Human Lung Adenocarcinoma Research Articles

Tris(aminomethyl)phosphines and Their Copper(I) (Pseudo)halide Complexes with Aromatic Diimines-A Critical Retrospection.

Metal complexes feature a wide range of available geometries, diversified lability, controllable hydrolytic stability, and easily available rich redox activity. These characteristics, combined with the specific properties of coordinated organic molecules, result in many different mechanisms of biological action, making each of the myriads of the classes of metal coordination compounds unique. This focused review presents combined and systematized results of the studies of a group of copper(I) (pseudo)halide complexes with aromatic diimines and tris(aminomethyl)phosphines of a general formula [CuX(NN)PR3], where X = I- or NCS-, NN = 2,2'-bipyridyl, 1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline or 2,2'-biquinoline, and PR3 = air-stable tris(aminomethyl)phosphines. The structural and electronic properties of the phosphine ligands and luminescent complexes are discussed. The complexes with 2,9-dimethyl-1,10-phenanthroline, apart from being air- and water-stable, exhibit a very high in vitro antimicrobial activity against the Staphylococcus aureus and Candida albicans. Moreover, some of these complexes also show a strong in vitro antitumor activity against human ovarian carcinoma cell lines: MDAH 2774 and SCOV 3, CT26 (mouse colon carcinoma), and A549 (human lung adenocarcinoma) cell lines. The tested complexes are moderately able to induce DNA lesions through free radical processes, however the trends do not reflect observed differences in biological activity.

PDE4D/cAMP/IL-23 axis determines the immunotherapy efficacy of lung adenocarcinoma via activating the IL-9 autocrine loop of cytotoxic T lymphocytes

Although immunotherapy has changed the prognosis of many advanced malignancies including lung adenocarcinoma (LUAD), many patients are insensitive to the drugs, with the mechanisms yet to be elucidated. Herein, we identified PDE4D as an immunotherapy efficacy-related gene through bioinformatics screening. By using a co-culture system of LUAD cells and tumor-cell-specific CD8+ T cells, a functional PDE4D/cAMP/IL-23 axis was further revealed in LUAD cells. Fluorescent multiplex immunohistochemistry analysis of patient-derived samples and the in vivo mouse LUAD xenograft tumors revealed not only the colocalization of IL-23 and CD8+ T cells but also the immune potentiating effect of IL-23 on cytotoxic T lymphocytes (CTLs) in LUAD tissues. Through transcriptome sequencing and functional validations, IL-23 was proven to up-regulate IL-9 expression in CTLs via activating the NF-κB signaling, leading to elevated productions of immune effector molecules and enhanced efficacy of antitumor immunotherapy. Interestingly, an autocrine loop of IL-9 was also uncovered during this process. In conclusion, PDE4D/cAMP/IL-23 axis determines the immunotherapy efficacy of human LUAD. This effect is mediated by the activation of an NF-κB-dependent IL-9 autocrine loop in CTLs.

Erlotinib-Loaded Dendrimer Nanocomposites as a Targeted Lung Cancer Chemotherapy.

Lung cancer is the main cause of cancer-related mortality globally. Erlotinib is a tyrosine kinase inhibitor, affecting both cancerous cell proliferation and survival. The emergence of oncological nanotechnology has provided a novel drug delivery system for erlotinib. The aims of this current investigation were to formulate two different polyamidoamine (PAMAM) dendrimer generations-generation 4 (G4) and generation 5 (G5) PAMAM dendrimer-to study the impact of two different PAMAM dendrimer formulations on entrapment by drug loading and encapsulation efficiency tests; to assess various characterizations, including particle size distribution, polydispersity index, and zeta potential; and to evaluate in vitro drug release along with assessing in situ human lung adenocarcinoma cell culture. The results showed that the average particle size of G4 and G5 nanocomposites were 200 nm and 224.8 nm, with polydispersity index values of 0.05 and 0.300, zeta potential values of 11.54 and 4.26 mV of G4 and G5 PAMAM dendrimer, respectively. Comparative in situ study showed that cationic G4 erlotinib-loaded dendrimer was more selective and had higher antiproliferation activity against A549 lung cells compared to neutral G5 erlotinib-loaded dendrimers and erlotinib alone. These conclusions highlight the potential effect of cationic G4 dendrimer as a targeting-sustained-release carrier for erlotinib.

