Non-small Cell Lung Cancer H460 Research Articles

A PD-L1-Targeted Probe Cy5.5-A11 for In Vivo Imaging of Multiple Tumors.

PD-L1 is an immune checkpoint molecule mediating cancer immune escape, and its expression level in the tumor has been used as a biomarker to predict response to immune checkpoint inhibitor (ICI) therapy. Our previous study reveals that an 11 amino acid-long ANXA1-derived peptide (named A11) binds and degrades the PD-L1 protein in multiple cancers and is a potential peptide for cancer diagnosis and treatment. Near-infrared fluorescence (NIF) optical imaging of tumors offers a noninvasive method for detecting cancer and monitoring therapeutic responses. In this study, an NIF dye Cy5.5 was conjugated with A11 peptide to develop a novel PD-L1-targeted probe for molecular imaging of tumors and monitor the dynamic changes in PD-L1 expression in tumors. In vitro imaging studies showed that intense fluorescence was observed in triple-negative breast cancer MDA-MB-231, nonsmall cell lung cancer H460, and melanoma A375 cells incubated with Cy5.5-A11, and the cellular uptake of Cy5.5-A11 was efficiently inhibited by coincubation with unlabeled A11 or knockdown of cellular PD-L1 by shRNA. In vivo imaging studies showed accumulation of Cy5.5-A11 in the MDA-MB-231, H460, and A375 xenografts with good contrast from 0.5 to 24 h after intravenous injection, indicating that Cy5.5-A11 possesses the strong ability for in vivo tumor imaging. Moreover, the fluorescent signal of A11-Cy5.5 in the xenografts was successfully blocked by coinjection of unlabeled A11 peptide or knockdown of cellular PD-L1 by shRNA, indicating the specificity of Cy5.5-A11 targeting PD-L1 in tumor imaging. Our data demonstrate that Cy5.5-A11 is a novel tool for tumor imaging of PD-L1, which has the potential for detecting cancer and predicting ICI therapeutic responses.

A Novel 3-Benzyloxychromone From Celastrus orbiculatus Thunb. Exhibits Anticancer Effects on Non-Small Cell Lung Cancer Cells via GSK-3β-Dependent c-Jun/ATF2 Pathway.

Celastrus orbiculatus Thunb. is a vine used as a traditional Chinese medicinal herb. In this study, we focused on the anticancer cytotoxicity and underlying mechanism of previously unreported 3-oxygen-substituted isoflavone analogue (3-benzyloxychromone, 3-Boc) from the herb. Initially, we established cell line-derived xenograft mouse model using H1299 non-small cell lung cancer (NSCLC) cells and found that the ethanol crude extracts of the stem part of C. orbiculatus (200 mg/kg) could substantially suppress the growth of xenograft tumors in athymic nu/nu mice. We compared 3-Boc with three other flavonoid analogues isolated from the stem part of C. orbiculatus. Among these, 3-Boc showed the most potent cytotoxicity against H1299 and H1975 NSCLC cells. Colony formation, EdU incorporation and Annexin V-FITC/PI apoptosis assays demonstrated that 3-Boc induced growth inhibition primarily by inhibiting DNA replication and inducing apoptotic death of NSCLC cells. Structure-based target prediction and MD simulation suggested that 3-Boc potentially suppressed the activity of glycogen synthase kinase-3β (GSK-3β) by interacting with the ATP-binding site. Western blot analysis indicated that 3-Boc triggered the phosphorylation of Serine 21 of GSK-3α or Serine 9 of GSK-3β in a time- and dose-dependent manner. To investigate the dependency of GSK-3β, we established GSK-3β knockout in H1299 cells. Depletion of GSK-3β enhanced 3-Boc-induced cytotoxicity compared with wild-type counterparts through activated c-Jun/ATF2 signaling pathway. Altogether, our study highlights the anticancer potential of C. orbiculatus and the discovery of novel 3-oxygen-substituted chromone from the herb, which may have important implications for screening promising modulators of GSK-3β and related signaling pathways in the treatment of cancer.

