Lung Adenocarcinoma Cell Line Research Articles

LncRNA PRKCA-AS1 promotes LUAD progression and function as a ceRNA to regulate S100A16 by sponging miR-508-5p.

Objective: This study aimed to elucidate the underlying mechanism of LncRNA PRKCA-AS1 in lung adenocarcinoma (LUAD). Methods: The expression of LncRNA PRKCA-AS1, miR-508-5p and S100A16, in LUAD tissues or cell lines (NCI-H520 and H1299) was analyzed with qRT-PCR. The clinical diagnostic value of LncRNA PRKCAAS1, miR-508-5p and S100A16 in LUAD were analyzed by receptor operating characteristic (ROC) curve. Then we knockdown or overexpression of PRKCAAS1 in NCI-H520 and H1299 cells, and the cell function test was applied to detect the activity and metastasis level of cells in different transfection groups. Then Pearson correlation analysis was used for the correlation between miR-508-5p and PRKCA-AS1. The dual-luciferase reporter experiment and CHIRP analysis was conducted to verify the target binding relationship of PRKCA-AS1, miR-508-5p or S100A16. FISH assay analyzed the colocalization of PRKCA-AS1 and miR-508-5p in NCI-H520 and H1299 cells. Rescue experiment and tumorigenesis experiment in nude mice further explore the regulatory mechanisms of LncRNA PRKCA-AS1, miR-508-5p and S100A16 on LUAD progression in vitro and in vivo. Results: From the results, PRKCA-AS1 and S100A16 were up-regulated in LUAD tissues, while miR-508-5p was downregulated compared with the adjacent tissues. And gain-of-function revealed that PRKCA-AS1 knock-down apparently suppressed the cell proliferation and metastasis, whereas miR-508-5p inhibitors or S100A16 overexpression showed a opposite effect. In addition, there is evidence that PRKCA-AS1, miR-508-5p and S100A16 have a targeted regulatory relationship. Moreover, rescue experiment and tumorigenesis experiment in nude mice further confirmed that LncRNA PRKCA-AS1 regulates S100A16 through sponging miR-508-5p to regulate LUAD progression in vitro and in vivo. Conclusion: These results demonstrated that LncRNA PRKCA-AS1 might regulate LUAD by acting as a ceRNA via sponging miR-508-5p and regulating S100A16 expression, indicating that manipulation of PRKCA-AS1 might be a potential therapeutic strategy in LUAD.

LncRNA-NEAT1 facilitates autophagy to boost pemetrexed resistance in lung adenocarcinoma via the mir-379-3p/HIF1A pathway.

As a primary chemotherapeutic agent for lung adenocarcinoma (LUAD), pemetrexed (PEM) faces the challenge of resistance development in cancer cells due to its chronic use, which compromises its therapeutic benefits. LncRNA-NEAT1, implicated in the promotion of cancer, is a key player in LUAD. The objective of this study is to explore the contribution of lncRNA-NEAT1 to PEM resistance in LUAD and to dissect the molecular mechanisms involved. The expression levels of lncRNA-NEAT1 in LUAD tissues and cells were deciphered using the TCGA database and qRT-PCR. To delve into the functional implications of lncRNA-NEAT1, we engineered plasmids to modulate its expression levels in PEM-resistant A549 cells. PEM resistance in the modified cells was then quantitatively assessed via a panel of assays including cell counting kit-8 (CCK-8), and colony formation, and flow cytometry. To predict the interaction sites between lncRNA-NEAT1 and miR-379-3p, along with the miR-379-3p and hypoxia-inducible factor (HIF1A), we referred to the StarBase and TargetScan databases. The interplay between these RNA molecules was further characterized by RNA immunoprecipitation (RIP) and dual-luciferase reporter assays, while the expression of autophagy-related proteins LC3I, LC3II, and Beclin1 was profiled using western blot (WB). Abundant lncRNA-NEAT1 expression was observed in LUAD tissues and cell lines. Its depletion resulted in impeded growth of A549/PEM cells, enhanced apoptotic rates, and a lowered threshold for PEM to exert a half-maximal inhibitory effect. The interplay between lncRNA-NEAT1 and miR-379-3p, as evidenced by dual-luciferase reporter assays, RIP, and qRT-PCR, led to the upregulation of HIF1A. WB and CCK-8 outcomes illustrated that the autophagy and PEM resistance were compromised when HIF1A expression was curtailed by miR-379-3p mimics in A549/PEM cells. The restoration of these effects was observed upon lncRNA-NEAT1-mediated downregulation of miR-379-3p. Our study illuminates the role of lncRNA-NEAT1 in LUAD, where it mediates resistance to PEM through the activation of autophagy via the miR-379-3p/HIF1A axis. This work paves the way for new therapeutic strategies for managing PEM resistance in LUAD patients.

