Calu-6 Cells Research Articles

Beyond its preferential niche: Brucella abortus RNA down-modulates the IFN-γ-induced MHC-I expression in epithelial and endothelial cells.

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance.

Immunomodulatory potential of sulfated xylated rhamnoglycan from an edible green seaweed Ulva lactuca: Regulation of cytokine expression in CALU-1 cells and prospects for therapeutic applications

Green seaweeds, particularly Ulva lactuca, commonly known as sea-lettuce, from the Ulvaceae class, constitute a significant proportion of the global edible seaweed population and serve as a rich storehouse of pharmacologically active compounds, including polysaccharides. In this study, a sulfated (1 → 4) linked xylated rhamnoglycan, (ULP-2), was extracted from Ulva lactuca. ULP-2 displayed significant immunomodulatory properties by modulating the expression of inflammatory cytokines in lipopolysaccharide (LPS)-induced CALU-1 non-small-cell lung cancer (NSCLC) cells. Treatment with ULP-2 at a concentration of 125 μg/mL resulted in a notable downregulation (63%, p < 0.05) of interferon (IFN)-γ gene expression compared to LPS-treated cells. Furthermore, the overexpression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β was significantly attenuated (p < 0.05) with ULP-2 treatment (125 μg/mL), showing substantial downregulation to 0.82% and 1.81%, respectively, compared to LPS-induced cells (29.28% and 94.89%, respectively). Additionally, a noteworthy 1.3-fold reduction in IL-33 expression was observed following the administration of ULP-2 at 125 μg/mL. An upregulation of IFN-α expression, was evident in LPS-induced CALU-1 cells following treatment with ULP-2 within the concentration range of 31.25–125 μg/mL. The structure-activity study reinforced the significance of the presence of polyanionic sulfate and the (1 → 4) linkage in ULP-2 for binding to their respective receptors, influencing the orientation and spatial arrangement of the molecule, and subsequently triggering intracellular signaling pathways linked to the activation of innate immunity. Consequently, this study highlights ULP-2 as a promising natural compound derived from edible seaweed with potential applications in the treatment of inflammatory disorders and immune enhancement.

S100A6 could not promote the differentiation of Calu-6 lung cancer cell line.

Our previous study demonstrated that S100 calcium binding protein A6 (S100A6) impairs tumorigenesis by Calu-6 lung cancer cells, as well as inhibit their growth. However, the role that S100A6 plays in tumor cell differentiation has not been previously explored. This study aimed to confirm the effect of S100A6 on the direction of differentiation in the human lung cancer cell linem Calu-6m based on our previous published research. A S100A6-overexpressing lentiviral vector was successfully constructed in our previous study. Nude mouse tumorigenicity was then applied successfully, and 15 mice were divided into three groups (Calu-6, Calu-6/neo, Calu-6/S100A6). After 5 weeks, we detected lung cancer markers with immunohistochemistry in mice tumor tissues, including the adenocarcinoma markers, TTF-1 and NapsinA, the squamous cell carcinoma markers, P40, CK5/6 and P63, and the small cell lung cancer markers CD56, Syn, CgA, TTF-1, CK, and Ki-67. Differences among the three groups were statistically compared. All the above-mentioned markers were positive in the tumor tissues of all three groups, and there were no significant differences. S100A6 cannot promote differentiation of the undifferentiated human lung cancer cell line, Calu-6, into adenocarcinoma, squamous, or small cell carcinoma cell lines.

Formyl-Peptide Receptor 2 Signaling Modulates SLC7A11/xCT Expression and Activity in Tumor Cells.

