Non-small Cell Lung Carcinoma Cells Research Articles

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia.

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

MiR-29b-3p targetedly regulates VEGF to inhibit tumor progression and cisplatin resistance through Nrf2/HO-1 signaling pathway in non-small cell lung cancer.

Non-small cell lung carcinoma (NSCLC) is a common type of lung cancer. Prior investigations have elucidated the pivotal role of miR-29b-3p in restraining tumor growth and metastasis. Nonetheless, it remains to be determined whether miR-29b-3p can effectively suppress NSCLC progression and enhance the sensitivity of NSCLC cells to cisplatin. This investigation sought to determine the mechanism by which miR-29b-3p inhibited the advancement of NSCLC and mitigated resistance to cisplatin. We initially assessed miR-29b-3p and VEGF levels in NSCLC tissues and cell lines. Next, miR-29b-3p expression was elevated in NSCLC cell lines H1975 and A549 by overexpression plasmid transfection. Following this, a sequence of molecular biology experiments was conducted to evaluate the impact of miR-29b-3p on the biological behaviors of NSCLC cells and their resistance to cisplatin. Additionally, we predicted VEGF was a target gene of miR-29b-3p by bioinformatics analysis. We next employed western blot to evaluate the protein expression of Nrf2 and HO-1 in NSCLC cells. Finally, we elucidated the effects of VEGF and Nrf2/HO-1pathway on NSCLC progression and cisplatin resistance by in vitro assays. In comparison to paracancerous tissues and human normal lung epithelial cells, the expression of miR-29b-3p was notably reduced and VEGF expression was clearly elevated in NSCLC tissues and cells. Moreover, miR-29b-3p upregulated obviously suppressed the biological activities of NSCLC cells and increased their sensitivity to cisplatin. Furthermore, in NSCLC cells, miR-29b-3p bound to VEGF and negatively regulate its transcription. Additionally, miR-29b-3p overexpression also inhibited the Nrf2/HO-1 signaling pathway. Finally, the overexpression of VEGF and the activation of the Nrf2/HO-1 pathway reversed miR-29b-3p-mediated inhibitory effect on biological behaviors of NSCLC cells and increased the cisplatin resistance. Our findings indicate that miR-29b-3p impedes NSCLC cells' biological behaviors and augments their sensitivity to cisplatin by targeting VEGF to modulate the Nfr2/HO-1 signaling pathway.

Abstract 631: Functional genetics of APR-246 rescuable TP53 mutants

Abstract TP53, often referred to as the guardian of the genome, encodes for the p53 tumor suppressor protein, and is one of the most frequently mutated genes in human cancers. Some cancer sub-types, such as non-small cell lung carcinoma (NSCLC) and high grade serous carcinoma (HGSC), have high TP53 mutation rates reaching up to 56% and 96%, respectively. Given its multi-faceted role in human cancers, there have been several emerging therapeutic approaches targeting mutant p53. APR-246 is a small molecule drug currently in clinical trials for the functional rescue of p53 missense mutants, the most common type of p53 mutant protein, across various cancer types. APR-246 is proposed to bind mutant p53 and promote thermodynamic stabilization of the protein allowing it to regain wild-type-like conformation and consequentially regain WT-like functionality. However, there has yet to be a functional genomic screening of p53 mutants to systematically catalog which mutants are rescuable by APR-246. To address this critical gap, we have performed functional genetic screenings using a TP53 mutagenesis library. This pooled lentiviral library contains ~8,000 amino acid variants, spanning the entirety of the TP53 gene. Following stable transduction of this library into p53-null NSCLC and HGSOC cell lines, we performed functional screenings against sublethal doses of APR-246. Next-generation sequencing of the untreated and treated cell population allows for comparison of variant representation and determination of APR-246-sensitivity of p53 mutants. Preliminary statistical analysis of our screens has identified a pool of 117 “hit” mutants which are significantly depleted following treatment, indicative of APR-246-sensitivity. Two of these mutants, C242F and G266V, are non-functional, damaging mutations which occur in patients. Identifying p53 mutants that can be rescued by APR-246 will help identify which patients may benefit most from treatment with the drug. Citation Format: Anais Saunders, Caili Tong, Anthony N. Karnezis, Gary S. Leiserowitz, Jeremy R. Chein. Functional genetics of APR-246 rescuable TP53 mutants [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 631.