Evaluation of t-DARPP Expression Alteration in Association with DDR1 Expression in Non-Small Cell Lung Cancer.

Discoidin domain receptor 1 (DDR1) signaling plays a critical role in various cellular functions. Increased DDR1 expression has been shown in different human cancers. t-DARPP is a truncated isoform of DARPP-32, and its upregulation promotes cell survival and migration. Most lung cancer patients have non-small cell lung cancer (NSCLC), and their survival rate is low. Therefore, it is necessary to study new and effective targeted therapies. Increased t-DARPP expression in NSCLC patients is associated with patient survival and can act as a prognostic marker correlated with increasing stages of NSCLC. The current study aimed to evaluate alteration in DDR1 expression and its effects on t-DARPP expression in NSCLC. Two human lung adenocarcinoma cell lines, A549 and Calu-3, were treated with collagen type I and transfected with DDR1 siRNA. The relative expression of DDR1 and t-DARPP was evaluated using qRT-PCR. The results indicated that collagen type I could stimulate DDR1 expression in NSCLC cells. Also, DDR1 upregulation resulted in a significant increase in t-DARPP expression. In contrast, suppression of DDR1 expression significantly decreased t-DARPP expression. Our findings propose that modification in the expression of DDR1, caused by collagen type I and siRNA, might influence the expression of t-DARPP in NSCLC that is linked to NSCLC progression. Moreover, this alteration could potentially serve as an innovative target for therapeutic intervention.

Study on the Synergistic Effects on the Induction of Apoptosis in A549 Human Lung Adenocarcinoma Cell Line by Gefitinib and TRAIL

Study on the Synergistic Effects on the Induction of Apoptosis in A549 Human Lung Adenocarcinoma Cell Line by Gefitinib and TRAIL

Arsenic-induced downregulation of BRWD3 suppresses proliferation and induces apoptosis in lung adenocarcinoma cells through the p53 and p65 pathways.

Bromodomain and WD-repeat domain-containing protein 3 (BRWD3) exhibits high expression in lung adenocarcinoma (LUAD) tissues and cells; however, its function in arsenic-induced toxicological responses remains unclear. This study aimed to investigate BRWD3 expression in response to arsenic-induced conditions and its impact on the proliferation and apoptosis of LUAD cell line SPC-A1 upon BRWD3 knockdown. The results revealed a decrease in BRWD3 expression in SPC-A1 cells treated with sodium arsenite (NaAsO2), but not sodium arsenite's metabolites. BRWD3 knockdown suppressed cell proliferation and induced apoptosis in SPC-A1 cells. Western blot analysis revealed that BRWD3 knockdown resulted in the upregulation of p53, phospho-p53-Ser392, and its downstream factors including MDM2, Bak, and Bax. Additionally, we observed the downregulation of p65, phospho-p65-Ser276, phospho-p65-Ser536, and its downstream factors, including IκBα, BIRC3, XIAP and CIAP1. Moreover, polymerase chain reaction analysis showed that BRWD3 knockdown also resulted in the downregulation of proliferation-related genes and upregulation of apoptosis-related genes. In conclusion, BRWD3 mediated proliferation and apoptosis via the p53 and p65 pathways in response to arsenic exposure, suggesting potential implications for LUAD treatment through BRWD3 downregulation by arsenic.

Effects and mechanism of anlotinib on radiosensitivity of non-small cell lung cancer cell lines a549

To investigate the effect and mechanism of Anlotinib on radio sensitization of human lung adenocarcinoma cell line A549. Human lung adeno carcinoma cell line A549 was teated with anlotinib and/or radiotherapy, then divided four groups, control group (Ctrl), Anlotinib treatment group (A), irradiation group (RT) and Anlotinib combined with irradiation group (A+RT) . CCK8 method was used to determine cell proliferation; the clone formation experiment was used to determine the inhibitory effect on cell growth; flow cytometry was used to determine cell cycle and apoptosis; immunofluorescence of γ-H2AX was used to determine DNA damage; expression of DNA-PKcs were detected by Western blot. Anlotinib inhibited proliferation and clonogenic survival following irradiation. The dose (Dq), the average lethal dose (D0) and the survival score (SF2) in the anlotinib combined radiotherapy group was significantly lower than those in the radiotherapy group. Anlotinib decreased G2/M phase arrest and promoted the cells apoptosis induced by in irradiation. The confocal microscopy results showed the average number of γ-H2AX foci in the A+RT group was more than that in RT group. The protein levels of DNA-PKcs were higher in A+RT group than that in RT group. Anlotinib enhances the radio sensitivity of A549 cells, which may be attributed to the delay DNA damage repair. It provides a rationale strategy by Anlotinib combined with irradiation for NSCLC.

