNAc in maintaining cellular redox balance and promoting cancer malignancy has been demonstrated, the potential mechanisms remain elusive. The immunoblotting, luciferase reporter, ROS assay, co-immunoprecipitation, and immunofluorescence was used to detect the effects of global cellular O-GlcNAcylation on NRF2. Mass spectrometry was utilised to map the O-GlcNAcylation sites on NRF2, which was validated by site-specific mutagenesis and O-GlcNAc enzymatic labelling. Human lung cancer samples were employed to verify the association between O-GlcNAc and NRF2. Subsequently, the impact of NRF2 O-GlcNAcylation in lung cancer malignancy and cisplatin resistance were evaluated in vitro and in vivo. NRF2 is O-GlcNAcylated at Ser103 residue, which hinders its binding to KEAP1 and thus enhances its stability, nuclear localisation, and transcription activity. Oxidative stress and cisplatin can elevate the phosphorylation of OGT at Thr444 through the activation of AMPK kinase, leading to enhanced binding of OGT to NRF2 and subsequent elevation of NRF2 O-GlcNAcylation. Both in cellular and xenograft mouse models, O-GlcNAcylation of NRF2 at Ser103 promotes the malignancy of lung cancer. In human lung cancer tissue samples, there was a significant increase in global O-GlcNAcylation, and elevated levels of NRF2 and its O-GlcNAcylation compared to paired adjacent normal tissues. Chemotherapy promotes NRF2 O-GlcNAcylation, which in turn decreases cellular ROS levels and drives lung cancer cell survival. Our findings indicate that OGT O-GlcNAcylates NRF2 at Ser103, and this modification plays a role in cellular antioxidant, lung cancer malignancy, and cisplatin resistance.

Optimization of whole slide imaging scan settings for computer vision using human lung cancer tissue.

Digital pathology has become increasingly popular for research and clinical applications. Using high-quality microscopes to produce Whole Slide Images of tumor tissue enables the discovery of insights into biological aspects invisible to the human eye. These are acquired through downstream analyses using spatial statistics and artificial intelligence. Determination of the quality and consistency of these images is needed to ensure accurate outcomes when identifying clinical and subclinical image features. Additionally, the time-intensive process of generating high-volume images results in a trade-off that needs to be carefully balanced. This study aims to determine optimal instrument settings to generate representative images of pathological tissue using digital microscopy. Using various settings, an H&E stained sample was scanned using the ZEISS Axio Scan.Z1. Next, nucleus segmentation was performed on resulting images using StarDist. Subsequently, detections were compared between scans using a matching algorithm. Finally, nucleus-level information was compared between scans. Results indicated that while general matching percentages were high, similarity between information from replicates was relatively low. Additionally, settings resulting in longer scanning times and increased data volume did not increase similarity between replicates. In conclusion, the scan setting ultimately deemed optimal combined consistent and qualitative performance with low throughput time.

Combination of arsenic trioxide and apatinib synergistically inhibits small cell lung cancer by down-regulating VEGFR2/mTOR and Akt/c-Myc signaling pathway via GRB10

BackgroundSmall cell lung carcinoma (SCLC) is characterized by -poor prognosis, -high predilection for -metastasis, -proliferation, and -absence of newer therapeutic options. Elucidation of newer pathways characterizing the disease may allow for development of targeted therapies and consequently favorable outcomes.MethodsThe current study explored the combinatorial action of arsenic trioxide (ATO) and apatinib (APA) in vitro and in vivo. In vitro models were tested using -H446 and -H196 SCLC cell lines. The ability of drugs to reduce -metastasis, -cell proliferation, and -migration were assessed. Using bioinformatic analysis, differentially expressed genes were determined. Gene regulation was assessed using gene knock down models and confirmed using Western blots. The in vivo models were used to confirm the resolution of pathognomic features in the presence of the drugs. Growth factor receptor bound protein (GRB) 10 expression levels of human small cell lung cancer tissues and adjacent tissues were detected by IHC.ResultsIn combination, ATO and APA were found to significantly reduce -cell proliferation, -migration, and -metastasis in both the cell lines. Cell proliferation was found to be inhibited by activation of Caspase-3, -7 pathway. In the presence of drugs, it was found that expression of GRB10 was stabilized. The silencing of GRB10 was found to negatively regulate the VEGFR2/Akt/mTOR and Akt/GSK-3β/c-Myc signaling pathway. Concurrently, absence of metastasis and reduction of tumor volume were confirmed in vivo. The immunohistochemical results confirmed that the expression level of GRB10 in adjacent tissues was significantly higher than that in human small cell lung cancer tissues.ConclusionsSynergistically, ATO and APA have a more significant impact on inhibiting cell proliferation than each drug independently. ATO and APA may be mediating its action through the stabilization of GRB10 thus acting as a tumor suppressor. We thus, preliminarily report the impact of GRB10 stability as a target for SCLC treatment.