Inhibition of MSH6 augments the antineoplastic efficacy of cisplatin in non-small cell lung cancer as autophagy modulator

The altered response to chemotherapeutic agents predominantly stems from heightened single-point mutations within coding regions and dysregulated expression levels of genes implicated in drug resistance mechanisms. The identification of biomarkers based on mutation profiles and expression levels is pivotal for elucidating the underlying mechanisms of altered drug responses and for refining combinatorial therapeutic strategies in the field of oncology. Utilizing comprehensive bioinformatic analyses, we investigated the impact of eight mismatch repair (MMR) genes on overall survival across 23 cancer types, encompassing more than 7500 tumors, by integrating their mutation profiles. Among these genes, MSH6 emerged as the most predictive biomarker, characterized by a pronounced mutation frequency and elevated expression levels, which correlated with poorer patient survival outcomes.The wet lab experiments disclosed the impact of MSH6 in mediating altered drug responses. Cytotoxic assays conducted revealed that the depletion of MSH6 in H460 non-small lung cancer cells augmented the efficacy of cisplatin, carboplatin, and gemcitabine. Pathway analyses further delineated the involvement of MSH6 as a modulator, influencing the delicate equilibrium between the pro-survival and pro-death functions of autophagy.Our study elucidates the intricate interplay between MSH6, autophagy, and cisplatin efficacy, highlighting MSH6 as a potential therapeutic target to overcome cisplatin resistance. By revealing the modulation of autophagy pathways by MSH6 inhibition, our findings offer insights into novel approaches for enhancing the efficacy of cisplatin-based cancer therapy through targeted interventions.

Cytotoxicity of Benzofuran-Containing Simplified Viniferin Analogues.

Within the huge class of plant secondary metabolites, resveratrol-derived stilbenoids show wide structural diversity and mediate a great number of biological responses relevant for human health, including cancer prevention and cytotoxicity. Resveratrol is known to modulate several pathways directly linked to cancer progression, as well as its analogue pterostilbene, characterized by an increased metabolic stability and significant pharmacological activities. To study the potential anticancer activity of other stilbenoids, a home-made collection of resveratrol dimers and simplified analogues was tested on melanoma A375, non-small cell lung cancer H460 and PC3 prostate cancer cell lines. The structural determinants responsible for the antiproliferative activity have been highlighted. Moreover, to investigate the DNA damage ability of the selected molecules, the expression of the γ-H2AX after compound exposure was evaluated.

Bazedoxifene Inhibits Cell Viability, Colony-Forming Activity, and Cell Migration in Human Non-Small Cell Lung Cancer Cells and Improves the Treatment Efficacy of Paclitaxel and Gemcitabine.

Bazedoxifene is a third-generation selective estrogen receptor modulator that inhibits the IL6/IL6R/GP130 signaling pathway by inhibiting IL6-induced homodimerization of GP130. Considering that the IL6/IL6R/GP130 signaling pathway is important in tumorigenesis and metastasis, bazedoxifene is thought to have an antitumor effect, which has been proven preliminarily in breast cancer and pancreatic cancer but has not yet been studied in non-small cell lung cancer (NSCLC). This study is aimed at evaluating the antitumor effect of bazedoxifene in NSCLC. A549 and H1299 NSCLC cell lines were employed and exposed to various concentrations of bazedoxifene, paclitaxel, gemcitabine, and their combinations for cell viability, colony formation, and wound healing assays to demonstrate the antitumor effect of bazedoxifene with or without paclitaxel or gemcitabine. MTT cell viability, colony formation, and wound healing assays showed that bazedoxifene was capable of inhibiting cell viability, colony formation, and cell migration in a dose-dependent manner. In addition, bazedoxifene was capable of working with paclitaxel or gemcitabine synergistically to inhibit cell viability, colony formation, and cell migration. This study demonstrated the potential antitumor effect of bazedoxifene and its ability to improve the treatment efficacy of paclitaxel and gemcitabine.