Neutrophil infiltration associated genes on the prognosis and tumor immune microenvironment of lung adenocarcinoma.

The neutrophils exhibit both anti-tumor and pro-tumor effects in cancers. The correlation between neutrophils and tumor development in lung adenocarcinoma (LUAD) is still uncertain, possibly due to a lack of specific neutrophil infiltration evaluation methods. In this study, we identified 30 hub genes that were significantly associated with neutrophil infiltration in LUAD through data mining, survival analysis, and multiple tumor-infiltrating immune cells (TICs) analysis, including TIMER, CIBERSORT, QUANTISEQ, XCELL, and MCPCOUNTER. Consensus clustering analysis showed that these 30 hub genes were correlated with clinical features in LUAD. We further developed a neutrophil scoring system based on these hub genes. The neutrophil score was significantly correlated with prognosis and tumor immune microenvironment (TIME) in LUAD. It was also positively associated with PD-L1 expression and negatively associated with tumor mutational burden (TMB). When combined with the neutrophil score, the predictive capacity of PD-L1 and TMB for prognosis was significantly improved. Thus, the 30 hub genes might play an essential role in the interaction of neutrophils and LUAD, and the neutrophil scoring system might effectually assess the infiltration of neutrophils. Furthermore, we verified the expression of these 30 genes in the LUAD tumor tissues collected from our department. We further found that overexpressed TNFAIP6 and TLR6 and downregulated P2RY13, SCARF1, DPEP2, PRAM1, CYP27A1, CFP, GPX3, and NCF1 in LUAD tissue might be potentially associated with neutrophils pro-tumor effects. The following in vitro experiments demonstrated that TNFAIP6 and TLR6 were significantly overexpressed, and P2RY13 and CYP27A1 were significantly downregulated in LUAD cell lines, compared to BEAS-2B cells. Knocking down TNFAIP6 in A549 and PC9 resulted in the upregulation of FAS, CCL3, and ICAM-1, and the downregulation of CCL2, CXCR4, and VEGF-A in neutrophils when co-culturing with the conditioned medium (CM) from LUAD cells. Knocking down TNFAIP6 in LUAD also led to an elevated early apoptosis rate of neutrophils. Therefore, overexpressed TNFAIP6 in LUAD cancer cells might lead to neutrophils "N2" polarization, which exhibited pro-tumor effects. Further research based on the genes identified in this pilot study might shed light on neutrophils' effects on LUAD in the future.

Vasculogenic mimicry-associated novel gene signature predicted prognosis and response to immunotherapy in lung adenocarcinoma

BackgroundsIt was highlighted by recent studies on the biological significance of vasculogenic mimicry (VM) in tumorigenicity and progression. However, it is unclear whether VM also plays a potential role in immune regulation and tumor microenvironment (TME) formation. MethodsTo identify patterns of VM alterations and VM-associated genetic features in non-small cell lung adenocarcinoma, we have screened 309 VM regulators and performed consensus molecular typing by the NMF algorithm. The ssGSEA and CIBORSORT algorithms were employed to measure the relative infiltration of distinct immune cell subpopulations. Individual tumors with immune responses were evaluated for alteration patterns of VM with typing-based differential genes. ResultsIn 490 LUAD samples, two distinctive VM alteration patterns connected to different clinical outcomes and biochemical pathways were established. TME characterization showed that the observed VM patterns were primarily saturated with cell proliferation and metabolic pathways and higher in immune cell infiltration of the C1 type. Vasculogenic mimicry-related genes (VMRG) risk scores were constructed to divide patients with lung adenocarcinoma into subgroups with high and low scores. Patients with lower scores had better immunological scores and longer survival times. Upon further investigation, higher scores were positively correlated with higher tumor mutation burden (TMB), M1-type macrophages and immune checkpoint molecules. Nevertheless, in two other immunotherapy cohorts, individuals with lower scores had enhanced immune responses and long-lasting therapeutic benefits. Finally, we monitored the ANLN gene from the VMRG model, which was highly expressed in lung adenocarcinoma tissues and negatively correlated with prognosis; it was also highly expressed in lung adenocarcinoma cell lines, and knockdown of ANLN elicited low expression of VEGFA, MMP2 and MMP9. ConclusionThis study highlights that VM modifications are significantly associated with the diversity and complexity of TME, revealing new features of the immune microenvironment in lung adenocarcinoma and providing a new strategy for immunotherapy. Screening ANLN as a critical target for vasculogenic mimicry in lung adenocarcinoma provides a novel perspective for the targeted treatment of lung adenocarcinoma.

