Non-small Cell Lung Cancer Cells Research Articles

Epsin3 promotes non-small cell lung cancer progression via modulating EGFR stability

BackgroundThe abnormal expression and overactivation of the epidermal growth factor receptor (EGFR), a typical cancer marker for non-small cell lung cancer (NSCLC), are closely related to the tumorigenesis and progression of NSCLC. However, the endocytosis mechanism of EGFR in lung cancer is not yet known. Epsin3 (EPN3), a member of the endocytic adaptor protein family, is essential for the endocytosis of multiple receptors. In this study, we aimed to investigate the role of EPN3 in modulating EGFR function, its effects on NSCLC progression, and its potential involvement in tyrosine kinase inhibitor (TKI) resistance, which remains a significant hurdle in NSCLC treatment.ResultsOur findings revealed that the expression of EPN3 is significantly up-regulated in NSCLC patients. Elevated EPN3 expression was proportional to shorter overall survival in patients with NSCLC. Functional analyses revealed that EPN3 directly interacts with EGFR, enhancing its recycling to the plasma membrane and preventing its degradation via the lysosomal pathway. This stabilization of EGFR led to sustained downstream signalling, promoting NSCLC cell proliferation and migration. Notably, mutations in the EGFR tyrosine kinase domain, which typically confer resistance to TKIs, did not alter the regulatory effect of EPN3.ConclusionsEPN3 enhances EGFR signalling by promoting its recycling and stability, contributing to NSCLC progression and TKI resistance. Targeting EPN3 could offer a novel therapeutic strategy to overcome drug resistance in EGFR-driven NSCLC.

The PDE4DIP-AKAP9 axis promotes lung cancer growth through modulation of PKA signalling

Phosphodiesterase 4D interacting protein (PDE4DIP) is a Golgi/centrosome-associated protein that plays critical roles in the regulation of microtubule dynamics and maintenance of the Golgi structure. However, its biological role in human cancer remains largely unknown. In this study, we showed that PDE4DIP is overexpressed in human non-small cell lung cancer (NSCLC) tissues and that upregulated PDE4DIP expression is associated with poor prognosis in patients with lung cancer. We demonstrated that PDE4DIP knockdown inhibits NSCLC cell proliferation in vitro and tumorigenicity in vivo. We further demonstrated that PDE4DIP knockdown triggers apoptosis and cell cycle arrest in NSCLC cells by activating the Protein kinase A (PKA) /CREB signalling pathway. PDE4DIP coordinates with A-kinase anchoring proteins 9 (AKAP9) to enhance the Golgi localization and stability of PKA RIIα. Depletion of PDE4DIP mislocalizes PKA RIIα from the Golgi and leads to its degradation, thereby compromising its negative regulatory effect on PKA signalling. Overall, our findings provide novel insights into the roles of the PDE4DIP-AKAP9 complex in regulating PKA signalling and NSCLC growth and highlight PDE4DIP as a promising therapeutic target for NSCLC.

Carnitine palmitoyltransferase 1C promotes EMT-associated cisplatin resistance in non-small cell lung cancer cells.

Lung cancer is the most common cause of cancer-related deaths worldwide. Platinum-based chemotherapy is one of the main treatment options for patients with non-small cell lung cancer (NSCLC) but the effectiveness of chemotherapy is encumbered by drug resistance. Therefore, understanding the molecular mechanisms underlying chemotherapy resistance is crucial in improving treatment outcomes and prognosis. The cell viability assay and apoptosis were used to analyze chemoresistance. Western blot analysis and wound healing testing were used to evaluate the epithelial-to-mesenchymal transition (EMT). Immunoprecipitation was used for analysis of protein modification. Promoter activity was determined using the luciferase reporter assay. Immunofluorescence staining was used to determine reactive oxygen species levels. The expression patterns of EMT markers and carnitine palmitoyltransferase 1C (CPT1C) were determined by Western blot analysis. CPT1C, which was shown to be highly expressed in lung cancer, is associated with cisplatin resistance in NSCLC cells. CPT1C depletion increased NSCLC cell sensitivity to cisplatin, while overexpression of CPT1C increased NSCLC cell resistance to cisplatin. Induction of EMT mediated CPT1C-induced cisplatin resistance. Ectopic expression of Snail reversed the increase in cisplatin sensitivity triggered by CPT1C knockdown. Moreover, CPT1C was shown to be regulated at the post-translational level and an E3-ubiquitin ligase, NEDD4L, was shown to be a major regulator of CPT1C stability and activity. These data provide evidence for the first time that the lipid metabolism enzyme, CPT1C, mediates resistance to chemotherapy. Therefore, the use of combination therapy with a CPT1C inhibitor may be a promising new avenue in lung cancer treatment.