Cancer cells exhibit high levels of oxidative stress and consequently require a high amount of cysteine for glutathione synthesis. Solute Carrier Family 7 Member 11 (SLC7A11), or xCT, mediates the cellular uptake of cystine in exchange for intracellular glutamate; imported extracellular cystine is reduced to cysteine in the cytosol through a NADPH-consuming reduction reaction. SLC7A11/xCT expression is under the control of stress-inducing conditions and of several transcription factors, such as NRF2 and ATF4. Formyl-peptide receptor 2 (FPR2) belongs to the FPR family, which transduces chemotactic signals mediating either inflammatory or anti-inflammatory responses according to the nature of its ligands and/or FPR2 binding with other FPR isoforms. The repertoire of FPR2 agonists with anti-inflammatory activities comprises WKYMVm peptide and Annexin A1 (ANXA1), and the downstream effects of the intracellular signaling cascades triggered by FPR2 include NADPH oxidase (NOX)-dependent generation of reactive oxygen species. Herein, we demonstrate that stimulation of CaLu-6 cells with either WKYMVm or ANXA1: (i) induces the redox-regulated activation of SLC7A11/xCT; (ii) promotes the synthesis of glutathione; (iii) prevents lipid peroxidation; and (iv) favors NRF2 nuclear translocation and activation. In conclusion, our overall results demonstrate that FPR2 agonists and NOX modulate SLC7A11/xCT expression and activity, thereby identifying a novel regulative pathway of the cystine/glutamate antiport that represents a new potential therapeutical target for the treatment of human cancers.

Apelin-13: a novel approach to suppressing renin production in RVHT.

Renovascular hypertension (RVHT) is characterized by renal artery stenosis and overactivated renin-angiotensin system (RAS). Apelin, known for its negative modulation of RAS, has protective effects against cardiovascular diseases. The role and mechanisms of the primary active form of apelin, apelin-13, in RVHT are unclear. In this study, male Sprague-Dawley rats were divided into control, two-kidney one-clip (2K1C) model, and 2K1C with apelin-13 treatment groups. Renin expression was analyzed using immunohistochemistry and molecular techniques. Full-length (pro)renin receptor (fPRR) and soluble PRR (sPRR) levels were assessed via Western blotting, and cAMP levels were measured using ELISA. Plasma renin content, plasma renin activity (PRA), angiotensin II (ANG II), and sPRR levels were determined by ELISA. Human Calu-6 and mouse As4.1 cells were used to investigate renin production mechanisms. The 2K1C model exhibited increased systolic blood pressure, plasma renin content, PRA, sPRR, and ANG II levels, while apelin-13 treatment reduced these elevations. Apelin-13 inhibited cAMP production, renin mRNA expression, protein synthesis, and PRR/sPRR protein expression in renal tissue. In Calu-6 cells, cAMP-induced fPRR and site-1 protease (S1P)-derived sPRR expression, which was blocked by cAMP-responsive element-binding protein (CREB) inhibition. Apelin-13 suppressed cAMP elevation, CREB phosphorylation, fPRR/sPRR protein expression, and renin production. Recombinant sPRR (sPRR-His) stimulated renin production, which was inhibited by the PRR decoy peptide PRO20 and S1P inhibitor PF429242. These findings suggest that apelin-13 inhibits plasma renin expression through the cAMP/PKA/sPRR pathway, providing a potential therapeutic approach for RVHT. Understanding the regulation of renin production is crucial for developing effective treatments.NEW & NOTEWORTHY Our research elucidated that apelin-13 inhibits renin production through the cAMP/PKA/soluble (pro)renin receptor pathway, presenting a promising therapeutic approach for renovascular hypertension (RVHT) by targeting renin expression mechanisms. These findings underscore the potential of apelin-13 as a novel strategy to address RVHT.

Abstract 6491: Epigenetic-based combinatorial therapy is synergistic in KRAS/LKB1 mutant non-small cell lung cancers