Antibody MYH9 and Antibiotic Lidamycin Inhibit the Growth and Proliferation of Lung Cancer Cells and Induce Their Apoptosis.

The aim of this study was to investigate the impact of the antibiotic lidamycin (LDM) and the targeted therapy with the antibody Myosin heavy chain 9 (MYH9) on cancer cells, aiming to provide insights for cancer treatment. In this study, antibiotics and targeted antibodies were used in cancer cells, and then their effects on cell growth, proliferation, apoptosis regulation, and related proteins were measured, and comparative analysis was conducted on the effects of different drug concentrations on the growth of cancer cells. In H460, the apoptotic effect of 2nM LDM on cells reached 70%. LDM had a downward trend on the levels of B-cell lymphoma-2 (Bcl-2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in cells. The inhibitory effects of LDM at different concentrations on human large cell lung cancer H460 transplanted tumor in nude mice reached 53.20% and 69.80%, and the inhibitory effects on the growth of lung adenocarcinoma transplanted tumor in nude mice reached 40.20% and 58.30%. The expression of MYH9 (myosin, heavy polypeptide 9, non-muscle) in human lung cancer tissues and adjacent tissues reached more than 80%. At the concentration of 300μM, antibody MYH9 inhibited cell growth by 30%, and the migration rate was also reduced by 25%. The inhibitory effect of siRNA after knocking out the MYH9 gene on cancer cells reached 70%. Antibiotic LDM and targeted antibody MYH9 can inhibit the growth, proliferation, and migration of cancer cells, promote cancer cell apoptosis, and have certain clinical significance for the treatment of cancer patients.

Neurotrophin-3 Facilitates Stemness Properties and Associates with Poor Survival in Lung Cancer

Introduction: Cancer stem cells (CSCs) shape the tumor microenvironment via neuroendocrine signaling and orchestrate drug resistance and metastasis. Cytokine antibody array demonstrated the upregulation of neurotrophin-3 (NT-3) in lung CSCs. This study aims to dissect the role of NT-3 in lung CSCs during tumor innervation. Methods: Western blotting, quantitative reverse transcription-PCR, and flow cytometry were used to determine the expression of the NT-3 axis in lung CSCs. NT-3-knockdown and NT-3-overexpressed cells were derived lung CSCs, followed by examining the stemness gene expression, tumorsphere formation, transwell migration and invasion, drug resistance, soft agar colony formation, and in vivo tumorigenicity. Human lung cancer tissue microarray and bioinformatic databases were used to investigate the clinical relevance of NT-3 in lung cancer. Results: NT-3 and its receptor tropomyosin receptor kinase C (TrkC) were augmented in lung tumorspheres. NT-3 silencing (shNT-3) suppressed the migration and anchorage-independent growth of lung cancer cells. Further, shNT-3 abolished the sphere-forming capability, chemo-drug resistance, invasion, and in vivo tumorigenicity of lung tumorspheres with a decreased expression of CSC markers. Conversely, NT-3 overexpression promoted migration and anchorage-independent growth and fueled tumorsphere formation by upregulating the expression of CSC markers. Lung cancer tissue microarray analysis revealed that NT-3 increased in patients with advanced-stage, lymphatic metastasis and positively correlated with Sox2 expression. Bioinformatic databases confirmed a co-expression of NT-3/TrkC-axis and demonstrated that NT-3, NT-3/TrkC, NT-3/Sox2, and NT-3/CD133 worsen the survival of lung cancer patients. Conclusion: NT-3 conferred the stemness features in lung cancer during tumor innervation, which suggests that NT-3-targeting is feasible in eradicating lung CSCs.