Transcriptional analysis of landmark molecular pathways in lung adenocarcinoma results in a clinically relevant classification with potential therapeutic implications.

Lung adenocarcinoma (LUAD) is a molecularly heterogeneous disease. In addition to genomic alterations, cancer transcriptional profiling can be helpful to tailor cancer treatment and to estimate each patient's outcome. Transcriptional activity levels of 50 molecular pathways were inferred in 4573 LUAD patients using Gene Set Variation Analysis (GSVA) method. Seven LUAD subtypes were defined and independently validated based on the combined behavior of the studied pathways: AD (adenocarcinoma subtype) 1-7. AD1, AD4, and AD5 subtypes were associated with better overall survival. AD1 and AD4 subtypes were enriched in epidermal growth factor receptor (EGFR) mutations, whereas AD2 and AD6 showed higher tumor protein p53 (TP53) alteration frequencies. AD2 and AD6 subtypes correlated with higher genome instability, proliferation-related pathway expression, and specific sensitivity to chemotherapy, based on data from LUAD cell lines. LUAD subtypes were able to predict immunotherapy response in addition to CD274 (PD-L1) gene expression and tumor mutational burden (TMB). AD2 and AD4 subtypes were associated with potential resistance and response to immunotherapy, respectively. Thus, analysis of transcriptomic data could improve patient stratification beyond genomics and single biomarkers (i.e., PD-L1 and TMB) and may lay the foundation for more personalized treatment avenues, especially in driver-negative LUAD.

Studies on desmodium velutinum (Willd.) DC. leaf extract's polyphenol profile, in-vitro antioxidant capacity, and anti-proliferative activity of A549 cell lines

In African and Asian ethnomedicinal studies, Desmodium velutinum (Willd) has been used in the treatment of tooth and head aches, diarrhoea, cancer, and haematuria and as repellent and aphrodisiac. The study is aimed at determining polyphenol profile and antioxidant activities of D. velutinum methanol leaf extract and its ethyl acetate and butanol fraction. The study also investigated the effect of this plant on lung adenocarcinoma (A549) cell line. The polyphenol constituents in D. velutinum were evaluated with the U-HPLC technique. While, the antioxidant activities of the methanol plant extract and its fractions were assessed with the DPPH, metal chelating, reducing power and DNA nicking assays. MTT assay was employed to analyse the effect of the plant on A549 cell line. Kaempferol is the principal polyphenol in the leaf extract/fraction of D. velutinum. Conversely, its ethyl acetate fraction possessed a higher concentration of catechin than kaempferol. Total phenolic content (TPC) result was directly proportional to the antioxidant activities of extract and fractions, a higher TPC value was obtained in ethyl-acetate fraction (787.1 ± 21.4 mg GAE/g) than the crude methanol extract (710.9 ± 24.5 mg GAE/g) and its n-butanol fraction (759.2 ± 1.3 mg GAE/g), thus, ethyl acetate fraction performed best in the antioxidant assays. D. velutinum also inhibited oxidative damage to supercoiled plasmid DNA and suppressed the proliferation of A549 cells in a dose-dependent manner. This study shows that D. velutinum does have potentials to protect against many diseases linked with reactive oxygen species.

NRIP1 regulates cell proliferation in lung adenocarcinoma cells.

Nuclear receptor interacting protein 1 (NRIP1) is a transcription cofactor that regulates the activity of nuclear receptors and transcription factors. Functional expression of NRIP1 has been identified in multiple cancers. However, the expression and function of NRIP1 in lung adenocarcinoma have remained unclear. Thus, we aimed to clarify the NRIP1 expression and its functions in lung adenocarcinoma cells. NRIP1 and Ki-67 were immunostained in the tissue microarray section consisting of 64 lung adenocarcinoma cases, and the association of NRIP1 immunoreactivity with clinical phenotypes was examined. Survival analysis was performed in lung adenocarcinoma data from The Cancer Genome Atlas (TCGA). Human A549 lung adenocarcinoma cell line with an NRIP1-silencing technique was used in vitro study. Forty-three of 64 cases were immunostained with NRIP1. Ki-67-positive cases were more frequent in NRIP1-positive cases as opposed to NRIP1-negative cases. Higher NRIP1 mRNA expression was associated with poor prognosis in the TCGA lung adenocarcinoma data. NRIP1 was mainly located in the nucleus of A549 cells. NRIP1 silencing significantly reduced the number of living cells, suppressed cell proliferation, and induced apoptosis. These results suggest that NRIP1 participates in the progression and development of lung adenocarcinoma. Targeting NRIP1 may be a possible therapeutic strategy against lung adenocarcinoma.