Abstract 2030: N-cadherin-mediated activation of PI3K/Akt pathway following application of tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) therapy is FDA-approved for the treatment of glioblastoma and unresectable pleural mesothelioma, and has recently demonstrated benefit (together with an immune checkpoint inhibitor) for the treatment of metastatic non-small cell lung carcinoma (NSCLC) progressing on or after platinum-based therapy. The current study aimed to uncover cellular response mechanisms to TTFields. Methods: A2780 ovarian cancer cells and H1299 NSCLC cells were treated with TTFields for 72 h (200 and 150 kHz, respectively; 1.7 V/cm RMS). Changes in signaling pathways were analyzed using the Luminex multiplex assay and validated by protein expression. Fluorescence microscopy, calcium switch assay, antibody neutralization assay, and immunoprecipitation were employed to investigate the mechanism of action. The efficacy of concomitant treatment with TTFields and a potential inhibitor was tested in cell lines (cytotoxic and clonogenic effects) and two orthotopic mouse models: MOSE-L-FFL ovarian cancer and LL/2 lung cancer. Immunohistochemistry was performed on tumor sections from both models. Results: Increased AKT phosphorylation was observed in the cancer cells treated with TTFields. N-cadherin, known to be involved in the activation of the PI3K/AKT pathway, was elevated in the membranes of cells treated with TTFields. Elimination of calcium ions required for N-cadherin homophilic ligation abrogated TTFields-induced AKT phosphorylation, whereas calcium supplementation restored AKT phosphorylation. Inhibition of N-cadherin-mediated cell-cell contacts by neutralizing antibody resulted in a significant reduction in TTFields-induced AKT phosphorylation. Pull-down assays with an anti-N-cadherin antibody demonstrated increased recruitment of the p85 regulatory subunit of PI3K to N-cadherin complexes following TTFields application. TTFields-induced activation of AKT could be mitigated in cell cultures as well as in animal models by application of the PI3K inhibitor alpelisib. Co-treatment with alpelisib also enhanced the effectiveness of TTFields in vitro and in vivo. Conclusions: The PI3K/AKT signaling pathway, activated via cell-cell N-cadherin interactions, plays a role in the cancer cell response to TTFields treatment. Inhibiting the PI3K/AKT pathway may potentially sensitize tumors to TTFields. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Kerem Wainer-Katsir, Alexandra Volodin, Bella Koltun, Boris Brant, Yiftah Barsheshet, Tal Kan, Cfir David, Tharwat Haj Khalil, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. N-cadherin-mediated activation of PI3K/Akt pathway following application of tumor treating fields (TTFields) [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 2030.

Abstract 351: Quaratusugene ozeplasmid mediated TUSC2 upregulation in EML4-ALK bearing non-small cell lung carcinoma can induce cellular apoptosis

Abstract Anaplastic Lymphoma Kinase (ALK), a potent oncogenic driver in Non-Small Cell Lung Carcinoma (NSCLC), is found to be rearranged and fused to Echinoderm microtubule-associated protein-like 4 (EML4), contributing to approximately 5% of NSCLC as a distinct clinicopathological subset. Tumors bearing the EML4-ALK fusion are sensitive to ALK Tyrosine Kinase Inhibitors (TKIs), that form the first and second line of treatment for these patients. However, ALK+ lung cancers develop resistance to ALK inhibitors, creating the need for newer treatment strategies in ALK+ NSCLC. Tumor Suppressor Candidate 2 (TUSC2) is a tumor suppressor gene that is known to have low endogenous expression in NSCLC in general but data on TUSC2 in ALK+ lung cancers are not available. Quaratusugene ozeplasmid, developed by Genprex, is an immunogene therapy that upregulates TUSC2 expression in cancer cells by delivering the functional TUSC2 gene. It consists of the TUSC2 gene expressing plasmid encapsulated in non viral lipid nanoparticles. We evaluated TUSC2 expression in 3 ALK+ cell lines, both before and after exposure to quaratusugene ozeplasmid and to a TUSC2-containing plasmid. Controls were non-ALK+ cell lines and transfection with a plasmid containing no insert. Our studies in ALK+ lung cancer reveal that overexpressing TUSC2 using quaratusugene ozeplasmid treatment of ALK+ lung cancer cell lines is able to suppress colony formation by 50%, the effect being more significant than using a TUSC2-containing plasmid. Furthermore, we have observed a robust pro-apoptotic response to TUSC2 expression in ALK+ NSCLC, as both quaratusugene ozeplasmid and TUSC2-containing plasmid induced an increase in caspase 3/7 activity in the cancer cells, accompanied by an increase in cleaved PARP expression. Taken together, our data indicate that overexpression of TUSC2 in ALK+ NSCLC cell lines with quaratusugene ozeplasmid or with TUSC2-containing plasmid is effective in decreasing growth and proliferation through the activation of apoptotic pathways and warrants further investigation as an anti-ALK NSCLC strategy. Citation Format: Ananya Banerjee, Neeke Busette, Mark S. Berger, Matthew B. Soellner, Angel Qin, Sofia D. Merajver, Nathan M. Merrill. Quaratusugene ozeplasmid mediated TUSC2 upregulation in EML4-ALK bearing non-small cell lung carcinoma can induce cellular apoptosis [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 351.