Study on the absolute configuration and biological activity of rotenoids from the leaves and twigs of Millettia pyrrhocarpa Mattapha, Forest & Hawkins, sp. Nov

BackgroundM. pyrrhocarpa is a new plant in the Fabaceae: Faboideae family that is found in Thailand. A literature search revealed that the Milletia genus is rich in bioactive compounds possessing a wide range of biological activities. In this study, we aimed to isolate novel bioactive compounds and to study their bioactivities.MethodsThe hexane, ethyl acetate, and methanol extracts from the leaves and twigs of M. pyrrhocarpa were isolated and purified using chromatography techniques. These extracts and pure compounds were tested in vitro for their inhibitory activities against nine strains of bacteria, as well as their anti-HIV-1 virus activity and cytotoxicity against eight cancer cell lines.ResultsThree rotenoids, named 6aS, 12aS, 12S-elliptinol (1), 6aS, 12aS, 12S-munduserol (2), dehydromunduserone (3), and crude extracts were evaluated for antibacterial, anti-HIV, and cytotoxic activities. It was found that compounds 1–3 inhibited the growth of nine strains of bacteria, and the best MIC/MBC values were obtained at 3/ > 3 mg/mL. The hexane extract showed anti-HIV-1 RT with the highest %inhibition at 81.27 at 200 mg/mL, while 6aS, 12aS, 12S-elliptinol (1) reduced syncytium formation in 1A2 cells with a maximum EC50 value of 4.48 μM. Furthermore, 6aS, 12aS, 12S-elliptinol (1) showed cytotoxicity against A549 and Hep G2 cells with maximum ED50 values of 2.27 and 3.94 μg/mL.ConclusionThis study led to the isolation of constituents with potential for medicinal application, providing compounds (1–3) as lead compounds against nine strains of bacteria. The hexane extract showed the highest %inhibition of HIV-1 virus, Compound 1 showed the best EC50 in reducing syncytium formation in 1A2 cells, and it also showed the best ED50 against human lung adenocarcinoma (A549) and human hepatocellular carcinoma (Hep G2). The isolated compounds from M. pyrrhocarpa offered significant potential for future medicinal application studies.

Circular RNA-mediated ceRNA network was identified in human lung adenocarcinoma by high-throughput sequencing.

Aberrantly expressed circular RNAs (circRNAs) have been detected in many types of tumors. Hence, they are currently investigated as candidate biomarkers for diagnostic and potential targets for therapy in cancers. The objective of this study was to assess the expression profile of circRNA in lung adenocarcinoma (LUAD). This study included 14 pairs of postoperative lung adenocarcinoma specimens, including cancer tissues and matched adjacent tissues. Second-generation sequencing was applied to the specimens to determine the circRNA expression in them among the 5242 distinct circRNAs detected. We identified a total of 18 significantly dysregulated circRNAs in the LUAD tissues: upregulation in four and downregulation in 14. ROC (The receiver operating characteristic curve) further suggested that hsa_circ_0120106, has_circ_0007342, has_circ_0005937, and circRNA_0000826 could potentially be used as biomarkers in the diagnosis of LUAD. Furthermore, study of the circRNA-microRNA (miRNA)-messenger RNA (mRNA) revealed interactions between the 18 dysregulated circRNA and several cancer-related miRNAs. Finally, a further Kyoto Encyclopedia of Genes and Genomes analysis showed that the cell cycle phase transition, p53 signaling pathway, AMP-activated protein kinase (AMPK) relative signaling pathway, and so on were key putative pathways in the process of LUAD. These findings demonstrated the correlation between abnormality in circRNA expression and LUAD, which lays the foundation of making CircRNAs candidate biomarkers in the diagnosis of LUAD.