Abstract KRAS and LKB1 mutations (KL) in non-small cell lung cancer (NSCLC) patients often results in poor prognosis, a high tumor mutational burden, and resistance to conventional therapy. The metabolic changes with these mutations can create unique vulnerabilities to be targeted therapeutically. HDAC inhibition has been reported as a promising approach to overcome MEK inhibitor resistance in animal models of melanoma, pancreatic cancer, and KL mutant NSCLC. Inhibition of HDAC3 and HDAC6 have independently been shown to have therapeutic activity in KL NSCLC. We now report on the activity of two novel HDAC3/6 inhibitors in cell culture and mouse models of human KL (A549) compared to KRAS/TP53-mutant NSCLC (KP) (CALU-6) alone and individually combined with the MEK inhibitor trametinib. GB-1101 inhibits HDAC3 and HDAC6 and GB-3103 inhibits HDAC1,3,6,10. SynergyFinder multi-dose combination data analytics showed synergistic interactions for both GB-1101 or GB-3103 when combined with trametinib in KL mutant A549 cells with maximum synergy scores of δ = 9.24 and 4.21, using zero interaction potency (ZIP) for synergy scoring. GB-1101 or GB-3103 when combined with trametinib in KP mutant CALU-6 cells did not demonstrate synergistic interaction with synergy scores of δ = -1.58 and -0.53, respectively. Mice were inoculated subcutaneously with 5.0x106 A549 or CALU-6 cells and treatment with GB-1101 (75 mg/kg qd, oral), GB-3103 (5.0 mg/kg qd, subcutaneous), trametinib (1.0 mg/kg qd, oral) and each drug in combination with trametinib was initiated when tumors reached an average of 120mm3. Single agent and combination treatments were well tolerated. GB-1101 exhibited tumor growth inhibition (TGI) of 33.0% in the A549 (KL) compared to 8.5% in CALU-6 (KP). In combination with trametinib, GB-1101 showed increased activity (TGI=92.7%) compared to trametinib alone (TGI=69.5%) in KL mutant A549. In KP mutant CALU-6 tumors, combination benefit was not observed; combination of GB-1101 and trametinib yielded a TGI=93% equivalent to trametinib alone (TGI=91%). GB-3103 resulted in a TGI of 66.7% in the KL mutant A549 and TGI= 55.9% in KP mutant CALU-6. In KL mutant A549, GB-3103 in combination with trametinib, showed increased inhibition (TGI=94.3%) compared to trametinib alone (TGI=69.5%). No combination benefit was seen for GB-3103 in KP mutant CALU-6 with the combination treatment TGI of 93.1% compared to trametinib alone TGI=90.7%. These results confirm and extend the finding that for KL mutant NSCLC, HDAC3/6 inhibitors show therapeutic benefit in combination with trametinib that is not observed in KP tumors. These data reveal a promising epigenetic-based combinatorial strategy for treating lung cancers harboring concurrent KRAS/LKB1 mutations, a path for clinical translation, and the potential to improve treatments for KRAS/LKB1 mutant NSCLC patients where there are currently no robust therapeutic strategies. Citation Format: Matthew Zucker, Kendra Devore, Logan Chesney, Gregg Hirschfeld, Annissa Reyna, Betsy Linnane, Joseph Mascarenhas, Maggie Murray, Alan Miller, Paul Gonzales, Stephen Gately. Epigenetic-based combinatorial therapy is synergistic in KRAS/LKB1 mutant non-small cell lung cancers [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 6491.

MiR-320a upregulation contributes to the effectiveness of pemetrexed by inhibiting the growth and invasion of human lung cancer cell line (Calu-6).

Lung cancer is a common and deadly disease. Chemotherapy is the most common treatment, which inhibits cancer cell growth. Pemetrexed (PMX) is often used with other drugs. Environmental stress can affect regulatory non-coding RNAs such as MicroRNAs that modify gene expression. This study investigates the effect of PMX on the hsa-miR-320a-3p expression in the Calu-6 lung cancer cell line. Calu-6 cells were cultured in an incubator with 37°C, 5% CO2, and 98% humidity. The MTT test was performed to determine the concentration of PMX required to inhibit 50% of cell growth. To examine growth inhibition and apoptosis, release of lactate dehydrogenase (LDH), cell assays and caspase 3 and 7 enzyme activity were used. Finally, molecular studies were conducted to compare the expression of hsa-miR-320a-3p and genes including VDAC1, DHFR, STAT3, BAX and BCL2 before and after therapy. According to a study, it has been observed that PMX therapy significantly increases LDH release after 24h. The study found that PMX's IC50 on Calu-6 is 8.870µM. In addition, the treated sample showed higher expression of hsa-miR-320a-3p and BAX, while the expression of VDAC1, STAT3, DHFR and BCL2 decreased compared to the control sample. According to the findings of the current research, hsa-miR-320a-3p seems to have the potential to play an important role in the development of novel approaches to the treatment of lung cancer.