TRIM14 suppressed the progression of NSCLC via hexosamine biosynthesis pathway.

Tripartite Motif 14 (TRIM14) is an oncoprotein that belongs to the E3 ligase TRIM family, which is involved in the progression of various tumors except for non-small-cell-lung carcinoma (NSCLC). However, little is currently known regarding the function and related mechanisms of TRIM14 in NSCLC. Here, we found that the TRIM14 protein was downregulated in lung adenocarcinoma tissues compared with the adjacent tissues, which can suppress tumor cell proliferation and migration both in vitro and in vivo. Moreover, TRIM14 can directly bind to glutamine fructose-6-phosphate amidotransferase 1 (GFAT1), which in turn results in the degradation of GFAT1 and reduced O-glycosylation levels. GFAT1 is a key enzyme in the rate-limiting step of the hexosamine biosynthetic pathway (HBP). Replenishment of N-Acetyl-D-glucosamine can successfully reverse the inhibitory effect of TRIM14 on the NSCLC cell growth and migration as expected. Collectively, our data revealed that TRIM14 suppressed NSCLC cell proliferation and migration through ubiquitination and degradation of GFAT1, providing a new regulatory role for TRIM14 on HBP.

Detection of MiRNA-126-5p in Non-Small Cell Lung Cancer Cells by Nano-Polymerase Chain Reaction and Its Regulatory Mechanism of Radiation Sensitivity to Cancer Cells

Radiotherapy resistance remains a huge hindrance in treating non-small cell lung carcinoma (NSCLC). Hyperthermia, a treatment method that raises the cell temperature to treat tumors, has been putting in clinical application combined with conventional chemotherapy and radiotherapy to enhance their effectiveness in NSCLC treatment. However, the specific mechanism of this combination therapy has not been extensively researched. In this study, we established a radiation-resistant NSCLC cell line by sequential radiation exposure. It was shown that the combination of hyperthermia and radiotherapy suppressed NSCLC xenograft tumor growth and increased radiation-induced apoptosis. Furthermore, downregulation of miR-126-5p was found in radio-resistant NSCLC cells. The results of bioinformatics analysis based on ENCORI showed that RAD50 is a direct target of miR-126-5p. Overexpressed miR-126-5p increased radiation-induced cell death by suppressing RAD50 expression. Hyperthermia treatment increases miR-126-5p and decreases RAD50, leading to more unrepaired DNA damage and greater cellular death. In conclusion, hyperthermia enhances NSCLC cells’ radio-sensitivity via miR-126-5p/RAD50 axis.

Progression of Non-Small-Cell Lung Carcinoma is Aggravated by LINC01410-Induced Upregulation of PGK1 via the Sponge Effect on miR-4523

This study investigates the role of LINC01410 in non-small-cell lung carcinoma (NSCLC) progression and the underlying mechanisms. Differential expressions of LINC01410, miR-4523, and PGK1 were examined in NSCLC and normal specimens using qRT-PCR. Prognostic and diagnostic potentials of LINC01410 were assessed via Kaplan-Meier and ROC analyses. Proliferation and migration rates in LINC01410-manipulated A549 and H1299 cells were evaluated using CCK-8 and transwell assays. Binding relationships between LINC01410/miR-4523 and miR-4523/PGK1 were determined through luciferase assays and correlation tests. Rescue experiments were conducted to elucidate the LINC01410/miR-4523/PGK1 axis’s role in NSCLC cell behavior regulation. LINC01410 and PGK1 were upregulated in NSCLC specimens, while miR-4523 was downregulated. High LINC01410 levels predicted poor NSCLC prognosis, with diagnostic potential confirmed by ROC curves. LINC01410 overexpression significantly enhanced A549 and H1299 cell proliferation and migration. LINC01410 acted as a miR-4523 sponge, and miR-4523 targeted PGK1. PGK1 overexpression partially attenuated LINC01410-induced NSCLC cell proliferation and migration. In conclusion, LINC01410 drives NSCLC cell proliferation and migration by upregulating PGK1 through miR-4523 sequestration.