Abstract B029: Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma

Abstract While KRAS is among the most frequently mutated oncogenes, our understanding of the mechanisms of KRAS-driven oncogenesis remains limited. A significant remaining gap is a lack of understanding of tissue-specific effectors of Ras activation and the role of specific KRAS mutations in determining downstream vulnerabilities. Previously, we used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells to identify a KRAS-specific vulnerability induced by the combined loss of RHOA and the long isoform of RAP1GDS1, suggesting a potentially novel approach for targeting KRAS-driven cancer. Here we use biochemical, proteomic, and genetic approaches to dissect the isoform-specific roles of RAP1GDS1 to elucidate its synthetic lethal interaction with RHOA in KRAS-driven LUAD. We performed AP/MS in KRAS-mutant A549 cells using both the long (RAP1GDS1-607) and the short (RAP1GDS1-558) isoforms of RAP1GDS1 as bait to identify overlapping and isoform-specific RAP1GDS1 interactors. We enriched this analysis with orthologous datasets from DepMap, published protein-protein interaction (PPI) data and RAP1GDS1-specific prenylome. This comprehensive analysis identified a cluster of RAB GTPases, proteins involved in vesicular transport and lysosomal function, as specific interactors of the long RAP1GDS1 isoform. We subsequently used the proximity ligation assay (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We also depleted either or both RAP1GDS1 isoforms in cells expressing GFP-tagged RAB7A, RAB22A or KRAS and used live cell imaging to determine how loss of RAP1GDS1 changes their subcellular localization. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of the long RAP1GDS1 isoform, significantly decreased KRAS localization at the plasma membrane. We also used PLA to confirm that RAP1GDS1-607 has a stronger interaction with KRAS compared to the short isoform. Moreover, we demonstrated that loss of the long RAP1GDS1 isoform significantly decreased the membrane localization levels of GTP-bound KRAS, compared to the control or RAP1GDS1-558 knock-down. Finally, to identify the molecular mechanism of R1G1/RhoA synthetic lethality with KRAS, we performed a Genome Wide-CRISPR screen in R1G1-607 and RhoA knock-out cells. We identified the strongest hits and designed a focused CRISPR library of approximately 400 genes to screen a larger panel of KRAS-mutant LUAD cell lines (H23, A549 and H358). The aim is to identify other genes that are synthetic lethal in combination with RhoA or R1G1-607 loss and genes that could mediate the lethal effect of their combined loss. Citation Format: Marta Roman Moreno, Kaja Kostyrko, Kari Herrington, Michael Bassik, Peter Jackson, Eric Alejandro Sweet-Cordero. Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B029.

Abstract IA20: The impact of RAS inhibition on tumor immune evasion

Abstract The approval of two KRAS G12C mutant-specific inhibitory drugs, sotorasib and adagrasib, has markedly improved the treatment of lung cancer patients harbouring KRAS G12C oncogenic mutations. However, development of acquired drug resistance is very common after initial responses, and impact on overall survival is small. Additional therapies will therefore need to be combined with KRASG12C inhibitors if long-term cures are to be achieved. Due to the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, targeted KRAS G12C inhibition can indirectly affect anti-tumour immunity. This has served as a rationale for combination with immune checkpoint blockade (ICB), which we have shown leads to additional therapeutic benefit in immunogenic/hot, but not immune evasive/cold, pre-clinical lung tumour models. However, in clinical trials in KRAS G12C mutant lung cancer, combination of KRAS G12C inhibitors with PD-(L)1 blockade has yet to show improved outcome, either due to toxicities or lack of efficacy, possibly linked to prior progression on immunotherapy. Study of the response of KRAS G12C mutant lung tumours to KRAS G12C inhibitory drugs using spatial and multi-omic technologies has highlighted the role of particular populations of immune cells in the tumor microenvironment in impeding immune attack on the tumor. In immune evasive lung cancer, following KRAS G12C inhibitor treatment a community of cells is identified within the tumor containing dendritic cells, CD8+ T cells and dying tumor cells, which appears to function as an antigen presentation hub. This community is restrained by T reg cells, whose coordinate targeting markedly improves the impact of KRAS G12C inhibitors. In an immunogenic/hot mouse model of KRAS G12C mutant lung cancer, tertiary lymphoid structures are seen along with tumor-binding antibodies, which target endogenous retrovirus (ERV) envelope glycoproteins on the tumor cells. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted KRAS G12C inhibition in the murine model. ERV-reactive antibodies exert anti-tumour activity that extend survival in the murine model and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Rational, mechanism-based exploitation of the impact of KRAS inhibition on tumor immune evasion provides opportunities for successful combination therapy of RAS mutant cancers. Citation Format: Julian Downward. The impact of RAS inhibition on tumor immune evasion [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA20.