AXL-Mediated Drug Resistance in ALK-Rearranged NSCLC Enhanced by GAS6 From Macrophages and MMP11 Positive Fibroblasts.

Anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer (NSCLC) shows marked tumor shrinkage by ALK-tyrosine kinase inhibitors (TKIs). However, tumors almost inevitably relapse owing to the development of acquired resistance. Resistance mechanisms include secondary ALK mutations and the activation of bypass pathways, such as cMET, cKIT, or EGFR, though some remain unknown. In this study, we analyzed alectinib-resistant patient samples and identified a significant increase in AXL expression in the tumor, and a high level of GAS6, the ligand for AXL, in the pleural effusion. AXL-overexpressing H3122 ALK-rearranged NSCLC cells exhibited partial resistance to alectinib, which was enhanced by GAS6 supplementation but could be overcome by the ALK/AXL inhibitor gilteritinib. Moreover, GAS6-overexpressing NIH3T3 cells and AXL-expressing H3122 cells were subcutaneously injected into the left and right sides of nude mice simultaneously, followed by alectinib treatment. The supply of GAS6 from NIH3T3 may have accelerated tumor relapse under alectinib treatment. However, even without GAS6-overexpressing NIH3T3, AXL-overexpressing H3122 tumor relapsed within 1 month possibly due to increased host mouse Gas6 expression. Single-cell RNA sequencing revealed that specific cancer-associated fibroblasts (CAFs) and a subset of tumor-associated macrophages (TAMs) are the primary sources of Gas6 in the tumor microenvironment (TME). During alectinib treatment, TAMs increased their infiltration into the TME, whereas CAFs altered their expression patterns, substantially upregulating Mmp11. These findings suggest that AXL expression in resistant cancer cells, combined with increased Gas6 production in the TME, contributes to enhanced ALK-TKI resistance.

Targeting HVEM-GPT2 axis: a novel approach to T cell activation and metabolic reprogramming in non-small cell lung cancer therapy.

The modulation of tumor microenvironments through immune checkpoint pathways is pivotal for the development of effective cancer immunotherapies. This study aims to explore the role of HVEM in non-small cell lung cancer (NSCLC) microenvironment. The lung cancer datasets for this study were directly downloaded from The Cancer Genome Atlas (TCGA). Single-cell data were sourced from the Tumor Immune Single-cell Hub (TISCH). Multiplex immunohistochemistry (mIHC) was used to explore the cellular composition and spatial distribution of HVEM in lung cancer immune microenvironment. Theimmunemicroenvironmentof HVEM KO mice bearing mouse lung cancer cell was also evaluated. Co-cultured system and phenotype assays facilitated the examination of Jurkat T cells' effect on A549 and H1299 lung cancer cells. Quantitative PCR and Western blotting determined gene and protein expression, respectively, cellular respiration was measured through oxygen consumption rate (OCR) assays. Lung cancer cells co-cultured with Jurkat T cells were xenografted into nude mice to evaluate tumor growth and metastatic potential. Next, RNA-seq, COIP, Dual-luciferasereporter experiment, and CHIP-seq were used to explore the potential underlying mechanism. In our study, we investigated the role of HVEM in the microenvironment of NSCLC and its implications in immunotherapy. Crucially, HVEM, part of the tumor necrosis factor receptor superfamily, influences T cell activation, potentially impacting immunotherapeutic outcomes. Using the TIDE algorithm, our results showcased a link between HVEM levels and immune dysfunction in NSCLC patients. Delving deeper into the NSCLC microenvironment, we found HVEM predominantly expressed in T cell subpopulations. CD8 + HVEM + and CD4 + HVEM + indicated better prognosis in lung adenocarcinoma tissue microarray using multiplex immunohistochemistry. Activated T cells, particularly from the Jurkat cell line, significantly inhibited NSCLC progression, reducing both proliferation and invasion capabilities of A549 and H1299 lung cancer cell lines. In vivo models reinforced these observations. Manipulating HVEM expression revealed its essential role in T cell survival and activation. In addition, animal experiments revealed the importance of HVEM in maintaining activated peripheral immunity and inflamed local tumor microenvironment. Furthermore, our data suggest that HVEM is pivotal in T cell metabolic reprogramming, transitioning from oxidative phosphorylation to aerobic glycolysis. RNA sequencing illuminated a potential relationship between HVEM and GPT2, an enzyme tied to amino acid metabolism and cellular energetics. Subsequent experiments confirmed that HVEM's influence on T cell activation and metabolism is potentially mediated through its regulation of GPT2. In addition, GATA1 was validated to regulate HVEM expression in activated Jurkat T cells. Our study establishes that HVEM significantly influences T cell functionality and NSCLC cell dynamics, pinpointing the HVEM-GPT2 axis as a promising target for NSCLC therapy.