Kaempferol Promotes Lung Cancer Cell Autophagy

Autophagy is a cellular process involved in the degradation and recycling of cellular components, and it plays a complex role in cancer. In the context of lung cancer, including lung cell cancer, the relationship between autophagy and cancer is multifaceted and can vary depending on the specific circumstances. Wang and colleagues investigated the molecular mechanism by which kaempferol (from Hedyotis diffusa) promotes autophagy in Non-Small Cell Lung Carcinoma (NSCLC) cells. Their results suggest that kaempferol significantly inhibits NSCLC cell proliferation. Kaempferol also caused NSCLC cell death by promoting NSCLC cell autophagy. Their experiments reveal that both protein and mRNA expression of Met was significantly inhibited. They also observed the inhibition of PI3K/AKT/mTOR signaling pathway. These results were validated by the overexpression of Met which resulted in the reversal of kaempferol effects on NSCLC cell viability and cell autophagy. They concluded that Kaempferol exerts its anti-NSCLC effect by promoting NSCLC cell autophagy which involves Met and its downstream PI3K/AKT/mTOR signaling pathway. Phytomedicine. 2023 Dec:121:155090. doi: 10.1016/j.phymed.2023.155090.

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance.

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5-2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed invivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.

Exosomes secreted by metastatic cancer cells promotes epithelial mesenchymal transition in small cell lung carcinoma: The key role of Src/TGF-β1 axis

Exosome-mediated epithelial mesenchymal transition (EMT) is key to cancer metastasis. c-Src is involved in the secretion of exosomes and initiation of EMT. Effects of exosomes from metastatic non-small cell lung carcinoma (NSCLC) cells on the EMT process in primary NSCLC cells were assessed. Levels of c-Src in NSCLC tissues were detected and the influence of exosomes from metastatic NSCLC cells on the exosome secretion and EMT process in primary NSCLC cells was assessed. The expression of c-Src was modulated, and the influence on the secretion of exosomes and EMT initiation was evaluated. The level of c-Src was higher in NSCLC specimen and NSCLC cells with promoted EMT process. The suppression of c-Src inhibited secretion of exosomes. Exosomes from metastatic NSCLC cells enhanced migration and invasion abilities of primary NSCLC cells, which had identical effects to c-Src overexpression. The suppression of c-Src inhibited growth and metastasis of solid tumors as well as secretion of exosomes, while the injection of exosomes with c-Src overexpression promoted lung metastasis. TGF-β1 restored the invasion and migration abilities even with c-Src knockdown. The exosomes from metastatic NSCLC cells with high c-Src expression of can increase c-Src level in primary NSCLC cells, contributing to the promoted EMT process through TGF-β1 pathway.

The effect of PLK1 inhibitor in Osimertinib resistant NCI-H1975 cell lines.