Fast and efficient isolation of human EGFR-positive cells using EasySep&[trade

Abstract Epidermal growth factor receptor (EGFR) is a cell surface receptor normally expressed on most epithelial cells and some hematopoietic cells. Mutation and overexpression of EGFR is a key driver of many human cancers, including lung, breast, as well as colorectal cancers. Circulating tumour cells (CTCs) can also express EGFR, where enrichment of EGFR+ CTCs can provide promising liquid biopsy and prognostic values. EGFR-positive cells can be difficult to isolate because they are present in many tissue types at a wide range of frequencies. We have developed a simple method to isolate human EGFR-positive cells to address this challenge. A549 human lung adenocarcinoma cells expressing EGFR were spiked into human PBMCs at defined frequencies as a model system. Starting with a single-cell suspension with 10% EGFR+ cells, the EGFR-expressing cells were labeled with an antibody complex that linked the EGFR-expressing cells to magnetic particles, then separated using an EasySep™ magnet. Using this method, EGFR+ cells were enriched from 9.9 ± 3.3% to 95.0 ± 2.8% (mean ± SD; n = 14). Protocols have been optimized for different sample sizes and different EGFR+ cell starting frequencies. EasySep™-isolated EGFR+ lung adenocarcinoma cells were viable and proliferative, with fold expansion and Ki67 expression comparable to untreated cells. EasySep™ Human EGFR Positive Selection Kit enables simple and easy isolation of EGFR-positive cells in 15 minutes, facilitating EGFR and cancer research.

ATF1 promotes the malignancy of lung adenocarcinoma cells by transcriptionally regulating ZNF143 expression.

The clinical oncogenic functions and mechanisms of activating transcription factor 1 (ATF1) in the progression of lung adenocarcinoma have not been completely elucidated. In this study, by employing human lung adenocarcinoma tissues and cells, we detect the correlation of ATF1 expression with the clinicopathological features and prognosis of patients with lung adenocarcinoma and find that ATF1 promotes lung adenocarcinoma cell proliferation and migration by transcriptionally enhancing zinc finger protein 143 (ZNF143) expression. ATF1 and ZNF143 are strongly expressed in lung adenocarcinoma tissues compared with those in the adjacent normal tissues, and high ATF1 and ZNF143 expressions are related to poor disease-free survival of lung adenocarcinoma patients. ATF1 overexpression results in increased proliferation and migration of lung adenocarcinoma cells, whereas knockdown of ATF1 inhibits cell proliferation and migration. Furthermore, ATF1 transcriptionally regulates the expression of ZNF143, and ATF1 and ZNF143 expressions are positively correlated in lung adenocarcinoma tissues. ZNF143 knockdown blocks lung adenocarcinoma cell migration, which is mediated by ATF1 upregulation. Hence, this study provides a potential therapeutic candidate for the treatment of lung adenocarcinoma.