Identification and validation of eight lysosomes-related genes signatures and correlation with immune cell infiltration in lung adenocarcinoma

Lung cancer is the leading cause of cancer-related death. Lysosomes are key degradative compartments that maintain protein homeostasis. In current study, we aimed to construct a lysosomes-related genes signature to predict the overall survival (OS) of patients with Lung Adenocarcinoma (LUAD). Differentially expressed lysosomes-related genes (DELYs) were analyzed using The Cancer Genome Atlas (TCGA-LUAD cohort) database. The prognostic risk signature was identified by Least Absolute Shrinkage and Selection Operator (LASSO)-penalized Cox proportional hazards regression and multivariate Cox analysis. The predictive performance of the signature was assessed by Kaplan–Meier curves and Time-dependent receiver operating characteristic (ROC) curves. Gene set variant analysis (GSVA) was performed to explore the potential molecular biological function and signaling pathways. ESTIMATE and single sample gene set enrichment analysis (ssGSEA) were applied to estimate the difference of tumor microenvironment (TME) between the different risk subtypes. An eight prognostic genes (ACAP3, ATP8B3, BTK, CAV2, CDK5R1, GRIA1, PCSK9, and PLA2G3) signature was identified and divided patients into high-risk and low-risk groups. The prognostic signature was an independent prognostic factor for OS (HR > 1, p < 0.001). The molecular function analysis suggested that the signature was significantly correlated with cancer-associated pathways, including angiogenesis, epithelial mesenchymal transition, mTOR signaling, myc-targets. The low-risk patients had higher immune cell infiltration levels than high-risk group. We also evaluated the response to chemotherapeutic, targeted therapy and immunotherapy in high- and low-risk patients with LUAD. Furthermore, we validated the expression of the eight gene expression in LUAD tissues and cell lines by qRT-PCR. LYSscore signature provide a new modality for the accurate diagnosis and targeted treatment of LUAD and will help expand researchers’ understanding of new prognostic models.

HDAC1-3 inhibition increases SARS-CoV-2 replication and productive infection in lung mesothelial and epithelial cells.

Despite the significant progress achieved in understanding the pathology and clinical management of SARS-CoV-2 infection, still pathogenic and clinical issues need to be clarified. Treatment with modulators of epigenetic targets, i.e., epidrugs, is a current therapeutic option in several cancers and could represent an approach in the therapy of viral diseases. Aim of this study was the analysis of the role of histone deacetylase (HDAC) inhibition in the modulation of SARS-CoV-2 infection of mesothelial cells (MCs).MeT5A cells, a pleura MC line, were pre-treated with different specific class I and IIb HDAC inhibitors. Unexpectedly, treatment with HDAC1-3 inhibitors significantly increased ACE2/TMPRSS2 expression, suggesting a role in favoring SARS-CoV-2 infection. We focused our analysis on the most potent ACE2/TMPRSS2 inducer among the inhibitors analysed, MS-275, a HDAC1-3 inhibitor. ACE2/TMPRSS2 expression was validated by Western Blot (WB) and immunofluorescence. The involvement of HDAC inhibition in receptor induction was confirmed by HDAC1/HDAC2 silencing. In accordance to the ACE2/TMPRSS2 expression data, MS-275 increased SARS-CoV-2 replication and virus propagation in Vero E6 cells.Notably, MS-275 was able to increase ACE2/TMPRSS2 expression and SARS-CoV-2 production, although to a lesser extent, also in the lung adenocarcinoma cell line Calu-3 cells.Mechanistically, treatment with MS-275 increased H3 and H4 histone acetylation at ACE2/TMPRSS2 promoters, increasing their transcription. This study highlights a previously unrecognized effect of HDAC1-3 inhibition in increasing SARS-CoV-2 cell entry, replication and productive infection correlating with increased expression of ACE2 and TMPRSS2. These data, while adding basic insight into COVID-19 pathogenesis, warn for the use of HDAC inhibitors in SARS-CoV-2 patients.

Evaluation the cytotoxic effect of Fe3O4@Glu-Gingerol on lung adenocarcinoma cell line (A549) with biological mechanisms