Efficacy of GluN2B-Containing NMDA receptor antagonist for antitumor and antidepressant therapy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer. Evidence suggests that the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA) receptor, a critical molecule in the central nervous system, is expressed in NSCLC. However, the specific expression patterns, subcellular localization, functional modulation, and pathological implications of NMDA receptor subtypes in NSCLC have not been fully elucidated. In this study, we employed a multi-disciplinary approach, combining biochemical and molecular biology with electrophysiological recordings and behavioral assays, to investigate these aspects. We reveal the expression of GluN2B-containing NMDA receptors in A549 and H460 NSCLC cell lines and the induction of NMDA receptor-mediated currents by glutamate in A549 cells. Furthermore, the GluN2B-specific inhibitors ifenprodil and Ro 25–6981 significantly reduced cell viability and migration, while promoting apoptosis. Importantly, intraperitoneal administration of ifenprodil in nude mice inhibited the growth of subcutaneous tumors derived from A549 and H460 cells and ameliorated depression-like behaviors. These findings underscore the potential antiproliferative effects of ifenprodil and Ro 25–6981 and suggest that GluN2B-containing NMDA receptors may represent novel therapeutic targets for NSCLC, with the added benefit of potential antidepressant action.

Adiponectin Receptor Agonist AdipoRon Inhibits Proliferation and Drives Glycolytic Dependence in Non-Small-Cell Lung Cancer Cells.

Despite the countless therapeutic advances achieved over the years, non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. To this primacy contribute both non-oncogene addicted and advanced NSCLCs, in which conventional therapies are only partially effective. The adiponectin receptor agonist AdipoRon has revealed antiproliferative action in different cancers, including osteosarcoma and pancreatic cancer. Herein, we investigated its potential anticancer role in NSCLC for the first time. We proved that AdipoRon strongly inhibits viability, growth and colony formation in H1299 and A549 NSCLC cells, mainly through a slowdown in cell cycle progression. Along with the biological behaviors, a metabolic switching was observed after AdipoRon administration in NSCLC cells, consisting of higher glucose consumption and lactate accumulation. Remarkably, both 2-Deoxy Glucose and Oxamate glycolytic-interfering agents greatly enhanced AdipoRon's antiproliferative features. As a master regulator of cell metabolism, AMP-activated protein kinase (AMPK) was activated by AdipoRon. Notably, the ablation of AdipoRon-induced AMPK phosphorylation by Compound-C significantly counteracted its effectiveness. However, the engagement of other pathways should be investigated afterwards. With a focus on NSCLC, our findings further support the ability of AdipoRon in acting as an anticancer molecule, driving its endorsement as a future candidate in NSCLC therapy.

Harnessing the CD44-targeted delivery of self-assembled hyaluronan nanogel to reverse the antagonism between Cisplatin and Gefitinib in NSCLC cancer therapy

The combination of the standard platinum-based chemotherapy with EGFR-tyrosine kinase inhibitor Gefitinib (Gef) principally boosts the anticancer efficacy of advanced non-small cell lung cancer (NSCLC) through non-overlapping mechanisms of action, however the clinical trials of cisplatin (Cis) and Gef combination failed to show a therapeutic improvement likely due to compromised cellular influx of Cis with the Gef interference. To overcome the antagonism between Cis and Gef in anti-NSCLC therapy, here we demonstrated a self-targeted hyaluronan (HA) nanogel to facilitate the anticancer co-delivery by utilizing the HA's intrinsic targeting towards CD44, a receptor frequently overexpressed on lung cancer cells. The co-assembly between HA, Cis and Gef generated a HA/Cis/Gef nanogel of 177.8 nm, featuring a prolonged drug release. Unlike the Gef inhibited the Cis uptake, the HA/Cis/Gef nanogel efficiently facilitated the drug internalization through CD44-targeted delivery as verified by HA competition and CD44 knocking down in H1975 NSCLC model both in vitro and in vivo. Moreover, the HA/Cis/Gef nanogel significantly improved the anticancer efficacy and meanwhile diminished the side effects in reference to the combination of free Cis and Gef. This CD44-targeted HA/Cis/Gef nanogel provided a potent strategy to advance the platinum-based combination therapy towards optimized NSCLC therapy.

ACTN4-dependent regulation of double-strand DNA break repair is independent of NF-Kb activity

α-Actinin-4 is an actin-binding protein that is involved in a wide range of cellular processes. Along with actin and other proteins of the actin cytoskeleton, α-actinin-4 was found not only in the cytoplasm, but also in the nucleus of various cells. As a nuclear protein, it is involved in regulation of certain transcription factors. In particular, it can regulate transcriptional activity of NF-kB, which largely determines the resistance of cancer cells to apoptosis and anticancer therapy. During our previous studies, it was found that α-actinin-4 can influence resistance of cancer cells to topoisomerase II inhibitors and determine the efficiency of DNA double-strand break repair. We have demonstrated that α-actinin-4 interferes with the assembly of complexes involved in DNA repair via NHEJ and HRR, which in turn leads to an imbalance between these pathways. In this study, we were answering to the question of how α-actinin-4 is involved in the regulation of the DNA double-strand breaks repair following genotoxic stress. Our results indicate that the effect of α-actinin-4 on repair progression in H1299 non-small cell lung cancer cells does not depend on the transcription factor NF-kB activity. We found that in the nucleus of H1299 cells, α-actinin-4 is localized not only in the nucleoplasm, but also reveals close association with chromatin.