Conserved transcriptional connectivity of regulatory T cells in the tumor microenvironment informs new combination cancer therapy strategies

While regulatory T (Treg) cells are traditionally viewed as professional suppressors of antigen presenting cells and effector T cells in both autoimmunity and cancer, recent findings of distinct Treg cell functions in tissue maintenance suggest that their regulatory purview extends to a wider range of cells and is broader than previously assumed. To elucidate tumoral Treg cell ‘connectivity’ to diverse tumor-supporting accessory cell types, we explored immediate early changes in their single-cell transcriptomes upon punctual Treg cell depletion in experimental lung cancer and injury-induced inflammation. Before any notable T cell activation and inflammation, fibroblasts, endothelial and myeloid cells exhibited pronounced changes in their gene expression in both cancer and injury settings. Factor analysis revealed shared Treg cell-dependent gene programs, foremost, prominent upregulation of VEGF and CCR2 signaling-related genes upon Treg cell deprivation in either setting, as well as in Treg cell-poor versus Treg cell-rich human lung adenocarcinomas. Accordingly, punctual Treg cell depletion combined with short-term VEGF blockade showed markedly improved control of PD-1 blockade-resistant lung adenocarcinoma progression in mice compared to the corresponding monotherapies, highlighting a promising factor-based querying approach to elucidating new rational combination treatments of solid organ cancers.

Lung Adenocarcinoma Expression of the Aryl Hydrocarbon Receptor Promotes PD-L1 and Immune Suppression

Abstract Immunotherapy has shown dramatic results in treating cancer, but only a minority of patients benefit. Therefore, there is an unmet medical need to understand the regulation of immune suppression in tumors. We show that the aryl hydrocarbon receptor (AhR) is a central player in regulating immune checkpoints in a mouse model of lung adenocarcinoma (LUAD). Within the murine malignant cell, AhR regulates expression of several genes important to suppressive immune signaling, including PD-L1. We find similar immune-related regulatory mechanisms in human LUAD cells. Notably, the presence of IFNγ boosts the expression of these AhR-regulated suppressive genes, suggesting a deleterious role for IFNγ in LUAD. Transplantation of mouse AhR-knockout CMT167 (CMT167 AhR-KO) LUAD cells leads to partial or complete tumor rejection and protection from wild type (WT) LUAD challenge. CMT167 AhR-KOtumors that do form have decreased PD-L1 expression and increased T cell infiltration relative to AhR +control tumors. To identify specific immune cell subsets involved in AhR-related tumor immunity, we employed an imaging mass cytometry (IMC) platform coupled with single cell RNA sequencing of tumor infiltrating CD45 +immune cells. Understanding the phenotypes and spatial orientation of the immune cells interacting with malignant cells will provide evidence of the key immune populations responsible for anti-tumor immunity and thus help pave the way for future therapies and biomarkers relevant to improving treatments for LUAD patients. Find the Cause Consortium Gifts

An In Vitro and In Vivo Assessment of Antitumor Activity of Extracts Derived from Three Well-Known Plant Species.

One of the objectives of this study consists of the assessment of the antitumor activity of several extracts from three selected plant species: Xanthium spinosum L., Trifolium pratense L., and Coffea arabica L. and also a comparative study of this biological activity, with the aim of establishing a superior herbal extract for antitumor benefits. The phytochemical profile of the extracts was established by HPLC-MS analysis. Further, the selected extracts were screened in vitro for their antitumor activity and antioxidant potential on two cancer cell lines: A549-human lung adenocarcinoma and T47D-KBluc-human breast carcinoma and on normal cells. One extract per plant was selected for in vivo assessment of antitumor activity in an Ehrlich ascites mouse model. The extracts presented high content of antitumor compounds such as caffeoylquinic acids in the case of X. spinosum L. (7.22 µg/mL-xanthatin, 4.611 µg/mL-4-O-caffeoylquinic acid) and green coffee beans (10.008 µg/mL-cafestol, 265.507 µg/mL-4-O-caffeoylquinic acid), as well as isoflavones in the case of T. pratense L. (6806.60 ng/mL-ononin, 102.78 µg/mL-biochanin A). Concerning the in vitro results, the X. spinosum L. extracts presented the strongest anticancerous and antioxidant effects. In vivo, ascites cell viability decreased after T. pratense L. and green coffee bean extracts administration, whereas the oxidative stress reduction potential was important in tumor samples after T. pratense L. Cell viability was also decreased after administration of cyclophosphamide associated with X. spinosum L. and T. pratense L. extracts, respectively. These results suggested that T. pratense L. or X. spinosum L. extracts in combination with chemotherapy can induce lipid peroxidation in tumor cells and decrease the tumor viability especially, T. pratense L. extract.