The use of nanotechnology products with supermagnetic properties for targeted delivery of drugs has gained attention recently. Due to the anticancer features of Gingerol, the major phenolic compound from Ginger, this study aims to prepare Fe3O4@Glucose-Gingerol nanoparticles (NPs) and investigate their anticancer potential in a lung adenocarcinoma cell line. The physical and chemical features of the nanoparticles were investigated by FT-IR, XRD, zeta potential, DLS, EDS mapping, VSM, and electron microscope imaging. Cytotoxic effects of the nanoparticles for the A549 (lung adenocarcinoma) and MRC-5 (normal) cell lines was investigated by MTT assay. Furthermore, the effects of Fe3O4@Glucose-Gingerol nanoparticles on the expression of the CASP8, BAX, and BCL2 genes and the activity of Caspase 3 were characterized. The flow cytometry assay (annexin V/PI) was employed to find out the percentage of apoptotic cells. The Fe3O4@Glu-Gingerol NPs were spherical (42–67 nm), without elemental impurity, and with surface charge, DLS size, and magnetic saturation of −47.7 mV, 154 nm, and 35 emu/g, respectively. Fe3O4@Glu-Gingerol NPs showed a remarkable greater toxicity in the A549 cells than normal cell line with the 50 % inhibition concentration (IC50) of 190 and 554 μg/mL, respectively. Treatment of lung adenocarcinoma cells with the Fe3O4@Glu-Gingerol NPs led to an increase in cell apoptosis from 4.6 to 39.48 %. Also, the CASP8 and BAX genes were upregulated by 2.49 and 2.8 folds, respectively, while a downregulation by 0.75 folds was noticed for the BCL2. Moreover, apoptotic features were observed in Fe3O4@Glu-Gingerol NPs treated cells by Hoechst staining, and activation of Caspase 3 by 2.8 folds. This study revealed that the Fe3O4@Glu-Gingerol NPs have antiproliferative effects on the lung adenocarcinoma cell line by activation of intrinsic and extrinsic apoptosis that is a promising feature in cancer treatment.

Abstract A115: mTOR targeting in STK11 deficient Non-Small Cell Lung Cancer (NSCLC): final results, pre-clinical rationale and biomarker analysis of a phase II trial of the mTORC1/2 inhibitor vistusertib in STK11 deficient lung adenocarcinoma (NLMT B2)

Abstract Background: STK11 loss of function is common in lung adenocarcinoma (LUAD). STK11 deficient LUAD demonstrates mTOR activation, increased HIF-1a expression and GLUT1 up-regulation, resulting in increased glucose metabolism. STK11 loss often co-occurs with KRAS mutation. KRAS and STK11 co-mutations synergise to drive metabolic re-programming and heightened glucose dependency, reversible with mTORC1/mTORC2 inhibition. mTORC2 is a central node driving enhanced glycolysis independent of Raptor and HIF-1α. mTORC1/2 inhibition suppresses pS6K, p4EBP1, HIF1a and GLUT1 in STK11/KRAS double mutant NSCLC cells and suppresses growth in vivo. Vistusertib (AZD2014) is a selective mTORC1/2 inhibitor. We report the results of outcomes of LUAD patients with STK11 loss+/-KRAS mutation treated with vistusertib, the first study to investigate the impact of mTOR inhibition in STK11 deficient LUAD. Results: 19 patients (17 per-protocol) had STK11 loss without concomitant KRAS mutation (cohort B2S). 30 patients had STK11 loss with concomitant KRAS mutation (26 per-protocol) (cohort B2D). The confirmed objective response rate (95% credible interval) was 3.8% (0.1 – 18.5) for B2S, 9.8% (2.4 – 24.3) for B2D. Durable clinical benefit ((DCB), defined as no progression at 4th 6 weekly on treatment CT assessment) rate was 14.6% (3.6 – 34.7) for B2S, 24.4% (11.1 – 42.3) for B2D. Tumour growth inhibition (median % change from baseline (range)) in those with DCB was -18.5% (-48.0, 3.0) and in those with progression &amp;lt;3 months/death +10.5% (-6.0, 42.0). Median PFS was 2.29 months (1.47 – 3.85) for B2S, 2.77 months (1.91 – 4.22) for B2D. Median OS was 9.84 months (6.23 – 16.85) for B2S, 6.08 months (4.20 – 9.26) for B2D. Clinically achievable concentrations of vistusertib achieve dose-dependent inhibition of STK11 deficient LUAD cell line viability in vitro. In contrast, IC50 for everolimus (mTORC1-only inhibition) exceeds pharmacologically achievable doses in all tested STK11 deficient cell lines. Rebound FOXO and Akt phosphorylation is observed after 48 hours of vistusertib treatment, with parallel activation of ERK. Longitudinal ctDNA from B2S/B2D were analysed using the Illumina TSO500 ctDNA platform. Using conventional p&amp;lt;0.05 for significance, 2 genes were enriched at progression versus baseline: SMARCA4 (3/36 pre, 11/27 post, p=0.005) and FOXP1 (0/36 pre, 4/27 post, p=0.029). TRAF2 mutations were non-significantly enriched post-treatment (1/36 pre, 5/27 post, P = 0.076). Conclusions: Despite strong rationale for mTORC1/2 inhibition in STK11 deficient LUAD, activity of vistusertib is modest. Pre-clinical work suggests that STK11 mutant LUAD cell lines are not exempt from relief of feedback inhibition during prolonged mTORC1/2 inhibition, driving Akt and FOXO phosphorylation. Further, longitudinal ctDNA analysis suggests that vistusertib treatment may drive selection for SMARCA4 mutations, which appears to be a common mechanism of resistance to targeted therapies. HLR and PF contributed equally. LB and GM contributed equally. Citation Format: Helen L Robbins, Peter Fletcher, Joshua Savage, Manita Mehmi, Yvonne Summers, Alastair Greystoke, Noelle O’Rourke, Sanjay Popat, Pooja Jain, James Spicer, Judith Cave, Paul Shaw, David Gilligan, Danielle Power, Dean Fennel, Andrew D Beggs, Lucinda Billingham, Gary Middleton. mTOR targeting in STK11 deficient Non-Small Cell Lung Cancer (NSCLC): final results, pre-clinical rationale and biomarker analysis of a phase II trial of the mTORC1/2 inhibitor vistusertib in STK11 deficient lung adenocarcinoma (NLMT B2) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A115.