Synergistic anticancer effects of SMYD2 inhibitor BAY-598 and doxorubicin in non-small cell lung cancer

BackgroundNon-small cell lung Cancer (NSCLC) persists as a lethal neoplastic manifestation, exhibiting a diminished 5-year survival rate, partially attributable to chemotherapeutic resistance. This investigative endeavor aimed to elucidate the synergistic antineoplastic effects and underlying mechanisms of the SMYD2 inhibitor BAY-598 and the chemotherapeutic agent doxorubicin (DOX) in NSCLC. MethodsThe human non-small cell lung cancer cell lines A549 and H460 were subjected to treatment regimens involving BAY-598 and/or DOX. Cellular viability, apoptotic events, invasive capacity, and migratory potential were evaluated through the implementation of CCK-8 assays, flow cytometric analyses, and Transwell assays, respectively. Protein expression levels were quantified via Western blot analyses. An in vivo xenograft murine model was established to assess therapeutic efficacy. ResultsBAY-598 and DOX synergistically suppressed the viability, invasiveness, and migratory capabilities of NSCLC cells. Co-treatment Promoting cell apoptosis and cell cycle arrest. Additionally, Furthermore, co-administration significantly inhibited cell migration and invasion. Mechanistic studies revealed coordinately inhibited JAK-STAT signaling upon combination treatment. In vivo study further validated the synergistic antitumor efficacy of BAY-598 and DOX against NSCLC xenografts. ConclusionsOur findings demonstrate that BAY-598 potentiates the anti-cancer effects of DOX in non-small cell lung cancer cells by modulating the JAK/STAT signaling pathway as a synergistic strategy. The combination holds promise as an emerging therapeutic strategy for NSCLC. Further optimization and validation are warranted to promote its translational potential.

Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner.

Several studies have shown an inverse correlation between the likelihood of developing a neurodegenerative disorder and cancer. We previously reported that the levels of amyloid beta (Aβ), at the center of Alzheimer's disease pathophysiology, are regulated by acetylcholinesterase (AChE) in non-small cell lung cancer (NSCLC). Here, we examined the effect of Aβ or its fragments on the levels of ACh in A549 (p53 wild-type) and H1299 (p53-null) NSCLC cell media. ACh levels were reduced by cell treatment with Aβ 1-42, Aβ 1-40, Aβ 1-28, and Aβ 25-35. AChE and p53 activities increased upon A549 cell treatment with Aβ, while knockdown of p53 in A549 cells increased ACh levels, decreased AChE activity, and diminished the Aβ effects. Aβ increased the ratio of phospho/total p38 MAPK and decreased the activity of PKC. Inhibiting p38 MAPK reduced the activity of p53 in A549 cells and increased ACh levels in the media of both cell lines, while opposite effects were found upon inhibiting PKC. ACh decreased the activity of p53 in A549 cells, decreased p38 MAPK activity, increased PKC activity, and diminished the effect of Aβ on those activities. Moreover, the negative effect of Aβ on cell viability was diminished by cell co-treatment with ACh.