Identification of prognostic biomarker of non-small cell lung cancer based on mitochondrial permeability transition-driven necrosis-related genes and determination of anti-tumor effect of ARL14

BackgroundMitochondrial permeability transition (MPT)-driven necrosis (MPTDN) is a non-apoptotic mode of cell death triggered by oxidative stress and cytosolic Ca2+ overload. Recent evidence suggests that activation of MPTND can effectively induce cancer cell death and may represent a novel therapeutic strategy for cancer. Yet, the role of MPTDN-related genes in non-small cell lung cancer (NSCLC) remains unrevealed. This study aimed to identify MPTDN-related biomarkers for predicting prognosis and guiding treatment in NSCLC.MethodsGene expression profiles and clinical information of NSCLC were collected from public databases, and MPTDN-related genes were obtained from published article. Differential expressed MPTDN-related genes in NSCLC and control were screened, and molecular clusters were obtained. Based on the differentially expressed genes (DGEs) between clusters, univariate Cox and LASSO regression analyses were performed to screen biomarkers, followed by nomogram construction. Correlations between these biomarkers and immune cell infiltration, immune checkpoints, and chemotherapeutic agents were observed. Expression levels of MPTDN-related biomarkers were detected using RT-qPCR in NSCLC tissues and cells. Moreover, the biological function of ARL14 in NSLCL was verified in vitro.ResultsThirty-five differential MPTDN-related genes were identified, and two molecular clusters were obtained. Three biomarkers with prognostic values were finally screened, including ARL14, ZDHHC11B, and HLF. Among them, ARL14 was significantly upregulated in tumor samples, while ZDHHC11B and HLF were downregulated. Nomogram containing three genes exhibited predictive accuracy in 1, 3, and 5-year survival rates. Three gene were strongly associated with most immune cells, immune checkpoints, and drugs sensitivity. RT-qPCR confirmed that expression levels of three genes in tissues or cells were consistent with the results of bioinformatics analysis. Finally, ARL14 knockdown inhibited the malignant phenotype of NSCLC cells.ConclusionWe first performed the comprehensive analysis of MPTDN in NSCLC and screened three NSCLC-related biomarkers as promising biomarkers. ARL14 might be a new potential target for therapy of NSCLC.

MFAP5 Strengthened the Stem Cell Features of Non-small Cell Lung Cancer Cells by Regulating the FBW/Sox9 Axis.

Non-small cell lung cancer (NSCLC) is a type of malignant tumor with high morbidity as well as mortality. The process of lung cancer may be driven by cancer stem cells. It was known that MFAP5 enhanced the occurrence of diverse types of cancer. Also, MFAP5 has the potential to induce the degradation of FBW7 which is a tumor suppressor. Lower levels of FBW7 enhance the stability of Sox9, which is the cancer stem cell-related protein. However, whether the MFAP5 can modulate the stem cell features of NSCLC cells by modulating the FBW7/Sox9 axis is unclear. Therefore, this study aimed to explore the role of MFAP5/FBW7/Sox9 axis on the stem cell features of NSCLC cells and develop a new treatment of this carcinoma. In this study, we explored the effects of MFAP5 on the stem cell features of NSCLC cells for the first time. We established MFAP5 overexpression and knockdown NSCLC cells. Clone formation assays and cell sphere culture assays were conducted for the exploration of the growth and stem cell features of these cells. Western blotting was applied for the detection of Sox9 and FBW7 expression in these cells. CHX was applied for the treatment of these cells for the detection of degradation of Sox9. Finally, we overexpressed the Sox9 in MFAP5 knockdown NSCLC cells. MFAP5 promoted the growth and stem cell features of these cells. Knockdown of MFAP5 induced higher levels of FBW7 while restricting the expression of Sox9. Knockdown of MFAP5 aggravated the degradation of Sox9. Overexpression of Sox9 abrogated the efficacy of MFAP5 inhibition on the growth as well as stem cell features of these cells. The results of this study clarified the role of MFAP5/FBW7/Sox9 axis on the development of non-small cell lung cancer cells, providing the potential therapeutic target for the clinical treatment of NSCLC. MFAP5 maintained the stem cell features of non-small cell lung cancer cells by modulating FBW7/Sox9 axis.

Acyclic Retinoid Inhibits the EGFR/AKT Signaling Pathway and Cancels Cisplatin-resistant Cell Characteristics.