Discriminative and prognostic significance of LINC00623 for lung adenocarcinoma.

This study was conducted to evaluate the diagnostic and prognostic values of LINC00623 in patients with lung adenocarcinoma (LUAD), as well as the promoting role in LUAD progression. LINC00623 levels were identified by RT-qPCR. Using ROC curve analysis, the discrimination value of LINC00623 for LUAD from lung squamous cell carcinoma (LUSC) and the health was assessed. Recurrence-free and overall survival were estimated using the Kaplan-Meier method and multivariate survival analysis. Cell biological properties were analyzed by growth curves, transwell migration, and invasion assays. The downstream miRNA and genes were retrieved and annotated by online databases. LINC00623 was significantly upregulated in the LUAD tissues and cell lines compared to LUSC tissues and the noncancerous tissues. LINC00623 showed a discriminating power for predicting LUAD. LUAD patients with a high level of LINC00623 tend to have poor overall survival and recurrence-free survival. Downregulation of LINC00623 reduced the proliferation, migration, and invasion of LUAD cells. MiR-1207-5p was a target of LINC00623 and its target genes were related to PI3K/Akt signaling pathway. The subtypes, recurrence-free survival, and overall survival of patients with LUAD might be well discriminated by LINC00623 levels. LINC00623 promoted LUAD progression through sponging miR-1207-5p.

Abstract B093: Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent

Abstract KRAS is the most frequently mutated oncoprotein in human cancers. Although long considered “undruggable”, recent breakthroughs in medicinal chemistry have led to FDA-approval of the KRAS(G12C) mutation-specific inhibitors sotorasib and adagrasib for the treatment of KRAS(G12C)-positive non-small cell lung cancer. However, illustrating the need to develop additional novel agents targeting mutated KRAS, the 5-year relative survival rate for pancreatic cancer, which is commonly associated with a variety of different KRAS mutations, is only 12% (all SEER stages) due to poor early detection and a lack of effective treatments. In particular, KRAS(G12D) is the most common KRAS mutation, being found in 37% of pancreatic ductal adenocarcinomas (PDACs), as well as in 12.5% of colorectal cancer and 4.9% of lung adenocarcinoma (LUAD) patients. MRTX1133 is a highly-selective, non-covalent KRAS(G12D) inhibitor that has recently entered a phase 1 clinical trial. However, here we show that exposure of KRAS(G12D)-mutated PDAC (PANC-1 and AsPC-1) and LUAD (SK-LU-1) cell lines and a patient-derived organoid (PDO) PDAC model (RPAN001) to MRTX1133 resulted in varying degrees of in vitro efficacy. Decreased downstream KRAS signaling in the form of reduced phospho-ERK1/2 levels was observed to rapidly recover within 24 hours of treatment with 500 nM MRTX1133. Moreover, this rebound coincided with increased expression of KRAS, NRAS andHRAS mRNAs. Concurrently, activation of the receptor tyrosine kinases (RTKs) EGFR and MET was observed in PANC-1 and SK-LU-1 cells that displayed innate resistance to MRTX1133, while the sensitive AsPC-1 cells showed no such RTK activation. These results suggest that targeting of both KRAS(G12D) and RTKs may be needed to treat KRAS(G12D) inhibitor-resistant cancers. To address this, we employed a novel hetero-bispecific CHAMP molecule, RNK08179, that simultaneously targets both KRAS(G12D) and HSP90, an RTK-regulating chaperone protein. RNK08179 treatment demonstrated a striking reduction in phospho-ERK1/2 levels, RTK activation and cell viability in MRTX1133-resistant PANC-1 and SK-LU-1 cells. Furthermore, similar efficacy was observed in the MRTX1133-resistant RPAN001 PDO model. In summary, RNK08179 displayed promising efficacy in cancer models harboring KRAS(G12D) by suppressing both mutated KRAS and HSP90-supported RTK signaling. Citation Format: Ines Pulido, Qiyue Luan, Chenghao Yin, Zimo Yang, Jinhua Lin, Yaya Wang, Yuetong Sun, Chuche Liu, Haoxin Zhou, Marek Massad, Ian Papautsky, Thomas L. Prince, Guoqiang Wang, Kevin P. Foley, Weiwen Ying, Takeshi Shimamura. Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B093.