VEGFR affects miR-3200-3p-mediated regulatory T cell senescence in tumour-derived exosomes in non-small cell lung cancer.

Numerous studies have demonstrated that regulatory T (Treg) cells play an important role in the tumour microenvironment (TME). The aim of this study was to investigate whether VEGFR2 affects the expression of miR-3200-3p in exosomes secreted by tumour cells, thereby influencing Treg senescence in the TME. The results showed that VEGFR2 expression level was the highest in Calu-1 cells, and after transfection with si-VEGFR2, the exosomes secreted from Calu-1 cells were extracted and characterised with no significant difference from the exosomes of the untransfected group, but the expression of miR-3200-3p in the exosomes of the transfected si-VEGFR2 group was elevated. The Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM) results suggested that exosomes highly expressing miR-3200-3p could inhibit Treg cell viability and promote apoptosis levels when treated with Treg cells. Detection of the senescence-associated proteins p16 INK4A and MMP3 by western blot (WB) revealed that exosomes highly expressing miR-3200-3p were able to elevate their protein expression levels. Tumour xenograft experiments demonstrated that exosomes with high miR-3200-3p expression promoted Treg cell senescence and inhibited subcutaneous tumour growth in nude mice. Dual-luciferase reporter assays and RNA pull-down assays showed that miR-3200-3p could be linked with DDB1. Overexpression of DDB1 reverses changes in DCAF1/GSTP1/ROS protein expression caused by exosomes with high miR-3200-3p expression. In conclusion, inhibition of VEGFR2 expression in tumour cells promotes the expression of miR-3200-3p in exosomes secreted by tumour cells. miR-3200-3p enters the TME through exosomes and acts on DDB1 in Treg cells to promote senescence of Treg cells to inhibit tumour progression.

Proline Dehydrogenase (PRODH) Is Expressed in Lung Adenocarcinoma and Modulates Cell Survival and 3D Growth by Inducing Cellular Senescence.

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence.

In vitro assessments of nanoplexes of polyethylenimine-coated graphene oxide-plasmid through various cancer cell lines and primary mesenchymal stem cells

Efficient gene therapy relies on an efficient gene delivery system. Viral gene delivery approaches excel in transferring and expressing external genes; however, their immunogenicity and difficulty in large-scale production limit their clinical applications. In contrast, nanoparticle-based gene delivery systems have gained increasing attention due to less immunogenicity and more convenience for large-scale production. Nevertheless, their poor transfection efficiency compared to viral systems remains a significant obstacle. In the present study, we investigated the transfection efficiency of our PEI-coated graphene oxides in HEK293T, Calu-3, Calu-6 cell lines, and primary human bone marrow mesenchymal stem cell (MSC). The high surface ratio and good biocompatibility of graphene oxide make it an appealing tool for gene delivery systems. However, the low dispersity of graphene oxide in aqueous environments is the first barrier that needs to be conquered. For this, we enhanced the dispersity and stability of graphene oxide in water by sonicating it for at least 5 hours at a pH of 7. Then, graphene oxide was conjugated with branched PEI (25 kDa) to have a positive charge, enabling it to condense nucleic acids with a naturally negative potential. The physio-chemical characteristics of our synthesized nano-carriers (GO-PEI) were determined by DLS, FT-IR, and AFM. The utilized plasmid in polyplexes contained a GFP gene, allowing us to verify transfection efficiency through fluorescent microscopy and flow cytometry. While GO-PEI carriers were highly efficient in transfecting HEK293T cells, the transfection efficiency in MSCs and Calu-3 cells was notably low. We suppose that the main reason for the low transfection efficiency of GO-PEI in these cells is due to its higher toxicity. Despite this, considering the various advantages of graphene oxide in drug delivery as well as its optical and electrical applications in biomedicine, we propose to functionalize graphene oxide with more biocompatible materials to enhance its potential as a successful gene carrier in these cell types.

Formyl-peptide receptor 2 signalling triggers aerobic metabolism of glucose through Nox2-dependent modulation of pyruvate dehydrogenase activity.