Lung cancer is among the most prevalent and lethal malignancies worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Overactivation of the EGFR/AKT signaling pathway contributes significantly to NSCLC progression and metastasis. Cisplatin, a widely used chemotherapeutic agent, faces limitations due to severe side effects and the emergence of resistant cancer cells. Acyclic retinoid (ACR), a synthetic derivative of vitamin A, has shown antitumor effects in hepatocellular carcinoma, but its efficacy against NSCLC and cisplatin-resistant cells remains unclear. This study aimed to investigate whether ACR could inhibit EGFR/AKT signaling and enhance therapeutic efficacy against NSCLC and cisplatin-resistant cells. Human NSCLC A549 cells, cisplatin-resistant A549 (A549CR) cells, and normal lung epithelial BEAS-2B cells were treated with ACR, alone or in combination with cisplatin. Cell viability, apoptosis, and changes in expression/phosphorylation of EGFR, AKT, and cell cycle regulators were assessed using cell viability assay, immunostaining, and immunoblotting. ACR selectively reduced viability of A549 cells with less toxicity to BEAS-2B cells and induced apoptosis via cleaved Caspase-3 activation. ACR inhibited EGFR/AKT signaling and up-regulated p27KIP1 in A549 cells. The combination of ACR and cisplatin synergistically reduced cell viability and suppressed AKT phosphorylation. Notably, ACR also inhibited EGFR/AKT signaling in A549CR cells, restoring sensitivity to cisplatin and reversing EMT-like characteristics. ACR effectively inhibits EGFR/AKT signaling and enhances cisplatin sensitivity in NSCLC and cisplatin-resistant cells, suggesting its potential as a promising therapeutic strategy for lung cancer.

Molecular mechanisms of phytoconstituents from selected Egyptian plants against non-small cell lung cancer using integrated in vitro network pharmacology and molecular docking approach.

Non-small cell lung cancer (NSCLC) is a widespread highly malignant type of lung cancer. Conventional chemotherapeutic drugs may be accompanied by both drug resistance and serious side effects in patients. Therefore, safer and more effective medications are urgently needed for the treatment of NSCLC. This study investigates the mode of action of 21 phytoconstituents previously isolated from the Amaryllidaceous plants Crinum bulbispermum (Burm.f.), Pancratium maritimum L., and Hippeastrum vittatum Herbert alongside the Asteraceous plant Centaurea scoparia Sieb. for therapy of NSCLC via in vitro cytotoxic, network pharmacology, and molecular docking analyses. Despite the in vitro and in vivo cytotoxic studies carried out on phytoconstituents from these plants in treating numerous cancer types, scarce information documenting their cytotoxic activity towards NSCLC cells is available. First, the compounds were tested for their in vitro cytotoxic activities and selectivity on human non-small cell lung cancer cells using disk diffusion assay. Compounds having significant potencies were promoted for network pharmacology analysis. Pharm mapper, Genecards, STRING, and KEGG databases were utilized for surfing target genes and pathways for these compounds, while for construction of compound-target-pathway (C-T-P) network, Cytoscape 3.7.1. freeware was used. Molecular docking and dynamics simulation were run for the top hit constituents against the most enriched molecular targets followed by in silico ADMET studies using Schrodinger® suite and Gromacs. In vitro cytotoxicity testing demonstrated that crinamine was the most potent compound followed by lycorine, hemanthidine, and haemanthamine. The network pharmacology approach revealed the enrichment of acetyllycoramine, pluviine, 5-hydroxy-7-methoxy-2-methylchromone, and ismine. Whereas, androgen receptor (AR), epidermal growth factor receptor (EGFR), and estrogen-sensitive receptor alpha (ESR1) were the most enriched target genes. Pathway analysis revealed that central carbon metabolism, EGFR tyrosine kinase inhibitor endocrine resistance, and non-small cell lung cancer were the most enriched cancer-related pathways. Ismine possessed the most stable ligand-protein interactions when docked to the three proteins, with MD simulations further confirming its strong and consistent binding to AR, moderate stability with ESR-1, and lower stability with EGFR over the 100ns trajectory. ADMET study conducted on the above compounds confirmed their excellent drug-likeness properties, oral bioavailability, and safety profiles highlighting the need for some structural modifications to pluviine to enhance its oral bioavailability. These integrated approaches showed that some constituents from the investigated plants interact synergistically against non-small cell lung cancer-related genes and pathways.