Effect of P. gingivalis on osimertinib resistance in EGFR-mutant lung cancer.

e20511 Background: While most patients with EGFR-mutant lung cancers respond to osimertinib, resistance inevitably develops. Mechanisms of osimertinib resistance remain inadequately understood. Recent studies suggest a potential link between intra-tumoral bacteria and therapy resistance in cancer. Our previous studies found that the pre-conditioned medium (PCM) of certain bacteria in the Bacteroidetes phylum conferred resistance of all EGFR-TKIs tested. In this study, we specifically investigate the effect of Porphyromonas gingivalis, a common component of the human oral microbiome and the main pathogen responsible for periodontal disease, on osimertinib sensitivity in EGFR mutant lung cancer. Methods: P. gingivalis PCM (P-CM) was applied to EGFR-mutant lung cancer cell lines for 3 days and cell viability was measured. Western blot and receptor tyrosine kinase (RTKs) assay were used to analyze the changes of cell signaling pathways, and the contribution of identified signaling pathway to P. gingivalis-mediated resistance to osimertinib were further validated by rescue experiments. To investigate the existence of P. gingivalis in human lung cancer tissues, we used Fluorescence In Situ Hybridization (FISH) probes specific to P. gingivalis and hybridized probes to a tissue microarray (TMA) containing 3 mm punches of lung adenocarcinoma tissue from 30 patients in duplicate. The underlying mechanisms of osimertinib resistance conferred by P. gingivalis were explored by a combination of bacterial genomic and cellular proteomic approaches. Results: P-PCM led to osimertinib resistance in all EGFR-mutant lung cancer cell lines tested. Both Western blot and RTKs assay showed that phosphorylated insulin-like growth factor 1 receptor (pIGF1R) level significantly increased when P-PCM was applied to PC9 EGFR mutant lung cancer cells. We found that both IGF1R inhibitor (linsitinib) and IGF1R knockdown with siRNA eliminated the effect of P-PCM-mediated osimertinib resistance. We found that two cysteine proteases secreted by P. gingivalis, lysine-specific gingipain (Kgp) and arginine-specific gingipain A (RgpA) led to osimertinib, whereas arginine-specific gingipain B (RgpB), which lacks haemagglutinin/adhesin (HA) region of RgpA had no effect on osimertinib response. We also found that gingipains-mediated osimertinib resistance is independent of enzymatic activity. We detected intra-tumoral P. gingivalis in tumor tissue of 9 (30%) patients by using FISH probes specific to P. gingivalis. Conclusions: Our studies demonstrate that P. gingivalis can induce osimertinib resistance in EGFR-mutant lung cancer cells through secreted gingipains that activate the IGF1R pathway through an enzymatic- independent mechanism. P. gingivalis is frequently detected in human lung cancer tissues , suggesting that therapies targeting P. gingivalis may improve EGFR-TKI response in certain patients with EGFR-mutant lung cancer.

Acetate reprogrammes tumour metabolism and promotes PD-L1 expression and immune evasion by upregulating c-Myc.