Regulation of VEGFR2 and AKT Signaling by Musashi-2 in Lung Cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.

Https://biotechnology.kiev.ua/index.php/en/journal-archive-en/2023-en/2023-no2-en/fibrinogen-specific-protease-in-the-vipera-renardi-snake-venom-k-v-baidakova-y-m-stohnii-o-m-platonov

Aim. To investigate the changes in MMPs expression and activity as well as invasiveness of human lung adenocarcinoma A549 cells with up-/down-regulation of Ruk/CIN85. Methods. There were used A549 cells with stable overexpression (subline RukUp) and knockdown of Ruk/CIN85 (subline RukDown), as well as corresponding vector control sublines Mock and Scr. Cells were cultured in the complete DMEM medium under standard conditions. mRNA expression levels were estimated by RT2-PCR, enzymatic activity was assessed using gelatin zymography. Invasiveness of cancer cells was studied using Boyden chambers coated with Matrigel. Results. Analysis of mRNA expression of MMPs in RukUp and RukDown cells revealed that MMP-2 and MMP-9 were preferentially overexpressed in RukDown cells, while RukUp subline did not exhibit significant difference compared with corresponding control. These findings were confirmed and complemented by study of enzyme activities. The gelatinolytic activities of both MMP-2 and MMP-9 were dramatically increased in RukDown subline, compared to respective control. we revealed that MMPs regulation was inversely correlated with invasion potential of Ruk/CIN85 up/down A549 cells. In particular, it was established that invasiveness of RukUp cells was 2 times higher in comparison with respective control subline. Alternatively, invasion ratio was significantly decreased in RukDown cells (0,5 times) in comparison with control. Conclusions. According to the data received, it is possible to suggest that up-regulation of adaptor protein Ruk/CIN85 in A549 cells can lead to the very aggressive MMP-independent mode of migration that rely on cycles of expansion and contraction of the cell body mediated by the cortically localized actin and myosin.

Physicochemical characterization and cancer cell antiproliferative effect of silver-doped magnesia nanoparticles

Silver-doped magnesia nanoparticles (Ag/MgO) were synthesized using the precipitation method and characterized by various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), Brunner-Emmett-Teller (BET) surface area measurements, and dispersive X-ray spectroscopy (EDX). The morphology of Ag/MgO nanoparticles was determined by transmission and scanning electron microscopy, which revealed cuboidal shaped nanoparticles with sizes ranging from 31 to 68 nm and an average size of 43.5 ± 10.6 nm. The anticancer effects of Ag/MgO nanoparticles were evaluated on human colorectal (HT29) and lung adenocarcinoma (A549) cell lines, and their caspase-3, -8, and -9 activities, as well as Bcl-2, Bax, p53, cytochrome C protein expressions were estimated. Ag/MgO nanoparticles showed selective toxicity towards HT29 and A549 cells while remaining relatively innocuous towards the normal human colorectal, CCD-18Co, and lung, MRC-5 cells. The IC50 values of Ag/MgO nanoparticles on the HT29 and A549 cells were found to be 90.2 ± 2.6 and 85.0 ± 3.5 μg/mL, respectively. The Ag/MgO nanoparticles upregulated caspase-3 and -9 activities, downregulated Bcl-2, upregulated Bax and p53 protein expressions in the cancer cells. The morphology of the Ag/MgO nanoparticle treated HT29 and A549 cells was typical of apoptosis, with cell detachment, shrinkage, and membrane blebbing. The results suggest that Ag/MgO nanoparticles induce apoptosis in cancer cells and exhibit potential as a promising anticancer agent.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

Macrophage CD5L is a target for cancer immunotherapy

Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p=0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages invitro. Its administration invivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. For a full list of funding bodies, please see the Acknowledgements.