The crosstalk between SND1 and PDCD4 is associated with chemoresistance of non-small cell lung carcinoma cells

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) is highly resistant to chemo- or radiation therapy, which poses a huge challenge for treatment of advanced NSCLC. Previously, we demonstrated the oncogenic role of Tudor Staphylococcal nuclease (TSN, also known as Staphylococcal nuclease domain-containing protein 1, SND1), in regulating chemoresistance in NSCLC cells. Here, we showed that silencing of SND1 augmented the sensitivity of NSCLC cells to different chemotherapeutic drugs. Additionally, the expression of PDCD4 (a tumor suppressor highly associated with lung cancer) in NSCLC cells with low endogenous levels was attenuated by SND1 silencing, implying that SND1 might function as a molecular regulator upstream of PDCD4. PDCD4 is differentially expressed in various NSCLC cells. In the NSCLC cells (A549 and H23 cells) with low expression of PDCD4, despite the downregulation of PDCD4, silencing of SND1 still led to sensitization of NSCLC cells to treatment with different chemotherapeutic agents by the inhibition of autophagic activity. Thus, a novel correlation interlinking SND1 and PDCD4 in the regulation of NSCLC cells concerning chemotherapy was revealed, which contributes to understanding the mechanisms of chemoresistance in NSCLC.

Lidocaine Inhibits the Proliferation of Non-Small Cell Lung Cancer and Exerts Anti-Inflammatory Effects Through the TLR-9/MyD88/NF-κB Pathway.

Lung cancer represents a significant global health burden, with non-small cell lung cancer (NSCLC) being the most common subtype. The current standard of care for NSCLC has limited efficacy, highlighting the necessity for innovative treatment options. Lidocaine, traditionally recognized as a local anesthetic, has emerged as a compound with potential antitumor and anti-inflammatory capabilities. This study was designed to explore the impact of lidocaine on NSCLC cell proliferation and inflammation, particularly focusing on the Toll-like receptor 9 (TLR)-9/MyD88/NF-κB signaling pathway. A nude mice model of NSCLC was employed, with animals receiving lidocaine at different concentrations. In vitro experiments on A549 cells involved exposure to lidocaine, followed by assessment of cell viability, cytokine expression, and TLR-9 levels using the 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, enzyme-linked immunosorbent assay, and Quantitative Real-time polymerase chain reaction (qPCR). Protein levels were evaluated via Western blot analysis. Additionally, A549 cells were transfected with a TLR-9-overexpressing lentivirus to dissect the role of TLR-9 in lidocaine's mechanism of action. Treatment with lidocaine led to a significant reduction in tumor dimensions and a decrease in inflammatory marker expression in the NSCLC mouse model. In cellular assays, lidocaine effectively suppressed A549 cell proliferation and the expression of inflammatory cytokines. The overexpression of TLR-9 partially negated the suppressive effects of lidocaine, underscoring the significance of the TLR-9/MyD88/NF-κB pathway in mediating lidocaine's effects. Lidocaine's inhibitory effects on NSCLC cell proliferation and its anti-inflammatory mechanisms are mediated through the TLR-9/MyD88/NF-κB pathway. The study's results offer promising insights into the therapeutic potential of lidocaine in NSCLC and pave the way for future investigations into its application in cancer therapy.

Design of ROS-Triggered Sesquiterpene Lactone SC Prodrugs as TrxR1 Covalent Inhibitors for the Treatment of Non-Small Cell Lung Cancer.

Thioredoxin reductase 1 (TrxR1) is an important therapeutic target for nonsmall cell lung cancer (NSCLC) treatment due to its overexpression in NSCLC cells. In this work, to address the deficiency that sesquiterpene lactone containing α-methylene-γ-lactone moiety was rapidly metabolized by endogenous nucleophiles, series of novel thioether derivatives were designed and synthesized based on a reactive oxygen species (ROS)-triggered prodrug strategy. Among them, prodrug 5u exhibited potent cytotoxicity against NSCLC cells and better release rates in response to ROS. The active compound 6a released from 5u covalently binds to Cys475 and Sec498 sites on TrxR1, resulting in inhibition on TrxR1 activity, which led to redox homeostasis disorder, and caused apoptosis and ferroptosis. Moreover, prodrug 5u exhibited significant antitumor efficiency in nude mice and NSCLC organoids. Our results deliver ROS-triggered prodrug 5u as a novel TrxR1 inhibitor for the treatment of NSCLC and provide a promising strategy of ROS-activated prodrug for covalent compounds in cancer therapy.