Acetate, a precursor of acetyl-CoA, is instrumental in energy production, lipid synthesis and protein acetylation. However, whether acetate reprogrammes tumour metabolism and plays a role in tumour immune evasion remains unclear. Here, we show that acetate is the most abundant short-chain fatty acid in human non-small cell lung cancer tissues, with increased tumour-enriched acetate uptake. Acetate-derived acetyl-CoA induces c-Myc acetylation, which is mediated by the moonlighting function of the metabolic enzyme dihydrolipoamide S-acetyltransferase. Acetylated c-Myc increases its stability and subsequent transcription of the genes encoding programmed death-ligand 1, glycolytic enzymes, monocarboxylate transporter 1 and cell cycle accelerators. Dietary acetate supplementation promotes tumour growth and inhibits CD8+ T cell infiltration, whereas disruption of acetate uptake inhibits immune evasion, which increases the efficacy of anti-PD-1-based therapy. These findings highlight a critical role of acetate promoting tumour growth beyond its metabolic role as a carbon source by reprogramming tumour metabolism and immune evasion, and underscore the potential of controlling acetate metabolism to curb tumour growth and improve the response to immune checkpoint blockade therapy.

Abstract LB347: PLA2G2D/PGD2-PTGDR signaling deficiency restricts the melanoma progression and lung metastasis

Abstract Normal 0 false false false EN-US KO X-NONE/* Style Definitions */table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-font-kerning:1.0pt; mso-ligatures:standardcontextual;} Melanoma is a serious form of skin cancer with an approximate incidence more than 160,000 cases and 41,000 melanoma-associated death per annual globally. Malignant Melanoma is more dangerous because of its rapid spread and metastasis if it were not treated at an early stage. The high mortality of malignant melanoma incites an urgent need to refine the current treatments. Phospholipase A2 group IID (PLA2G2D) is an inducible enzyme expressed in myeloid cells, positively associated with immune infiltration through producing anti-inflammatory prostaglandin D2 (PGD2). The aging-related increase of PLA2G2D and PTGDR, a PGD2 receptor, makes them attractive targets for treating aged-related diseases, such as chronic inflammation and cancer diseases. In this study, by using Pla2g2d-/- and Ptgdr-/- mice, we found that PLA2G2D or PTGDR deficiency effectively restricted the in-situ growth and lung metastasis of subcutaneously implanted melanoma B16F10. These benefits are associated with the early enrichment of γδ-T cells in the tumor mass. The activation and accumulation of γδ-T cells can be induced in B16F10-bearing WT mice by the adoptive transfer of Pla2g2d-/- or Ptgdr-/- DCs. Pro-inflammatory characteristics of Pla2g2d-/- or Ptgdr-/- DCs, especially the production of IL-1b, contribute to γδ-T cells recruitment. These tumor-restraining effects were further confirmed in DC-specific PTGDR deficient (zDCcrePtgdrfloxp ) mice. Taken together with the prominent expression of PLA2G2D and PTGDR in human lung cancer tissue, this study demonstrates the potential of PLA2G2D-PGD2/PTGDR signaling-targeted therapy in treating melanoma and lung metastasis. Citation Format: Hima Dhakal, Jian Zheng. PLA2G2D/PGD2-PTGDR signaling deficiency restricts the melanoma progression and lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB347.

Tumor Exosomal ENPP1 Hydrolyzes cGAMP to Inhibit cGAS-STING Signaling.

To evade immune surveillance, tumor cells express ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) on the surface of their membrane, which degrades extracellular cyclic GMP-AMP (cGAMP), thereby inhibiting the cyclic GMP-AMP synthase (cGAS) stimulator of interferon gene (STING) DNA-sensing pathway. To fully understand this tumor stealth mechanism, it is essential to determine whether other forms of ENPP1 with hydrolytic cGAMP activity also are present in the tumor microenvironment to regulate this innate immune pathway. Herein, it is reported that various tumor-derived exosomes carry ENPP1, and can hydrolyze synthetic 2'3'-cGAMP and endogenous 2'3'-cGAMP produced by cells to inhibit cGAS-STING pathway in immune cells. Moreover, tumor exosomal ENPP1 also can hydrolyze 2'3'-cGAMP bound to LL-37 (an effective transporter of 2'3'-cGAMP) to inhibit STING signaling. Furthermore, high expression of ENPP1 in exosomes is observed isolated from human breast and lung cancer tissue, and tumor exosomal ENPP1 inhibited the immune infiltration of CD8+ T cells and CD4+ T cells. The results elucidate the essential function of tumor exosomal ENPP1 in the cGAS-STING pathway, furthering understanding of the crosstalk between the tumor cells and immune system.

CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis in lung cancer cells via the PERK-ATF4 and IRE1α-JNK pathways.

Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomic analysis revealed that CYB5R3 overexpression increased the production of nicotinamide adenine dinucleotide (NAD+) and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress signaling is induced via activation of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent intrinsic cell death. Our findings highlight the importance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.

Simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine at specific genomic loci by engineered deaminase-assisted sequencing.

Cytosine modifications, particularly 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), play crucial roles in numerous biological processes. Current analytical methods are often constrained to the separate detection of either 5mC or 5hmC, or the combination of both modifications. The ability to simultaneously detect C, 5mC, and 5hmC at the same genomic locations with precise stoichiometry is highly desirable. Herein, we introduce a method termed engineered deaminase-assisted sequencing (EDA-seq) for the simultaneous quantification of C, 5mC, and 5hmC at the same genomic sites. EDA-seq utilizes a specially engineered protein, derived from human APOBEC3A (A3A), known as eA3A-M5. eA3A-M5 exhibits distinct deamination capabilities for C, 5mC, and 5hmC. In EDA-seq, C undergoes complete deamination and is sequenced as T. 5mC is partially deaminated resulting in a mixed readout of T and C, and 5hmC remains undeaminated and is read as C. Consequently, the proportion of T readouts (P T) reflects the collective occurrences of C and 5mC, regulated by the deamination rate of 5mC (R 5mC). By determining R 5mC and P T values, we can deduce the precise levels of C, 5mC, and 5hmC at particular genomic locations. We successfully used EDA-seq to simultaneously measure C, 5mC, and 5hmC at specific loci within human lung cancer tissue and their normal counterpart. The results from EDA-seq demonstrated a strong concordance with those obtained from the combined application of BS-seq and ACE-seq methods. EDA-seq eliminates the need for bisulfite treatment, DNA oxidation or glycosylation and uniquely enables simultaneous quantification of C, 5mC and 5hmC at the same genomic locations.

Single-cell spatial metabolomics with cell-type specific protein profiling for tissue systems biology

Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet’s ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology.

Epigenetic downregulation of O6-methylguanine-DNA methyltransferase contributes to chronic hexavalent chromium exposure-caused genotoxic effect and cell transformation

Hexavalent chromium [Cr(VI)] is a common environmental pollutant and chronic exposure to Cr(VI) causes lung cancer and other types of cancer in humans, although the mechanism of Cr(VI) carcinogenesis remains elusive. Cr(VI) has been considered as a genotoxic carcinogen, but accumulating evidence indicates that Cr(VI) also causes various epigenetic toxic effects that play important roles in Cr(VI) carcinogenesis. However, it is not clear how Cr(VI)-caused epigenetic dysregulations contributes to Cr(VI) carcinogenesis. This study investigates whether Cr(VI) epigenetic toxic effect has an impact on its genotoxic effect. It was found that chronic low dose of Cr(VI) exposure time-dependently down-regulates the expression of a critical DNA damage repair protein O6-methylguanine-DNA methyltransferase (MGMT), leading to the increases of the levels of the highly mutagenic and carcinogenic DNA lesion O6-methylguanine (O6-MeG) in human bronchial epithelial BEAS-2B cells. Moreover, the levels of MGMT and O6-MeG in chronic Cr(VI) exposure-caused human lung cancer tissues are also significantly lower and higher than that in the adjacent normal lung tissues, respectively. It was further determined that chronic low dose of Cr(VI) exposure-transformed BEAS-2B cells display impaired DNA damage repair capacity and a high sensitivity to the toxicity of the alkylating chemotherapeutic drug Temozolomide. In contrast, stably overexpressing MGMT in parental BEAS-2B cells reverses chronic low dose of Cr(VI) exposure-caused DNA damage repair deficiency and significantly reduces cell transformation by Cr(VI). Further mechanistical studies revealed that chronic low dose of Cr(VI) exposure down-regulates MGMT expression through epigenetic mechanisms by increasing DNA methylation and histone H3 repressive modifications. Taken together, these findings suggest that epigenetic down-regulation of a crucial DNA damage repair protein MGMT contributes significantly to the genotoxic effect and cell transformation caused by chronic low dose of Cr(VI) exposure.