Abstract LB176: Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids

Abstract A major barrier in lung cancer pre-clinical research is the lack of simple, replicative and reproducible cell culture models. Most studies are still focused on 2D, completely ignoring cell-to-cell interactions, in vivo tissue architecture and the surrounding microenvironment. To address this void in cancer research, we aimed to develop a biomimetic 3D lung cancer spheroid model that can be used as a more reliable tool for pharmacological studies. Therefore, 3D spheroids of A549 (adenocarcinoma), H460 (large cell carcinoma) and H520 (squamous cell carcinoma) non-small cell lung cancer (NSCLC) cells were optimized and consequently submitted to AZD2014 treatment, a dual mTOR inhibitor targeting mTORC1 and mTORC2, members of the PI3K/Akt/mTOR pathway, which is commonly deregulated in lung cancer. Each cell line was seeded at 2000, 5000 or 10000 cells/spheroid in agarose micro-molds for eight days. The 3D NSCLC spheroids were further characterized for their tumor features, namely 3D dynamics and morphology, including diameter, circularity and compactness, viability properties, as well as proliferative and necrotic areas. Based on this characterization, the 2000 cells/spheroid condition was selected for further treatments and spheroid treatment time-points were selected. For each cell line, spheroids were treated with AZD2014, using concentrations based on the IC30 values (sensitization concentration) defined under 2D conditions: 10 μM, 10 μM and 0.2 μM for A549, H460 and H520 spheroids, respectively. Treated and untreated spheroids were followed during six days post-treatment, and their morphological characteristics, cell viability, necrotic core formation and proliferative areas, as well as ATP production, were assessed. Spheroids of all cell lines exhibited a significant inhibition of spheroid growth with AZD2014 treatment, which was maintained over the six days. Moreover, a reduction in spheroid intracellular ATP was observed at days three and six post-treatment. An additional treatment regimen was studied by re-treating spheroids with the same concentration of AZD2014 after 48hrs from the first treatment. Spheroids exposed to this dual treatment regimen exhibited a significant inhibition of spheroid growth, while the respective controls exhibited intensified growth. While AZD2014 did not induce significant reductions in 3D cell viability, a clear inhibition of spheroid growth was observed. Having a more intricate cellular organization, this study alleviates the increased complexity of pharmacological responses in 3D spheroids when compared to 2D models. Further studies are being carried out to define the enhanced biomimetic ability of these NSCLC spheroids and the underlying mechanisms involved when targeting the PI3K/Akt/mTOR pathway using AZD2014 as mono-therapy or in combination with two novel anti-sense oligonucleotides (ASO). Citation Format: Nathan Vella, Diogo Estêvão, Tânia Cruz, Maria José Oliveira, Anthony George Fenech, Vanessa Petroni Magri. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids [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 LB176.

Abstract 3279: MUC1 is necessary for the inflammatory memory response to osimertinib resistance in NSCLC cells

Abstract Emergence of resistance is an invariable outcome in cancer progression that may involve memory of cancer cells to treatment. The oncogenic MUC1-C protein confers pleotropic resistance of cancer cells to diverse cytotoxic and targeted agents by unclear mechanisms. We demonstrate that MUC1-C regulates the interferon (IFN) type I/II pathways in H1975 non-small cell lung cancer (NSCLC) cells selected for osimertinib resistance (H1975-OR). Targeting MUC1-C in H1975-OR cells suppressed STAT1 and inflammatory IFN signaling in association with reversing the osimertinib-resistant phenotype. Studies of H1975-OR cells selected for growth in the absence of osimertinib demonstrate that revertant H1975-RT cells retain MUC1-C-dependent memory necessary for recalling osimertinib resistance. Mechanistically, osimertinib treatment of H1975-RT cells activates the MUC1 gene at a (i) promoter-like signature (PLS) by MUC1-C and STAT1, and (ii) proximal enhancer-like signature (pELS) by PBAF and JUN/AP-1. Targeting MUC1-C in osimertinib-treated H1975-RT and MGH170 NSCLC MET-amplified cells isolated from a patient refractory to osimertinib treatment suppressed induction of STAT1 and downstream IFN-stimulated genes (ISGs) encoding MX1, OAS1 and ISG15. Targeting MUC1-C, STAT1, PBRM1/PBAF and JUN/AP-1 further confirmed their dependencies for reemergence of osimertinib resistance in H1975-RT and MGH170 cells. In support of these results, high MUC1-C protein expression analyzed by IHC is associated with shorter progression free survival for patients treated with osimertinib. These findings and the demonstration that MUC1-C is upregulated in NSCLC cells from patients refractory to osimertinib indicate that MUC1-C drives osimertinib resistance in NSCLC cells by promoting an inflammatory memory response. Citation Format: Naoki Haratake, Atrayee Bhattacharya, Ayako Nakashoji, Hiroki Ozawa, Mototsugu Shimokawa, Donald Kufe. MUC1 is necessary for the inflammatory memory response to osimertinib resistance in NSCLC cells [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 3279.