The human formyl-peptide receptor 2 (FPR2) is activated by an array of ligands. By phospho-proteomic analysis we proved that FPR2 stimulation induces redox-regulated phosphorylation of many proteins involved in cellular metabolic processes. In this study, we investigated metabolic pathways activated in FPR2-stimulated CaLu-6 cells. The results showed an increased concentration of metabolites involved in glucose metabolism, and an enhanced uptake of glucose mediated by GLUT4, the insulin-regulated member of GLUT family. Accordingly, we observed that FPR2 transactivated IGF-IRβ/IRβ through a molecular mechanism that requires Nox2 activity. Since cancer cells support their metabolism via glycolysis, we analysed glucose oxidation and proved that FPR2 signalling promoted kinase activity of the bifunctional enzyme PFKFB2 through FGFR1/FRS2- and Akt-dependent phosphorylation. Furthermore, FPR2 stimulation induced IGF-IRβ/IRβ-, PI3K/Akt- and Nox-dependent inhibition of pyruvate dehydrogenase activity, thus preventing the entry of pyruvate in the tricarboxylic acid cycle. Consequently, we observed an enhanced FGFR-dependent lactate dehydrogenase (LDH) activity and lactate production in FPR2-stimulated cells. As LDH expression is transcriptionally regulated by c-Myc and HIF-1, we demonstrated that FPR2 signalling promoted c-Myc phosphorylation and Nox-dependent HIF-1α stabilization. These results strongly indicate that FPR2-dependent signalling can be explored as a new therapeutic target in treatment of human cancers.

Ebselen Inhibits the Growth of Lung Cancer Cells via Cell Cycle Arrest and Cell Death Accompanied by Glutathione Depletion.

Ebselen is a glutathione (GSH) peroxidase (GPx) mimic originally developed to reduce reactive oxygen species (ROS). However, little is known about its cytotoxicological effects on lung cells. Therefore, this study aimed to investigate the effects of Ebselen on the cell growth and cell death of A549 lung cancer cells, Calu-6 lung cancer cells, and primary normal human pulmonary fibroblast (HPF) cells in relation to redox status. The results showed that Ebselen inhibited the growth of A549, Calu-6, and HPF cells with IC50 values of approximately 12.5 μM, 10 μM, and 20 μM, respectively, at 24 h. After exposure to 15 μM Ebselen, the proportions of annexin V-positive cells were approximately 25%, 65%, and 10% in A549, Calu-6, and HPF cells, respectively. In addition, Ebselen induced arrest at the S phase of the cell cycle in A549 cells and induced G2/M phase arrest in Calu-6 cells. Treatment with Ebselen induced mitochondrial membrane potential (MMP; ΔΨm) loss in A549 and Calu-6 cells. Z-VAD, a pan-caspase inhibitor, did not decrease the number of annexin V-positive cells in Ebselen-treated A549 and Calu-6 cells. Intracellular ROS levels were not significantly changed in the Ebselen-treated cancer cells at 24 h, but GSH depletion was efficiently induced in these cells. Z-VAD did not affect ROS levels or GSH depletion in Ebselen-treated A549 or Ebselen-treated Calu-6 cells. In conclusion, Ebselen inhibited the growth of lung cancer and normal fibroblast cells and induced cell cycle arrest and cell death in lung cancer cells with GSH depletion.

Dynamics of TERT regulation via alternative splicing in stem cells and cancer cells.

Part of the regulation of telomerase activity includes the alternative splicing (AS) of the catalytic subunit telomerase reverse transcriptase (TERT). Although a therapeutic window for telomerase/TERT inhibition exists between cancer cells and somatic cells, stem cells express TERT and rely on telomerase activity for physiological replacement of cells. Therefore, identifying differences in TERT regulation between stem cells and cancer cells is essential for developing telomerase inhibition-based cancer therapies that reduce damage to stem cells. In this study, we measured TERT splice variant expression and telomerase activity in induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs), and non-small cell lung cancer cells (NSCLC, Calu-6 cells). We observed that a NOVA1-PTBP1-PTBP2 axis regulates TERT alternative splicing (AS) in iPSCs and their differentiation into NPCs. We also found that splice-switching of TERT, which regulates telomerase activity, is induced by different cell densities in stem cells but not cancer cells. Lastly, we identified cell type-specific splicing factors that regulate TERT AS. Overall, our findings represent an important step forward in understanding the regulation of TERT AS in stem cells and cancer cells.