To investigate the effects of PLK1 inhibitors on Osimertinib-resistant NCI-H1975 cell lines and the anti-tumor effect combined with Osimertinib. Osimertinib resistant NCI-H1975 cell lines were constructed by the methods of concentration-increasing.; The classical tumor pathway inhibitor library compounds were combined with Osimertinib on Osimertinib-resistant cells to screen compounds with synergistic effects with Osimertinib; The gene set enrichment analysis (GSEA) was used to investigate the activations of signaling pathways in Osimertinib-resistant cells; Sulforhodamine B (SRB) staining was used to investigate the inhibitory effect of PLK1 inhibitors on Osimertinib-resistant cells and the anti-tumor effect of PLK1 inhibitors combined with Osimertinib. Osimertinib-resistant cells lines H1975-resistance (resistance index=43.45) were successfully established. The PLK1 inhibitors BI 2536 and GSK461324 have synergistic effect with Osimertinib. Compared with Osimertinib-sensitive cells, PLK1 regulatory pathway and cell cycle pathway were significantly activated in Osimertinib-resistant cells. In a cohort of NSCLC patients with epidermal growth factor receptor mutations treated with Osimertinib, PLK1 mRNA levels were negatively correlated with progression free survival of patients (R=0.62, P<0.05), confirming that excessive activation of PLK1 in NSCLC cells may cause cell resistance to Osimertinib. Further vitro experiments showed that, The IC50 of PLK1 inhibitors BI 6727 and GSK461324 in Osimertinib-resistant cells were lower than those in sensitive ones. Compared to the mono treatment of Osimertinib, PLK1 inhibitors combined with Osimertinib behave significantly stronger effect on the proliferation of Osimertinib-resistant cells. PLK1 inhibitors combined with Osimertinib behave stronger anti-tumor effect to Osimertinib-resistant NSCLC cells and may be used for intervention and treatment of Osimertinib resistance.

Deubiquitinating enzyme JOSD2 affects susceptibility of NSCLC cells to anti-cancer drugs through DNA damage repair.

To investigate the effects of deubiquitinating enzyme Josephin domain containing 2 (JOSD2) on susceptibility of non-small cell lung carcinoma (NSCLC) to anti-cancer drugs and its mechanism. The transcriptome expression matrix and clinical data of NSCLC were downloaded from the Gene Expression Omnibus database. Principal component analysis and limma analysis were used to investigate the deubiquitinating enzymes up-regulated in NSCLC tissues. Kaplan-Meier analysis was used to investigate the relationship between the expression of deubiquitinating enzymes and overall survival of NSCLC patients. Gene ontology enrichment and gene set enrichment analysis (GSEA) were used to analyze the activation of signaling pathways in NSCLC patients with high expression of JOSD2. Gene set variation analysis and Pearson correlation analysis were used to investigate the correlation between JOSD2 expression levels and DNA damage response (DDR) pathway. Western blotting was performed to examine the expression levels of JOSD2 and proteins associated with the DDR pathway. Immunofluorescence and quantitative analysis were used to detect the sub-localization of JOSD2. Sulforhodamine B staining was used to examine the sensitivity of JOSD2-knock-down NSCLC cells to DNA damaging drugs. Compared with adjacent tissues, the expression level of JOSD2 was significantly up-regulated in NSCLC tissues (P<0.05); and the expression of JOSD2 was significantly correlated with poor prognosis in NSCLC patients (P<0.05). Compared to tissues with low expression of JOSD2, the DDR-related pathways were significantly activated in NSCLC tissues with high expression of JOSD2 (P<0.05). In addition, the expression of JOSD2 was positively correlated with the activation of DDR-related pathways (P<0.01). Compared with control group, overexpression of JOSD2 significantly promoted the DDR in NSCLC cells. In addition, DNA damage agent significantly increase the nuclear localization of JOSD2, whereas depletion of JOSD2 significantly enhanced the sensitivity of NSCLC cells to DNA damaging agents (P<0.05). Deubiquitinating enzyme JOSD2 regulates the malignant progression of NSCLC by promoting DNA damage repair pathway, and depletion of JOSD2 significantly enhances the sensitivity of NSCLC cells to DNA damage agents.

MicroRNA‑27a‑3p regulates the proliferation and chemotaxis of pulmonary macrophages in non‑small cell lung carcinoma tissues through CXCL2.

The present study aimed to investigate microRNA (miRNA)-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with early non-small cell lung carcinoma (NSCLC) and its regulatory effect on the infiltration of pulmonary macrophages into cancer tissues and invasion of NSCLC cells. Blood specimens were withdrawn from 36 patients with NSCLC and 29 healthy subjects. NSCLC tissues and cancer-adjacent tissues were both obtained from patients with NSCLC; furthermore, certain tissue samples were used to extract macrophages. The levels of miRNA-27a-3p and C-X-C motif ligand chemokine 2 (CXCL2) mRNA were detected by reverse transcription-quantitative PCR and the levels of CXCL2 protein were measured by ELISA and western blot analysis. A dual-luciferase reporter assay was performed to determine the interactions between miRNA and mRNA. An MTT assay was employed to examine the viability of transfected cells and macrophages and a Transwell assay was performed to assess chemotaxis. The differential expression of miRNA-27a-3p in NSCLC tissues, pulmonary macrophages and peripheral blood indicated that miRNA-27a-3p exerted different roles in these specimens. CXCL2 was upregulated in NSCLC tissues at both transcriptional and translational levels. In addition, the untranslated region of CXCL2 was confirmed to be directly targeted by miRNA-27a-3p prior to its transcriptional activation. Furthermore, miRNA-27a-3p regulated CXCL2 expression, thereby affecting the proliferation of human pulmonary macrophages. The present study highlights that miRNA-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with NSCLC is downregulated, while its target gene CXCL2 is upregulated. miRNA-27a-3p may regulate the viability and chemotaxis of macrophages in tumor tissues of patients with NSCLC through CXCL2 and is expected to become a genetic marker of this disease.