DAP3-mediated cell cycle regulation and its association with radioresistance in human lung adenocarcinoma cell lines.

Mitochondria play important roles in the cellular response to various types of stress, including that triggered by ionizing radiation. We have previously reported that the mitochondrial ribosomal protein death-associated protein 3 (DAP3) regulates the radioresistance of human lung adenocarcinoma (LUAD) cell lines A549 and H1299. However, the underlying mechanism of this regulation remains to be elucidated. To this end, we have herein investigated the role of DAP3 in the cell cycle regulation after irradiation. Notably, the DAP3 knockdown attenuated the radiation-induced increase of the G2/M cell population. Furthermore, western blotting analysis has revealed that the DAP3 knockdown decreased the expression of proteins related to the G2/M arrest, such as those of the phosphorylated cdc2 (Tyr15) and the phosphorylated checkpoint kinase 1 (Ser296), in irradiated A549 cells and H1299 cells. Moreover, by using a chk1 inhibitor, we were able to demonstrate that chk1 is involved in the radiation-induced G2/M arrest in both A549 and H1299 cells. Notably, the chk1 inhibitor was able to enhance the radiosensitivity of H1299 cells, while both chk1 inhibitor-abolished G2 arrest and inhibition of chk2-mediated events such as downregulation of radiation-induced p21 expression were required for enhancing radiosensitivity of A549 cells. Collectively, our findings reveal a novel role of DAP3 to regulate G2/M arrest through pchk1 in irradiated LUAD cells and suggest that chk1-mediated G2/M arrest regulates the radioresistance of H1299 cells, whereas both the chk1-mediated G2/M arrest and the chk2-mediated events contribute to the radioresistance of A549 cells.

Abstract 5752: Enhancer amplification defines lineage addiction in human lung adenocarcinoma

Abstract Lung cancer is the most common cause of death from cancer worldwide, and lung adenocarcinoma (LUAD) is the most common type of lung cancer. NKX2-1 (also known as TTF-1, TITF-1) is a lineage defining transcription factor for normal lung development. In LUAD, NKX2-1 is a highly specific and sensitive marker for both primary and metastatic LUADs, with 85-90% expressing NKX2-1. We and others identified that NKX2-1 as the most significantly amplified gene in LUAD, with 22.5% of primary LUADs exhibiting copy number gains. NKX2-1 is amplified as the earliest stages of LUAD development, and NKX2-1 amplification is a truncal event in multi-region LUAD evolution. Despite strong evidence for an oncogenic role for NKX2-1, little is known about the mechanisms of NKX2-1 activation, or how its oncogenic regulation drives LUAD. Here, we identify recurrent focal amplification targeting a super-enhancer (SE) of NKX2-1 as a driving event in LUAD. Using epigenomic data from LUAD cell lines and tumors, we identify this region as a alveolar lineage super-enhancer (NKX2-1 SE) that is specifically active in NKX2-1(+) LUAD cell lines and primary tumors. Notably, this region is co-amplified with NKX2-1 in 96-100% of NKX2-1-amplified samples, suggesting this region a critical component of the NKX2-1 amplicon. Using endogenous ChIP-seq and exogenous luciferase assays, we show the enhancer activity of the NKX2-1 SE is comprised of three constituent enhancers, and demonstrate that the key enhancer elements can activate transcription of the NKX2-1 promoter. Using CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) in LUAD cell lines expressing high or low levels of NKX2-1, we show that activity of the NKX2-1 SE defines endogenous NKX2-1 expression. Using RNA-seq, ChIP-seq, and ATAC-seq, we find that NKX2-1 controls the transcriptional and epigenomic landscape of lung adenocarcinoma. NKX2-1 knockdown activates an epithelial-mesenchymal transition (EMT) gene signature and downregulates an alveolar differentiation signature, including critical markers of LUAD differentiation, such as NAPSA, SFTPA1/2, and HOPX, at which NKX2-1 mediates lineage enhancer accessibility to regulate target gene expression. Global clustering of LUAD cell lines by RNA-seq or H3K27ac ChIP-seq identifies a distinct NKX2-1(+) cluster, and we find that NKX2-1 directly regulates the genes (57/97) and enhancers that distinguish this subpopulation. Using genome-wide shRNA and CRISPR screens, we identify a NKX2-1 dependency in NKX2-1(+) LUAD cell lines, and find that the majority of NKX2-1(+) LUAD cell lines (11/14) assayed are dependent on NKX2-1. Our data demonstrates that enhancer amplification is a hallmark of oncogenic NKX2-1 activation in LUAD, through which NKX2-1 drives a lineage addicted state and oncogenic cell proliferation. This suggests that NKX2-1 is a critical defining oncogene for LUAD, and that targeting of NKX2-1 or its enhancer may suppress LUAD. Citation Format: John Louis Pulice, Matthew Meyerson. Enhancer amplification defines lineage addiction in human lung adenocarcinoma. [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 5752.