The effects of cetuximab and cisplatin anti-cancer drugs on the mechanical properties of the lung cancerous cells using atomic force microscope.

Each anti-cancer drug has special effects on the target cells. One of the most important reasons to recommend an anti-cancer drug is related to the influences of it on the mechanical properties of the target cells. In this study, the effects of cetuximab and cisplatin anti-cancer drugs on the mechanical properties of A-549 and Calu-6 cells as the cancerous lung cells have been investigated. For both of the cells and anti-cancer drugs, MTT assessment has been used to define the convenient dosages for 24 and 48 h incubations based on IC50 concentration for the cell line viability. The mechanical specifications of the cells before and after treatment were obtained using nanoindentation by the JPK Instruments' NanoWizard3 atomic force microscope. The results show that cetuximab increases the stiffness of A-549 cell from 1225 to 3403 and 12 690 Pa for 24 and 48 h incubations. The influence of cetuximab on the Calu-6 shows that the elastic modulus after 24 and 48 h culture times increases about cisplatin anti-cancer drug, for A-549 cell indicates that the elastic modulus rises from 1225 to 1506 and 2375 Pa for 24 and 48 h, respectively. For Calu-6 cell, cisplatin has an important role to increase the stiffness of the cell. Applying cisplatin increases the elastic modulus from 33 to 682.8 Pa for 24 h and 1105 Pa after 48 h incubations.

GNAQ-Regulated ZO-1 and ZO-2 Act as Tumor Suppressors by Modulating EMT Potential and Tumor-Repressive Microenvironment in Lung Cancer.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in the development and progression of lung cancer by promoting its invasiveness and metastasis. Using integrative analyses of the public lung cancer database, we found that the expression levels of the tight junction proteins, zonula occluden (ZO)-1 and ZO-2, were lower in lung cancer tissues, including both lung adenocarcinoma and lung squamous cell carcinoma than in normal lung tissues analyzed using The Cancer Genome Atlas (TCGA). Although the ectopic expression or knockdown of ZO-1 and ZO-2 did not affect the growth of lung cancer cells, they significantly regulated cell migration and invasion. When M0 macrophages were co-cultured with ZO-1 or ZO-2 knockdown Calu-1 cells, M2-like polarization was efficiently induced. Conversely, co-culture of M0 THP-1 cells with A549 cells stably expressing ZO-1 or ZO-2 significantly reduced M2 differentiation. We also identified G protein subunit alpha q (GNAQ) as a potential ZO-1- and ZO-2-specific activator through analysis of correlated genes with the TCGA lung cancer database. Our results suggest that the GNAQ-ZO-1/2 axis may play a tumor-suppressive role in lung cancer development and progression and highlight ZO-1 and ZO-2 as key EMT- and tumor microenvironment-suppressive proteins. These findings provide new insights for the development of targeted therapies for lung cancer.

Abstract 4893: Evaluation of bystander antitumor effect of Dato-DXd in TROP2 (+) and TROP2 (-) lung cell lines admixed tumor xenograft mouse models