Upregulation of MCL-1 by LUCAT1 through interacting with SRSF1 promotes the migration and invasion in non-small cell lung carcinoma.

The onset of non-small cell lung carcinoma (NSCLC) still be in the fog. LUCAT1 is potentially capable of modulating MCL-1-involved NSCLC pathogenesis via targeting SRSF1. Also, MCL-1 can regulate Wnt/β-catenin pathway to affect the tumorigenesis of NSCLC. Thus, this paper aims to uncover an intriguing and novel role of LUCAT1/SRSF1/MCL-1 axis in NSCLC based on Wnt/β-catenin pathway. A549 and NCI-H1650, two cell lines of NSCLC, were used to mimic NSCLC in vitro. MCL-1 siRNA (si-MCL-1) and LUCAT1 siRNA (si-LUCAT1) were used to downregulate MCL-1 and LUCAT1 in NSCLC cells, respectively. The overexpression vector of SRSF1 based on pcDNA 3.1 was constructed to upregulate SRSF1 expression. 40 μM SKL2001 was used to activate Wnt/β-catenin pathway. Transwell assay was used for migrative and invasive tests. The effect of LUCAT1 on tumor metastasis was verified in nude mice. MCL-1 downregulation led to the decrease of EMT, invasion, and migration in NSCLC, while Wnt/β-catenin pathway agonist partially reversed the effects of MCL-1 downregulation. Mechanistic investigations revealed that LUCAT1 and MCL-1 mRNA were enriched in SRSF1; LUCAT1 silence decreased MCL-1, whereas SRSF1 enhancement elevated MCL-1; Importantly, SRSF1 overexpression significantly reversed MCL-1 alteration due to LUCAT1 silence. In NSCLC cells, SRSF1 overexpression offset the si-LUCAT1-induced changes, and si-MCL-1 reversed the SRSF1-induced cellular changes. Further, LUCAT1 inhibition reduced lung metastasis of cancer cells. LUCAT1 can interact with SRSF1 to regulate MCL-1 expression that targets the Wnt/β-catenin pathway-mediated NSCLC cell migration, invasion, and EMT.

EGFR suppression contributes to growth inhibitory activity of G-quadruplex ligands in non-small cell lung cancers

Non-small cell lung carcinomas (NSCLCs) commonly harbor activating mutations in the epidermal growth factor receptor (EGFR). Drugs targeting the tyrosine kinase activity of EGFR have shown effectiveness in inhibiting the growth of cancer cells with EGFR mutations. However, the development of additional mutations in cancer cells often leads to the persistence of the disease, necessitating alternative strategies to overcome this challenge. We explored the efficacy of stabilizing the G-quadruplex structure formed in the promoter region of EGFR as a means to suppress its expression and impede the growth of cancer cells with EGFR mutations. We revealed that the carbazole derivative BMVC-8C3O effectively suppressed EGFR expression and demonstrated significant growth inhibition in EGFR-mutated NSCLC cells, both in cell culture and mouse xenograft models. Importantly, the observed repression of EGFR expression and growth inhibition were not exclusive to carbazole derivatives, as several other G-quadruplex ligands exhibited similar effects. The growth-inhibitory activity of BMVC-8C3O is attributed, at least in part, to the repression of EGFR, although it is possible that additional cellular targets are also affected. Remarkably, the growth-inhibitory effect was observed even in osimertinib-resistant cells, indicating that BMVC-8C3O holds promise for treating drug-resistant NSCLC. Our findings present a promising and innovative approach for inhibiting the growth of NSCLC cells with EGFR mutations by effectively suppressing EGFR expression. The demonstrated efficacy of G-quadruplex ligands in this study highlights their potential as candidates for further development in NSCLC therapy.