Abstract 3920: Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma

Abstract While KRAS is among the most frequently mutated oncogenes, our understanding of the mechanisms of KRAS-driven oncogenesis remains limited. A significant remaining gap is a lack of understanding of tissue-specific effectors of Ras activation and the role of specific KRAS mutations in determining downstream vulnerabilities. Previously, we used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells to identify a KRAS-specific vulnerability induced by the combined loss of RHOA and the long isoform of RAP1GDS1, suggesting a potentially novel approach for targeting KRAS-driven cancer. Here we use biochemical, proteomic, and genetic approaches to dissect the isoform-specific roles of RAP1GDS1 to elucidate its synthetic lethal interaction with RHOA in KRAS-driven LUAD. We performed AP/MS in KRAS-mutant A549 cells using both the long (RAP1GDS1-607) and the short (RAP1GDS1-558) isoforms of RAP1GDS1 as bait to identify overlapping and isoform-specific RAP1GDS1 interactors. We enriched this analysis with orthologous datasets from DepMap, published protein-protein interaction (PPI) data and RAP1GDS1-specific prenylome. This comprehensive analysis identified a cluster of RAB GTPases, proteins involved in vesicular transport and lysosomal function, as specific interactors of RAP1GDS1-607. We subsequently used the proximity ligation assay (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We also depleted either or both RAP1GDS1 isoforms in cells expressing GFP-tagged RAB7A, RAB22A or KRAS and used live cell imaging to determine how loss of RAP1GDS1 changes their subcellular localization. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of RAP1GDS1-607, significantly decreased KRAS localization at the plasma membrane. We also used PLA to confirm that RAP1GDS1-607 has a stronger interaction with KRAS compared to the short isoform. Moreover, we demonstrated that loss of the long RAP1GDS1 isoform significantly decreased the membrane localization levels of GTP-bound KRAS, compared to the control or RAP1GDS1-558 knock-down. Finally, to identify the molecular mechanism of R1G1/RhoA synthetic lethality with KRAS, we performed a Genome Wide-CRISPR screen in R1G1-607 and RhoA knock-out cells. We identified the strongest hits and designed a focused CRISPR library of approximately 400 genes to screen a larger panel of KRAS-mutant LUAD cell lines (H23, A549 and H358). The aim is to identify other genes that are synthetic lethal in combination with RhoA or R1G1-607 loss and genes that could mediate the lethal effect of their combined loss. In conclusion, these data demonstrate a specific role of the long RAP1GDS1 isoform in the membrane-localization and activation of KRAS, which supports its role as a potential therapeutic target for KRAS mutant tumors. Citation Format: Marta Roman Moreno, Kaja Kostyrko, Kari Herrington, Michael Bassik, Peter Jackson, Alejandro Sweet-Cordero. Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma. [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 3920.