M6A -mediated lncRNA SCIRT stability promotes NSCLC progression through binding to SFPQ and activating the PI3K/Akt pathway

Non-small cell lung cancer (NSCLC) has emerged as one of the most prevalent malignancies worldwide. N6-methyladenosine (m6A) methylation, a pervasive epigenetic modification in long noncoding RNAs (lncRNAs), plays a crucial role in NSCLC progression. Here, we report that m6A modification and the expression of the lncRNA stem cell inhibitory RNA transcript (SCIRT) was significantly upregulated in NSCLC tissues and cells. Functional analysis revealed that SCIRT enhanced NSCLC cell proliferation, migration, invasion, and epithelial‒mesenchymal transition. The m6A modification of SCIRT can be installed by METTL3, which enhanced the stability of this lncRNA. Notably, SCIRT overexpression in response to DNA double-strand breaks (DSBs) sensitized cells to camptothecin (CPT) and impairs DNA homologous recombination repair. SCIRT directly interacted with SFPQ in vitro and was primarily localized in the nucleus. Furthermore, ectopic SCIRT expression upregulated SFPQ and activated the PI3K/Akt pathway following CPT treatment, suggesting an unexpected role of SCIRT in facilitating SFPQ-mediated DSB repair. In brief, our findings highlight the oncogenic role of SCIRT in NSCLC by binding SFPQ and activating PI3K/Akt signaling, presenting a promising therapeutic target for personalized NSCLC treatment.

Utilizing TP53 hotspot mutations as effective predictors of gemcitabine treatment outcome in non-small-cell lung cancer

TP53 mutations are recognized to correlate with a worse prognosis in individuals with non-small cell lung cancer (NSCLC). There exists an immediate necessity to pinpoint selective treatment for patients carrying TP53 mutations. Potential drugs were identified by comparing drug sensitivity differences, represented by the half-maximal inhibitory concentration (IC50), between TP53 mutant and wild-type NSCLC cell lines using database analysis. In addition, clinical data from NSCLC patients were collected to evaluate both their TP53 status and their response to gemcitabine, thereby facilitating further validation. Subsequently, NSCLC cell lines with different TP53 status (A549 and H1299) were subjected to gemcitabine treatment to investigate the association between TP53 mutations and gemcitabine response. According to the dataset, NSCLC cell lines carrying TP53 mutations displayed heightened sensitivity to gemcitabine. From a clinical standpoint, patients exhibiting TP53 hotspot mutations demonstrated prolonged overall survival upon gemcitabine treatment. In vitro, overexpressing various hotspot TP53 mutations significantly sensitized H1299 cells to gemcitabine. Moreover, the knockdown of TP53 in A549 cells notably augmented sensitivity to gemcitabine treatment, as evidenced by cell viability and reproductive cell death assays. Conversely, the overexpression of wild-type TP53 in H1299 cells led to an increased resistance against gemcitabine. Gemcitabine is a treatment option for patients with non-small cell lung cancer (NSCLC) who carry TP53 hotspot mutations. This potential effectiveness might arise from its ability to disrupt DNA damage repair processes, leading to G2/M phase cell cycle arrest or an augmentation of mitotic abnormalities, eventually cause cell death. As a result, when planning treatment strategies for NSCLC patients possessing TP53 hotspot mutations, gemcitabine should be considered to incorporate into the indication.

Abstract B025: Identifying and exploiting a novel role of FTO in promoting cysteine metabolism for NSCLC therapy

Abstract The RNA demethylase enzyme FTO is emerging as an important oncogenic factor and therapeutic target in a variety of solid tumors. However, its effect on cancer metabolism remains largely unknown. Here we show that FTO promotes cysteine metabolism, a hallmark of metabolic rewiring in non-small cell lung cancer (NSCLC) cells. Mechanistically, FTO inhibition reduces the expression of multiple targets in the cysteine metabolism pathway including the cystine transporter SLC7A11 involved in exogenous cystine uptake as well as two key enzymes cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CTH) involved in de novo cysteine synthesis. Functionally, FTO inhibition reduces cystine uptake and NSCLC growth and survival in a homocysteine-dependent manner. As cysteine plays a crucial role in glutathione biosynthesis and maintenance of cellular redox homeostasis, FTO inhibition decreased glutathione levels and increased reactive oxygen species (ROS) levels in NSCLC cells. To therapeutically exploit the increased oxidative stress in NSCLC cells treated with FTO inhibitors, we combined FTO inhibition with radiation therapy. FTO inhibition enhanced the radiosensitivity of NSCLC cells. Our findings reveal a novel role for FTO in NSCLC cysteine metabolism and oxidative stress, highlighting the therapeutic potential of FTO inhibition as a strategy to enhance the efficacy of radiation therapy for the treatment of NSCLC. Citation Format: Nishanth Kuganesan, Stavros Melemenidis, Edward E. Graves, Erinn B. Rankin. Identifying and exploiting a novel role of FTO in promoting cysteine metabolism for NSCLC therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B025.