Histone demethylase KDM6A coordinating with KMT2B regulates self-renewal and chemoresistance of non-small cell lung cancer stem cells

Background and aimsWnt signaling is essential for the maintenance of cancer stem cells (CSCs), but mutations in the β-catenin and APC genes are less common in non-small cell lung carcinoma (NSCLC). Thus, the mechanism underlying the constitutive activation of Wnt signaling in lung CSCs is still unknown. Materials and methodsGene set enrichment analysis and immunohistochemistry were performed to establish the correlation between KDM6A/KM2B and CSC stemness. Human NSCLC cell lines were genetically manipulated for functional studies. Sphere formation assay and stemness gene expression profiling were examined to investigate the role of KDM6A/KMT2B in lung CSCs. Tumor xenograft assay were used to identify the function of KDM6A/KMT2B on tumorigenicity and tumor recurrence in vivo. Western blot analysis, coimmunoprecipitation and chromatin immunoprecipitation were performed to understand KDM6A/KMT2B mediated epigenetic regulation of Histone 3 lysine 4 methylation (H3K4me) on Wnt signaling pathway. ResultsWe discovered that the expression of Histone demethylase KDM6A and methyltransferase KMT2B correlate with the stemness of CSCs in NSCLC. KDM6A coordinates with KMT2B to activate the Wnt/β-catenin signaling pathway by regulating the H3K4me3 level and promotes the tumorigenicity and maintenance of CSC stemness. Furthermore, KDM6A/ KMT2B overexpression promotes the CSC chemoresistance and tumor recurrence both in vitro and in vivo. Inhibition of KDM6A and KMT2B potently suppress tumor initiation and recurrence in xenografted animal models. ConclusionOur findings suggest that KDM6A and KMT2B mediate the constitutive activation of Wnt/β-catenin signaling in lung CSCs, potentially providing a therapeutic target for NSCLC.

FSP1 confers ferroptosis resistance in KEAP1 mutant non-small cell lung carcinoma in NRF2-dependent and -independent manner

Ferroptosis, a type of cell death induced by lipid peroxidation, has emerged as a novel anti-cancer strategy. Cancer cells frequently acquire resistance to ferroptosis. However, the underlying mechanisms are poorly understood. To address this issue, we conducted a thorough investigation of the genomic and transcriptomic data derived from hundreds of human cancer cell lines and primary tissue samples, with a particular focus on non-small cell lung carcinoma (NSCLC). It was observed that mutations in Kelch-like ECH-associated protein 1 (KEAP1) and subsequent nuclear factor erythroid 2-related factor 2 (NRF2, also known as NFE2L2) activation are strongly associated with ferroptosis resistance in NSCLC. Additionally, AIFM2 gene, which encodes ferroptosis suppressor protein 1 (FSP1), was identified as the gene most significantly correlated with ferroptosis resistance, followed by multiple NRF2 targets. We found that inhibition of NRF2 alone was not sufficient to reduce FSP1 protein levels and promote ferroptosis, whereas FSP1 inhibition effectively sensitized KEAP1-mutant NSCLC cells to ferroptosis. Furthermore, we found that combined inhibition of FSP1 and NRF2 induced ferroptosis more intensely. Our findings imply that FSP1 is a crucial suppressor of ferroptosis whose expression is partially dependent on NRF2 and that synergistically targeting both FSP1 and NRF2 may be a promising strategy for overcoming ferroptosis resistance in cancer.

Exosomal ERBB2IP contributes to tumor growth via elevating PSAT1 expression in non-small cell lung carcinoma.