Abstract Background: Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I inhibitor payload (DXd) via a stable, tumor-selective, tetrapeptide-based cleavable linker (Nakada T. et al. Bioorg Med Chem Lett., 2016). Dato-DXd demonstrated encouraging efficacy in clinical studies in TROP2 expressing tumors, including NSCLC and TNBC (NCT03401385). Clinical efficacy of Dato-DXd in heterogeneous tumors is hypothesized to be derived not only via effective selective delivery of DXd to TROP2 (+) tumor cells but also DXd diffusion from TROP2 (+) tumor cells to nearby TROP2 (-) tumor cells due to high membrane permeability of the DXd payload, an effect called the bystander antitumor effect. In this preclinical study, the bystander antitumor effect was tested by establishing tumor xenograft mouse models containing admixed TROP2 (+) and TROP2 (-) lung cell lines. The antitumor activity of Dato-DXd was tested in these models to demonstrate the bystander antitumor effect of Dato-DXd. Materials and Methods: EBC-1 cells and NCI-H526 cells or Calu-6 cells were used as models of TROP2 (+) cells and TROP2 (-) cells, respectively. TROP2 (+) cells and TROP2 (-) cells were mixed in various ratios and inoculated subcutaneously in mice to establish heterogeneous xenograft tumor models with various levels of TROP2 expression. Tumor growth inhibition in each model was evaluated by tumor volume after Dato-DXd or control-ADC administration. Immunohistochemistry (IHC) of DXd and TROP2 were performed in Dato-DXd treated tumors to analyze distribution in TROP2 (+) and (-) tumor cells. Tumor cells that responded to Dato-DXd treatment were identified by IHC assay of γH2AX (an indicator of DNA damage). Results: TROP2 expression analysis showed that various TROP2 positivity tumor models were established by inoculating various ratios of admixed EBC-1 cells with NCI-H526 cells or Calu-6 cells. Dato-DXd did not inhibit growth of tumors consisting of only TROP2 (-) cells, as expected. In contrast, Dato-DXd showed strong tumor growth inhibition effect on all tumors in which TROP2 (+) cells were inoculated in various levels with TROP2 (-) cells. DXd-IHC analysis showed that Dato-DXd distributed to only TROP2 (+) tumor cells in admixed tumors, however, an increase of γH2AX was observed not only in TROP2 (+) tumor cells but also TROP2 (-) tumor cells, suggesting TROP2 (-) tumor cells responded to DXd generated and distributed from Dato-DXd bound to TROP2 (+) tumor cells. Conclusion: Dato-DXd is effective not only in TROP2 (+) tumor cells but also TROP2 (-) tumor cells adjacent to TROP2 (+) tumor cells, thereby demonstrating the bystander antitumor effect of Dato-DXd. This property suggests Dato-DXd should be active in the clinical setting even given potential heterogeneity of TROP2 expression. Citation Format: Satoru Yasuda, Takanori Maejima, Tadashi Toki, Tsuyoshi Karibe, Daisuke Okajima, Penny Phillips. Evaluation of bystander antitumor effect of Dato-DXd in TROP2 (+) and TROP2 (-) lung cell lines admixed tumor xenograft mouse models. [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 4893.

Identification and characterization of the CDK1-BMAL1-UHRF1 pathway driving tumor progression

The abnormal regulation of BMAL1 could lead to the occurrence and progression of various tumors. However, the mechanism of phosphorylation regulation of BMAL1 in tumorigenesis remains poorly understood. In this study, we report a previously unrecognized BMAL1 dephosphorylation pathway that promotes tumor progression. BMAL1 accelerates cell proliferation, migration, and invasion of HT1080 and Calu1 cells. CDK1 binds to BMAL1 through a conserved domain and regulates the dephosphorylation of BMAL1 on Ser42 residues, but not on Ser78 or Thr224, thereby enhancing the oncogenic activity of BMAL1. Dephosphorylation of BMAL1 Ser42 promotes tumor growth and metastasis in mouse subcutaneous transplantation tumor and lung metastatic tumor models. Moreover, UHRF1 is recognized as an important target gene of BMAL1 in cancer cells. Consequently, UHRF1 depletion mimics BMAL1 deficiency with respect to tumor suppression, whereas transfection-enforced re-expression of UHRF1 restores tumor growth in BMAL1-deficient cells. These findings suggest a link between the circadian clock regulator and cancer progression.

PD-L1/PD-1 blockage enhanced the cytotoxicity of natural killer cell on the non-small cell lung cancer (NSCLC) by granzyme B secretion