Nitrogen isotopic composition as a gauge of tumor cell anabolism-to-catabolism ratio.

Studies have suggested that cancerous tissue has a lower 15N/14N ratio than benign tissue. However, human data have been inconclusive, possibly due to constraints on experimental design. Here, we used high-sensitivity nitrogen isotope methods to assess the 15N/14N ratio of human breast, lung, and kidney cancer tissue at unprecedented spatial resolution. In lung, breast, and urothelial carcinoma, 15N/14N was negatively correlated with tumor cell density. The magnitude of 15N depletion for a given tumor cell density was consistent across different types of lung cancer, ductal in situ and invasive breast carcinoma, and urothelial carcinoma, suggesting similar elevations inthe anabolism-to-catabolism ratio. However, tumor 15N depletion was higher in a more aggressive metaplastic breast carcinoma. These findings may indicate the ability of certain cancers to more effectively channel N towards growth. Our results support 15N/14N analysis as a potential tool for screening biopsies and assessing N metabolism in tumor cells.

Factor inhibiting HIF (FIH) promotes lung cancer progression.

Factor inhibiting HIF (FIH) is an asparagine hydroxylase that acts on hypoxia inducible factors (HIFs) to control cellular adaptation to hypoxia. FIH is expressed in several tumor types, but its impact in tumor progression remains largely unexplored. We observed that FIH was expressed on human lung cancer tissue. Deletion of FIH in mouse and human lung cancer cells resulted in an increased glycolytic metabolism, consistent with increased HIF activity. FIH-deficient lung cancer cells exhibited decreased proliferation. Analysis of RNA-seq data confirmed changes in the cell cycle and survival, and revealed molecular pathways that were dysregulated in the absence of FIH including the upregulation of angiomotin (Amot), a key component of the Hippo tumor suppressor pathway. We show that FIH-deficient tumors were characterized by higher immune infiltration of NK and T cells compared to FIH competent tumor cells. In vivo studies demonstrated that FIH deletion resulted in reduced tumor growth and metastatic capacity. Moreover, high FIH expression correlated with poor overall survival in non-small cell lung cancer (NSCLC). Together, our data unravel FIH as a therapeutic target for the treatment of lung cancer.

ADPGK‐AS1 long noncoding RNA switches macrophage metabolic and phenotypic state to promote lung cancer growth

Long noncoding RNAs (lncRNAs) influence the transcription of gene networks in many cell types, but their role in tumor‐associated macrophages (TAMs) is still largely unknown. We found that the lncRNA ADPGK‐AS1 was substantially upregulated in artificially induced M2‐like human macrophages, macrophages exposed to lung cancer cells in vitro, and TAMs from human lung cancer tissue. ADPGK‐AS1 is partly located within mitochondria and binds to the mitochondrial ribosomal protein MRPL35. Overexpression of ADPGK‐AS1 in macrophages upregulates the tricarboxylic acid cycle and promotes mitochondrial fission, suggesting a phenotypic switch toward an M2‐like, tumor‐promoting cytokine release profile. Macrophage‐specific knockdown of ADPGK‐AS1 induces a metabolic and phenotypic switch (as judged by cytokine profile and production of reactive oxygen species) to a pro‐inflammatory tumor‐suppressive M1‐like state, inhibiting lung tumor growth in vitro in tumor cell‐macrophage cocultures, ex vivo in human tumor precision‐cut lung slices, and in vivo in mice. Silencing ADPGK‐AS1 in TAMs may thus offer a novel therapeutic strategy for lung cancer.