Both exosomes and circular RNAs (circRNAs) are involved in tumor growth. Hsa_circ_0001492 (circERBB2IP) has been reported to be overexpressed in plasma exosomes from patients with lung adenocarcinoma, but the biological role of exosomal circERBB2IP in non-small cell lung carcinoma (NSCLC) is indistinct. Exosomes isolated from serums and medium samples were validated by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. Relative expression of circERBB2IP was detected by RT-qPCR. Loss-of-function was done to determine the effect of circERBB2IP on NSCLC cell proliferation and migration. Molecular mechanisms associated with circERBB2IP were predicted by bioinformatic analysis and validated by dual-luciferase reporter, RIP, and RNA pulldown assays. In vivo experiments were performed to identify the function of circERBB2IP in NSCLC. We discovered that circERBB2IP expression was correlated with TNM grade, lymph node metastasis and tumor size of NSCLC patients. Upregulation of circERBB2IP was observed in exosomes derived from NSCLC patient's serum and circERBB2IP might be a potential diagnostic biomarker for NSCLC. CircERBB2IP was transmitted between carcinoma cells through exosomes. Knockdown of circERBB2IP lowered cell growth in mouse models and restrained NSCLC cell proliferation and migration. CircERBB2IP could mediate PSAT1 expression via sponging miR-5195-3p. In conclusion, circERBB2IP may drive NSCLC growth by the miR-5195-3p/PSAT1 axis in NSCLC, shedding light on a diagnostic biomarker and therapeutic target for NSCLC.

Abstract B026: Overcoming KRAS G12C inhibitor resistance with chaperone-mediated protein degrader in NSCLC

Abstract KRAS-G12C targeting inhibitors (G12Cis), such adagrasib and sotorasib, have shown specificity and promising anti-tumor potential in non-small cell lung carcinoma (NSCLC). However, acquired resistance to these inhibitors has recently been observed in the clinic. Mechanisms of resistance to G12Cis are associated with genetic alterations of several pro-growth signaling pathways. A strategy to overcome the multifarious resistance mechanisms to G12Cis that does not require further molecular characterization is needed. To develop an all-round strategy to overcome G12Ci resistance mechanisms, we exposed NCI-H358, NCI-H1373, and MIAPaCa2 cell lines harboring KRAS-G12C mutations to increasing concentrations of sotorasib or adagrasib over 6 months. The resulting resistant cells proliferate normally in the presence of 10 μM sotorasib or adagrasib. Sotorasib-resistant cells designated as NCI-H358S-R, NCI-H1373S-R, and MIAPaCa2S-R remarkably remain sensitive to adagrasib, suggesting but not proving that the resistant cells remain dependent on mutant KRAS for survival. Adagrasib-resistant cells designated as NCI-H358A-R, NCI-H1373A-R, and MIAPaCa2A-R in contrast remain resistant to both sotorasib and adagrasib. Treatment of adagrasib-resistant cells with 500 nM adagrasib attenuated KRAS activity, however, this was compensated by activation of varied receptor tyrosine kinases (RTKs) to restore phospho-ERK levels, a surrogate marker of RAS activity. Moreover, treatment of de novo resistant NSCLC cells lines NCI-H2030, NCI-H1792, and NCI-H23 or NSCLC patient derived organoids (PDOs) with 500 nM adagrasib resulted in sustained phospho-ERK levels through activation of RTKs. These results indicate the need to simultaneously reduce both KRAS G12C and compensating RTK activity to effectively treat NSCLC. The majority of oncogenic RTKs are dependent on the molecular chaperone HSP90 to maintain stability. Therefore, a heterobifunctional chaperone mediated protein (CHAMP) degrader, RNK07421, was designed to specifically target both KRAS G12C and HSP90. RNK07421 induces an artificial ternary complex between KRAS G12C and HSP90, while also inhibiting the activity of both proteins. The RNK07421 coordinated KRAS G12C-HSP90 interaction promotes the proteasome degradation of KRAS G12C likely via HSP90 associated E3-ligases. Treatment of adagrasib resistant NSCLC cells and PDOs with RNK07421 resulted in reduced levels of KRAS G12C and phospho-ERK along with resistance enabling RTKs: EGFR, BRAF and FGFR2. Furthermore, in vivo treatment of resistant NSCLC cells with RNK07421 demonstrated dramatic tumor reduction as compared to G12Ci. Together these observations indicate that the novel mechanisms of action of RNK07421 may provide several advantages to G12Cis and possibly other targeted protein degradation agents to effectively treat KRAS G12C dependent NSCLC. Citation Format: Ines Pulido, Jennifer M. Graeber, Brianna C. Borger, Kevin P. Foley, Weiwen Ying, Chenghao Ying, Jinhua Li, Yaya Wang, Yuetong Sun, Yan Dai, Guoqiang Wang, Thomas L. Prince, Takeshi Shimamura. Overcoming KRAS G12C inhibitor resistance with chaperone-mediated protein degrader in NSCLC [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 B026.