ObjectiveTo explore the role of PD-L1/PD-1 blockage in the cytotoxicity of natural killer cell in NSCLC.MethodsTwo NSCLC cell lines, Calu-1 and H460, were tested for susceptibility to the cytolytic activity of freshly isolated healthy donor NK cells by a non-radioactive cellular cytotoxicity assay kit. Western blot analysis, FACS, ELISA and antibody blockage experiments were conducted to determine the mechanisms. NK cells isolated from NSCLC patients were also collected for functional assays.ResultsCalu-1 and H460 cells were lysed by NK cells in a dose-dependent manner. H460 cells showed less susceptibility to NK cell-mediated lysis than Calu-1 cells at all ratios. The expression of PD-L1 on H460 cells was higher than that on Calu-1 cells, as determined by FACS and western blot analysis. The specific lysis of H460 cells by NK cells was enhanced when the PD-L1/PD-1 interaction was blocked by anti-PD-L1 antibody. This finding was also demonstrated in NK cells isolated from NSCLC patients.ConclusionsThe present study revealed that PD-L1/PD-1 blockage enhanced the cytotoxicity of natural killer cells in NSCLC via granzyme B secretion. This study will greatly facilitate the precise treatment of lung cancer through determination of PD-L1 expression in tumors.

ER ribosomal-binding protein 1 regulates blood pressure and potassium homeostasis by modulating intracellular renin trafficking

BackgroundGenome-wide association studies (GWASs) have linked RRBP1 (ribosomal-binding protein 1) genetic variants to atherosclerotic cardiovascular diseases and serum lipoprotein levels. However, how RRBP1 regulates blood pressure is unknown.MethodsTo identify genetic variants associated with blood pressure, we performed a genome-wide linkage analysis with regional fine mapping in the Stanford Asia–Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) cohort. We further investigated the role of the RRBP1 gene using a transgenic mouse model and a human cell model.ResultsIn the SAPPHIRe cohort, we discovered that genetic variants of the RRBP1 gene were associated with blood pressure variation, which was confirmed by other GWASs for blood pressure. Rrbp1- knockout (KO) mice had lower blood pressure and were more likely to die suddenly from severe hyperkalemia caused by phenotypically hyporeninemic hypoaldosteronism than wild-type controls. The survival of Rrbp1-KO mice significantly decreased under high potassium intake due to lethal hyperkalemia-induced arrhythmia and persistent hypoaldosteronism, which could be rescued by fludrocortisone. An immunohistochemical study revealed renin accumulation in the juxtaglomerular cells of Rrbp1-KO mice. In the RRBP1-knockdown Calu-6 cells, a human renin-producing cell line, transmission electron and confocal microscopy revealed that renin was primarily retained in the endoplasmic reticulum and was unable to efficiently target the Golgi apparatus for secretion.ConclusionsRRBP1 deficiency in mice caused hyporeninemic hypoaldosteronism, resulting in lower blood pressure, severe hyperkalemia, and sudden cardiac death. In juxtaglomerular cells, deficiency of RRBP1 reduced renin intracellular trafficking from ER to Golgi apparatus. RRBP1 is a brand-new regulator of blood pressure and potassium homeostasis discovered in this study.Graphical

Conditioned media from human pluripotent stem cell-derived cardiomyocytes inhibit the growth and migration of lung cancer cells.

Conditioned media (CM) from various cell types contain significant levels of paracrine factors. Recently, therapeutic properties of CM derived from stem cells have been revealed. Based on the fact that heart cancer is extremely rarely, we hypothesized that the CM obtained from human pluripotent stem cell-derived cardiomyocytes might inhibit cancer cell growth and survival. To this end, lung cancer cell line A549 along with human foreskin fibroblasts (HFF) were treated with serial concentrations of cardiomyocyte CM (CCM) or fibroblast CM (FCM). We found that CCM markedly reduced the viability of lung cancer cells, while FCM did not compromise the viability of neither cancer cells nor HFF cells. Furthermore, we determined an optimized CCM concentration, 30 mg/mL, at which the growth, clonogenicity, and migration of A549 and Calu6 lung cancer cell lines were substantially impaired, whereas FCM did not influence these properties. Moreover, lung cancer cells exhibited cell cycle regulation upon treatment with CCM and the rate of apoptosis was markedly increased by cardiomyocyte CM in both lung cancer cell lines tested. Finally, in response to CCM treatment, A549 and Calu6 cells expressed lower levels of antiapoptotic and stemness genes, but higher levels of proapoptotic genes. In conclusion, this study provides cellular and molecular evidence for the antitumor ability of secretome obtained from stem cell-derived cardiomyocytes.