Abstract B015: PLK4 inhibition as a strategy to enhance non-small cell lung cancer radiosensitivity

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer and comprises 85% of cases. Despite treatment advances, local control after curative-intent chemoradiation for NSCLC remains suboptimal. Polo-like kinase 4 (PLK4) is a serine-threonine kinase that plays a critical role in the regulation of centrosome duplication and cell cycle progression and is overexpressed in NSCLC, thus, making it a potential therapeutic target. CFI-400945 is an orally available PLK4 inhibitor currently undergoing clinical trial evaluation. As radiation causes cell death primarily by mitotic catastrophe, a process enhanced by alterations in centrosome amplification, we hypothesized that disruption of the mitotic machinery by inhibition of PLK4 would enhance the effects of radiation in NSCLC. PLK4 inhibition by CFI-400945 resulted in radiosensitization of NSCLC cell lines. In contrast, CFI-400945 had no effect on the radiosensitivity of normal lung fibroblasts. PLK4 inhibition did not affect cell-cycle phase distribution prior to radiation, but rather the combination of CFI-400945 and radiation resulted in increased G2/M cell cycle arrest, increased centrosome amplification, and a concomitant increase in cell death through mitotic catastrophe. Lastly, CFI-400945 treatment enhanced the radiation-induced tumor growth delay of NSCLC tumor xenografts. These data indicate that targeting PLK4 is a novel approach to enhance the radiation sensitivity of NSCLC in vitro and in vivo through potentiation of centrosome amplification and cell death through mitotic catastrophe. Citation Format: Irma G. Dominguez-Vigil, Kishore Banik, Marta John Baro, Joseph N. Contessa, Thomas J. Hayman. PLK4 inhibition as a strategy to enhance non-small cell lung cancer radiosensitivity. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B015.

Intervention of a Communication Between PI3K/Akt and β-Catenin by (-)-Epigallocatechin-3-Gallate Suppresses TGF-β1-Promoted Epithelial-Mesenchymal Transition and Invasive Phenotype of NSCLC Cells.

The epithelial-mesenchymal transition (EMT) assists in the acquisition of invasiveness, relapse, and resistance in non-small cell lung cancer (NSCLC) and can be caused by the signaling of transforming growth factor-β1 (TGF-β1) through Smad-mediated or Smad-independent pathways. (-)-Epigallocatechin-3-gallate (EGCG), a multifunctional cancer-preventing bioconstituent found in tea polyphenols, has been shown to repress TGF-β1-triggered EMT in the human NSCLC A549 cell line by inhibiting the activation of Smad2 and Erk1/2 or reducing the acetylation of Smad2 and Smad3. However, its impact on the Smad-independent pathway remains unclear. Here, we found that EGCG, similar to LY294002 (a specific inhibitor of phosphatidylinositol 3-kinase [PI3K]), downregulated Akt activation and restored the action of glycogen synthase kinase-3β (GSK-3β), accompanied by TGF-β1-caused changes in hallmarks of EMT such as N-cadherin, E-cadherin, vimentin, and Snail in A549 cells. EGCG inhibited β-catenin expression and its nuclear localization caused by TGF-β1, suggesting that EGCG blocks the crosstalk between the PI3K/Akt/GSK-3β route and β-catenin. Furthermore, it was shown that EGCG suppressed TGF-β1-elicited invasive phenotypes of A549 cells, including invading and migrating activities, matrix metalloproteinase-2 (MMP-2) secretion, cell adhesion, and wound healing. In summary, we suggest that EGCG inhibits the induction of EMT by TGF-β1 in NSCLC not only through a Smad-dependent pathway, but also through the regulation of the PI3K/Akt/β-catenin signaling axis.