Abstract 1935: The FAK/SRC axis mediates resistance to KRAS G12C inhibitors and its blockade can overcome KRAS inhibitor resistance

Abstract Activating mutations in KRAS are found in approximately 30% of non-small cell lung cancer (NSCLC), and among all KRAS mutations, KRAS G12C is the most frequent variant in NSCLC, with a prevalence of approximately 14%. KRAS G12C inhibitors, sotorasib and adagrasib are FDA approved for locally advanced or metastatic KRAS G12C-positive NSCLC. However, clinical evidence suggests that there are various factors limiting the efficacy of KRAS G12C inhibitors in patients with KRAS G12C-positive NSCLC, highlighting the urgent need for novel treatment strategies or combinations to improve efficacy. Therefore, we sought to identify effective combinations to enhance the efficacy of KRAS G12C inhibitors using in vitro and in vivo models of KRAS G12C-positive NSCLC. Employing a high throughput in vitro drug screening effort using KRAS mutant NSCLC cell lines, we observed that Focal Adhesion Kinase (FAK) and Steroid Receptor Coactivator (SRC) inhibitors produced a synergistic effect when combined with KRAS G12C inhibitors adagrasib or sotorasib. As determined by reverse phase array and Western blotting, in H358, H23, HCC44, and H2122 cells, KRAS inhibitor treatment alone resulted in an initial suppression of RAS/MAPK and AKT pathway activation which was followed by upregulation of multiple receptor tyrosine kinases and integrins which contributed to the re-activation of the RAS/MAPK and AKT pathways after 24-48 hours. We identified FAK as a key downstream mediator of these bypass pathways. FAK/SRC inhibitors enhanced the efficacy of KRAS G12C inhibitors by preventing this feedback activation. Moreover, we observed increased FAK activation in H358 NSCLC cell lines with acquired resistance to adagrasib or sotorasib as compared to parental cells, and FAK/SRC inhibitors re-sensitized resistant cells to KRAS G12C inhibitors. Using a xenograft model of human KRAS mutant NSCLC, we determined that the combination of repotrectinib, a FAK/SRC inhibitor, and a KRAS G12C inhibitor significantly suppressed tumor growth as compared to KRAS G12C inhibitors alone, and this combination was well tolerated. Overall, our findings indicate that activation of FAK is a key mechanism of adaptive feedback and acquired resistance to KRAS G12C inhibitors in KRAS G12C-positive NSCLC and highlight the therapeutic potential of FAK/SRC inhibitors in combination with KRAS G12C inhibitors. These data support the clinical testing of the combination of FAK/SRC inhibitors and KRAS G12C inhibitors in patients with KRAS G12C-positive NSCLC. Citation Format: Yuji Shibata, Hibiki Udagawa, Monique B. Nilsson, Jacqulyne Robichaux, Ana Galan-Cobo, Marcelo Negrao, David P. Molkentine, Junqin He, Alissa Poteete, Yu Qian, Qian Huang, Ferdinandos Skoulidis, John V. Heymach. The FAK/SRC axis mediates resistance to KRAS G12C inhibitors and its blockade can overcome KRAS inhibitor resistance [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 1935.

Abstract 4297: Interrogating metabolic heterogeneity and cooperation in lung cancer collective cell invasion