PKC-ζ mediated reduction of the extracellular vesicles-associated TGF-β1 overcomes radiotherapy resistance in breast cancer

BackgroundRadiotherapy is widely applied in breast cancer treatment, while radiotherapy resistance is inevitable. TGF-β1 has been considered to be an endogenous factor for the development of radiotherapy resistance. As a large portion of TGF-β1 is secreted in an extracellular vesicles-associated form (TGF-β1EV), particularly in radiated tumors. Thus, the understanding of the regulation mechanisms and the immunosuppressive functions of TGF-β1EV will pave a way for overcoming the radiotherapy resistance in cancer treatment.MethodsThe superoxide-Zinc-PKC-ζ-TGF-β1EV pathway in breast cancer cells was identified through sequence alignments of different PKC isoforms, speculation and experimental confirmation. A series of functional and molecular studies were performed by quantitative real-time PCR, western blot and flow cytometry analysis. Mice survival and tumor growth were recorded. Student’s t test or two-way ANOVA with correction was used for comparisons of groups.ResultsThe radiotherapy resulted in an increased expression of the intratumoral TGF-β1 and an enhanced infiltration of the Tregs in the breast cancer tissues. The intratumoral TGF-β1 was found mainly in the extracellular vesicles associated form both in the murine breast cancer model and in the human lung cancer tissues. Furthermore, radiation induced more TGF-β1EV secretion and higher percentage of Tregs by promoting the expression and phosphorylation of protein kinase C zeta (PKC-ζ). Importantly, we found that naringenin rather than 1D11 significantly improved radiotherapy efficacy with less side effects. Distinct from TGF-β1 neutralizing antibody 1D11, the mechanism of naringenin was to downregulate the radiation-activated superoxide-Zinc-PKC-ζ-TGF-β1EV pathway.ConclusionsThe superoxide-zinc-PKC-ζ-TGF-β1EV release pathway was elucidated to induce the accumulation of Tregs, resulting in radiotherapy resistance in the TME. Therefore, targeting PKC-ζ to counteract TGF-β1EV function could represent a novel strategy to overcome radiotherapy resistance in the treatment of breast cancer or other cancers.Trial registration: The using of patient tissues with malignant Non-Small Cell Lung Cancer (NSCLC) was approved by the ethics committees at Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (NCC2022C-702, from June 8th, 2022).

Abstract 594: Effects of interstitial pressure on lung cancer cells

Abstract Chronic inflammation is a risk factor for various types of cancer. The pressure in the interstitial tissues has been reported to be elevated in chronic inflammation and cancer. However, little is known about the biological effect of pressure from the basal side on epithelia, and a significance of the elevated interstitial fluid pressure in cancer biology is still poorly understood. In this study, we investigated the effect of hydrostatic pressure on cultured lung cancer cells grown on Transwell permeable filters by changing the height of the surfaces of culture medium in the apical and basal sides. A gradient of the hydrostatic pressure from the basal to the apical side induced stratification of epithelia. The elimination of the hydrostatic pressure gradient restored the stratification of epithelia. Interestingly, cavities were found within the stratified epithelia, and the characteristics of apical cell polarity including microvilli and tight junctions were formed in the surfaces the cavities. The cavities with similar aberrant cell polarity were also observed in human lung cancer tissues. The hydrostatic pressure gradient from the basal side also promoted cell proliferation and suppressed apoptosis. On the other hand, the expression levels of phosphorylated ERK and AKT were not significantly affected by the hydrostatic pressure. These results indicate that the pressure from the basal side to the epithelia promotes cell proliferation, suppresses apoptosis and induces aberrant cell polarity, which results in epithelial stratification. The findings in this study suggest that the elevated interstitial fluid pressure in chronic inflammation and cancer tissues facilitates cancer growth and plays a significant role in cancer biology. Citation Format: Shinsaku Tokuda, Keisuke Onoi, Tadaaki Yamada, Koichi Takayama. Effects of interstitial pressure on lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 594.