Abstract P017: Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer

Abstract Introduction: Definitive chemoradiation therapy (CRT) is essential for controlling locoregionally advanced non-small cell lung cancer (NSCLC); however, up to 30% of patients experience local recurrences within the radiation field. Analyzing favorable responders to treatment could enable identification of genetic alterations associated with radiosensitivity, but standard imaging cannot distinguish residual disease from inflammation and fibrosis during treatment to assess treatment response. We hypothesized that circulating tumor DNA (ctDNA) kinetics during CRT could identify genetic alterations associated with radiosensitivity that could be validated in preclinical models and harnessed to develop new combination therapies with radiotherapy (RT). Methods: We applied cancer personalized profiling by deep sequencing (CAPP-Seq) ctDNA analysis to pre-CRT and mid-treatment (mid-CRT) plasma samples from 61 patients with Stage II-III NSCLC. We identified ctDNA rapid responders as the 10 patients with the largest decrease in ctDNA concentration mid-CRT without local progression and determined the prevalence of genetic alterations in rapid responders versus slow responders using Fisher’s exact tests corrected for multiple hypothesis testing. To investigate the therapeutic potential of targeting PDYN during RT, we performed clonogenic assays on isogeneic PDYN CRISPR-Cas9 knockout and wildtype H1299 and SW1573 human NSCLC cell lines and H1299 cells treated with prodynorphin-neutralizing antibodies. Results: Mid-CRT log-fold change in ctDNA concentration was significantly associated with progression-free survival (P=0.02) in Stage II-III NSCLC. Mutations in PDYN, which encodes the opioid peptide precursor protein prodynorphin, were observed in 30% of ctDNA rapid responders and 0% of ctDNA slow responders (adjusted P=0.04). In NSCLC patients from TCGA treated with RT, the cumulative incidence of local failure was significantly lower in tumors with PDYN mutations (0% vs. 17% at 3 years, P<0.001). PDYN knockout (PDYNNULL) radiosensitized human NSCLC cancer cells by clonogenic assay, and extracellular administration of prodynorphin or its cleavage product dynophin A partially restored radioresistance in PDYNNULL cells. Prodynorphin cleavage products have been reported to signal through opioid receptors and to antagonize N-methyl-D-aspartate receptor (NMDAR) signaling. Although treatment of wildtype H1299 cells with opioid receptor antagonists did not affect radiosensitivity, the NMDAR antagonist MK-801 partially restored RT resistance in PDYNNULL cells. Finally, prodynorphin-neutralizing antibodies significantly reduced clonogenic survival of human NSCLC cells. Conclusions: ctDNA kinetics enable the identification of patients responding favorably to treatment and putative targets to enhance the efficacy of RT. Targeting prodynorphin may enhance the radiosensitivity of NSCLC by increasing NMDAR signaling. Citation Format: Ziwei Wang, Angela B. Hui, Ash A. Alizadeh, Maximilian Diehn, Everett J. Moding.Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P017

Tumor Microenvironment-Responsive Lipid Nanoparticle for Blocking Mitosis and Reducing Drug Resistance in NSCLC.

Blocking mitosis is a promising strategy to induce tumor cell death. However, AMPK- and PFKFB3-mediated glycolysis can maintain ATP supply and help tumor cells overcome antimitotic drugs. Inhibiting glycolysis provides an opportunity to decrease the resistance of tumor cells to antimitotic drugs. Meanwhile, increased glutathione (GSH) expression in cancer cells due to glycolysis becomes a target for developing microenvironment-responsive drugs. Herein, a novel cationic lipid with disulfide bonds in hydrophobic tails was synthesized and used to prepare a GSH-triggered lipid nanoparticle named 2-DG@SLNP(siR) encapsulating both Plk1 siRNA and 2-deoxyglucose (2-DG) for blocking mitosis and reducing drug resistance of nonsmall cell lung cancer (NSCLC) cells in vivo. Experimental results showed that the NSCLC cell cycle was arrested at the G2/M phase by Plk1 siRNA and glycolysis was effectively inhibited by 2-DG, demonstrating the potential of 2-DG@SLNP(siR) as an efficient platform for blocking mitosis and reducing drug resistance of cancer cells.

In situ RAS:RAF binding correlates with response to KRASG12C inhibitors in KRASG12C--mutant non-small cell lung cancer.

Therapeutic efficacy of KRASG12C(OFF) inhibitors (KRASG12Ci) in KRASG12C-mutant non-small cell lung cancer (NSCLC) varies widely. The activation status of RAS signaling in tumors with KRASG12C mutation remains unclear, as its ability to cycle between the active GTP-bound and inactive GDP-bound states may influence downstream pathway activation and therapeutic responses. We hypothesized that the interaction between RAS and its downstream effector RAF in tumors may serve as indicators of RAS activity, rendering NSCLC tumors with a high degree of RAS engagement and downstream effects more responsive to KRASG12Ci compared to tumors with lower RAS---RAF interaction. We developed a method for measuring in situ RAS binding to RAF in cancer samples using proximity ligation assays (PLAs) designed to detect panRAS-CRAF interactions. The panRAS-CRAF PLA signal correlated with levels of both RAS-GTP and phosphorylated ERK protein, suggesting that this assay can effectively assess active RAS signaling. We found that elevated panRAS-CRAF PLA signals were associated with increased sensitivity to KRASG12Ci in KRASG12C-mutant NSCLC cell lines, xenograft models, and patient samples. Applying a similar PLA approach to measure the interactions between EGFR and its adaptor protein GRB2 as a surrogate for EGFR activity, we found no relationship between EGFR activity and response to KRASG12Ci in the same samples. Our study highlights the importance of evaluating in situ RAS-RAF interactions as a potential predictive biomarker for identifying NSCLC patients most likely to benefit from KRASG12Ci. The PLA developed for quantifying these interactions represents a valuable tool for guiding treatment strategies.