Abstract Tumor heterogeneity contributes to tumor progression and a variable response to treatment. We have previously shown that tumor heterogeneity - including metabolic, transcriptional and mutational - underlies non-small cell lung cancer (NSCLC) collective invasion. Using stable subpopulations derived with our Spatiotemporal Cellular and Genomic Analysis (SaGA) technique, we found that two distinct subpopulations of the H1299 NSCLC line cooperate during collective tumor invasion in vitro; while H1299 pack leaders are heavily dependent on oxidative phosphorylation, H1299 pack followers are more reliant on glycolysis. To further dissect how intratumoral heterogeneity enables cancer progression to metastasis, we incorporate high resolution imaging, stable fluorescent labeling, gene editing, and in vitro tumor invasion models to interrogate mechanisms of metabolic cooperation between heterogeneous NSCLC subpopulations. We hypothesize that cooperation and communication between these distinct metabolic subpopulation drives metastatic tumor progression. To determine mechanisms of metabolic cooperativity between subpopulations, we stably transduced our subpopulations to fluorescently label either the mitochondria (through TOM20 labeling) or a key enzyme in glycolysis (through glucose 6-phosphate isomerase GPI labeling), cultured labeled subpopulations with unlabeled subpopulations, and observed metabolic changes and exchange via time-lapse, fluorescence microscopy and flow cytometry in 2D culture conditions. Next steps include visualizing the metabolic cooperation that occurs in conditions mimicking the tumor microenvironment (i.e., 3D invasion assays) using super-resolution microscopy. Taken together, we have developed a method of temporally visualizing metabolic exchange with high spatial resolution between NSCLC subpopulations to investigate the underlying mechanisms enabling successful cell invasion in a variety of tumor microenvironments. Thus far, we have discovered multiple broad mechanisms of exchange including tunneling nanotubes and extracellular vesicles, both of which are inducible with certain stressors common to the tumor microenvironment. Citation Format: Isaac E. Robinson, Veronika Matsuk, Mala Shanmugam, Janna K. Mouw, Adam I. Marcus. Interrogating metabolic heterogeneity and cooperation in lung cancer collective cell invasion [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 4297.

Abstract 4687: Primary study on the anticancer role of BAM15 in lung cancer cells

Abstract Background: Lung cancer remains the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancer cases. BAM15, a mitochondrial uncoupler, is a potential therapeutic agent for many diseases, including obesity, diabetes, nonalcoholic fatty liver disease, Sepsis and cancers. However, there is no report on the anti-tumor role of BAM15 in NSCLC. Methods: To investigate the direct anti-cancer effects of BAM15 in NSCLC, we examined its inhibitory role on cell proliferation of NSCLC cells using the Cell Counting Kit-8(CCK8), colony forming assays and EdU staining assays. The impact of BAM15 on the cell cycle and cell apoptosis of NSCLC cells was analyzed by flow cytometric. A xenotransplanted NSCLC model in mice were used to test the anticancer role of BAM15. Result: Using the CCK8 assay, we found that the inhibitory role of BAM15 on the proliferation and colony formation of NSCLC cells is dose-dependent. The half maximal inhibitory concentration (IC50) values for cell proliferation were 2.71, 4.74, and 9.50 μM for A549, PC9, and H1299 NSCLC cells, respectively. The EdU staining assay demonstrated that BAM15 treatment significantly inhibited DNA synthesis in NSCLC cells. Flow cytometric analysis revealed that BAM15 arrested cell cycle at G2 phase in NSCLC cells and caused a striking increase in the percentage of apoptotic cells in NSCLC cells. The xenotransplanted NSCLC experiment indicated that BAM15 could significantly suppressed the growth of NSCLC tumors compared with the control tumors. Conclusions: Our study demonstrates that BAM15 significantly inhibits cell proliferation and promotes apoptosis in NSCLC cells, indicating that BAM15 is a potential therapeutic candidate for the treatment of NSCLC. Citation Format: Mei-yin Zhang, Yue-Ning Wang, Yu-Feng Zhou, Shuo-Cheng Wang, Shi-Juan Mai, Hui-Yun Wang. Primary study on the anticancer role of BAM15 in lung cancer cells [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 4687.

Sufentanil Suppresses Cell Carcinogenesis Via Targeting miR-186-5p/HMGB1 Axis and Wnt/β-Catenin Pathway in Non-Small-Cell Lung Cancer.

Sufentanil is a common opioid anesthetic agent, which exerts anti-cancer properties in several cancer types. However, its action mechanisms in non-small cell lung cancer (NSCLC) are unclear. Therefore, the present study investigated the pharmacological effect of sufentanil on miRNAs in NSCLC treatment. In this study, after treatment with sufentanil, the proliferation, migration, invasion and apoptosis of A549 and H1299 NSCLC cell lines were measured by cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assays and flow cytometry. Quantitative real time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of miR-186-5p and high mobility group box-1 (HMGB1), and their interaction was analyzed using luciferase reporter assay. The proteins of HMGB1, and apoptosis- and Wnt/β-catenin pathway-related factors were detected by western blot. It was demonstrated that sufentanil significantly upregulated miR‑186‑5p to restrict NSCLC cell proliferation, migration, invasion, and boost apoptosis in vitro. Mechanically, miR-186-5p interacted with HMGB1 and negatively regulated HMGB1 in NSCLC cells. Furthermore, rescue assay showed that sufentanil exerted antitumor activities by upregulating miR-186-5p, which targeted HMGB1 and restrained Wnt/β-catenin signal pathway in NSCLC cells. In conclusion, these results suggested that sufentanil disrupts the oncogenicity of NSCLC cells by regulating miR-186-5p/HMGB1/β-catenin axis, providing a promising implication for the anti-oncogenic effect of sufentanil.

Knockdown of EIF4G1 in NSCLC induces CXCL8 secretion.

Non-small cell lung cancer (NSCLC) is the most common type of lung tumor; however, we lack effective early detection indicators and therapeutic targets. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is vital to initiate protein synthesis, acting as a scaffolding protein for the eukaryotic protein translation initiation factor complex, EIF4F, which regulates protein synthesis together with EIF4A, EIF4E, and other translation initiation factors. However, EIF4G1's function in NSCLC cancer is unclear. Herein, transcriptome sequencing showed that knockdown of EIF4G1 in H1299 NSCLC cells upregulated the expression of various inflammation-related factors. Inflammatory cytokines were also significantly overexpressed in NSCLC tumor tissues, among which CXCL8 (encoding C-X-C motif chemokine ligand 8) showed the most significant changes in both in the transcriptome sequencing data and tumor tissues. We revealed that EIF4G1 regulates the protein level of TNF receptor superfamily member 10a (TNFRSF10A) resulting in activation of the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) pathways, which induces CXCL8 secretion, leading to targeted chemotaxis of immune cells. We verified that H1299 cells with EIF4G1 knockdown showed increased chemotaxis compared with the control group and promoted increased chemotaxis of macrophages. These data suggested that EIF4G1 is an important molecule in the inflammatory response of cancer tissues in NSCLC.

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Acacetin inhibited non-small-cell lung cancer (NSCLC) cell growth via upregulating miR-34a in vitro and in vivo

Acacetin, one of the flavonoid compounds, is a natural product found in various plants, including Silver birch, and Damiana. Previous studies showed that acacetin has anti-cancer effects on many kinds of cancer cells, however, the role of and the mechanisms of actions of acacetin on non-small cell lung cancer (NSCLC) cells is still not fully understood. Herein, we found that, in vitro, acacetin inhibited the proliferation, invasion, and migration of NSCLC cells, A549 and H460, in a dose-dependent manner. Meanwhile, flow cytometry assay results showed that acacetin induced G2/M phase cell cycle arrest, and apoptosis of NSCLC cells. In vivo, acacetin suppressed tumor formation of A549-xenografted nude mice model with no obvious toxicities. Western blotting results showed that the protein levels of cell cycle-related proteins cyclin B1, cyclin D, and anti-apoptotic protein Bcl-2 had decreased, while the apoptosis-related protein Bak had increased both in NSCLC cells and in A549-xenografted tumor tissues. For investigating the molecular mechanism behind the biological effects of acacetin on NSCLC, we found that acacetin induced the expression levels of tumor suppressor p53 both in vitro and in vivo. MicroRNA, miR-34a, the direct target of p53, has been shown anti-NSCLC proliferation effects by suppressing the expression of its target gene programmed death ligand 1 (PD-L1). We found that acacetin upregulated the expression levels of miR-34a, and downregulated the expression levels of PD-L1 of NSCLC cells in vitro and of tumors in vivo. In vitro, knockdown p53 expression by siRNAs reversed the induction effects of acacetin on miR34a expression and abolished the inhibitory activity of acacetin on NSCLC cell proliferation. Furthermore, using agomir and antagomir to overexpress and suppress the expression miR-34a in NSCLC cells was also examined. We found that miR-34a agomir showed similar effects as acacetin on A549 cells, while miR-34a antagomir could partially or completely reverse acacetin’s effects on A549 cells. In vivo, intratumor injection of miR-34a antagomir could drastically suppress the anti-tumor formation effects of acacetin in A549-xenografted nude mice. Overall, our results showed that acacetin inhibits cell proliferation and induces cell apoptosis of NSCLC cells by regulating miR-34a.