Abstract B003: Inhibition of cell growth and migration by polyisoprenylated cysteinyl amide inhibitors of a mutant KRAS Black American lung cancer cell line involves MAPK pathway activation and disruption of the cytoskeleton

Abstract KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl Amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, as caused by mutated RAS proteins. Here we investigate the effects of PCAIs on the viability, G-protein levels, downstream mediators, cytoskeletal structure, focal adhesion protein and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48-hour exposure to 5 μM of the PCAIs, 57 ± 3.6 to 150 ± 4.1 % increases of BRAF, MEK1/2, ERK1/2, and p90RSK phosphorylation along with a 60 ± 6.5 to 78 ± 4.0 % decrease of pRAF1 phosphorylation were observed, indicating significant changes in RAS signaling. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 ± 5.5 and 76 ± 2.0 %, respectively. The depletion of these key cytoskeletal proteins, and collapse of F-actin and vinculin punctates, may account for the observed inhibition of cell migration after 72-hour exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46, respectively. The invasion area of 3D NCI-H23 spheroids after treatment with 10 μM of NSL-YHJ-2-27 and NSL-YHJ-2-46 decreased by 87 ± 0.1% and 92 ± 0.3 %, respectively. Treatment with 0.5 μM NSL-YHJ-2-27 and NSL-YHJ-2-46 caused cell rounding and reduction of mean cell areas by 30 ± 5.0 and 42 ± 0.6 %, respectively. Mean cell area occupied by vinculin punctates decreased by 51 ± 2.6 % and 31 ± 2.8 % after exposure to 0.5 μM NSL-YHJ-2-27 and NSL-YHJ-2-46, respectively. NSL-YHJ-2-27 (5 μM) depleted full-length inactive caspase 3 and 7 levels by 72 ± 5.8 and 91 ± 7.0 %, respectively. These findings show the direct effects of the PCAIs on the KRAS signaling pathway thereby supporting their continuous development as potential targeted therapies for cancers with RAS mutations. Citation Format: Kweku Ofosu-Asante. Inhibition of cell growth and migration by polyisoprenylated cysteinyl amide inhibitors of a mutant KRAS Black American lung cancer cell line involves MAPK pathway activation and disruption of the cytoskeleton [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B003.

Abstract C093: Increasing racial diversity of lung adenocarcinoma cell lines to mitigate lung cancer health disparities

Abstract Lung cancer is the leading cause of cancer death in the United States and worldwide, and a significant source of cancer health disparities. Black/African American (B/AA) men exhibit the highest rate of lung cancer death compared to any other racial group. Compared to White men, B/AA men have a 12% higher lung cancer incidence rate and a 15% higher lung cancer death rate. One essential tool in the study of lung cancer is the use of lung cancer cell lines, which can be applied to molecular studies of lung cancer development and progression and pre-clinical drug testing. To minimize disparities in lung cancer research, it is important that cell lines representing different lung cancer histological subtypes, carrying different cancer driver genes, and representing different races and ethnicities be available. This is particularly relevant for lung cancer cell lines from B/AA men. We reviewed the availability of lung cancer cell lines and observed a considerable imbalance in the representation of cell lines from different population groups. We identified over 800 lung cancer cell lines, with the majority of lung cancer cell lines derived from White and Asian patients, and only 31 lung cancer cell lines from B/AA patients. In addition, we did not find any cell lines from Hispanic/Latin(x) (H/L), American Indian/American Native (AI/AN), or Native Hawaiian or other Pacific Islander (NHOPI) patients. Focusing on lung adenocarcinoma, the most common type of lung cancer, we aim to increase the representation of B/AA cell lines by creating new cell lines from B/AA lung cancer patients, as well as by generating isogenic cell line variants from existing lung cancer cell lines, such as NCI-H23. NCI-H23 was derived from a Black male patient with a KRAS G12C heterozygous mutation. We use CRISPR/Cas9-based genome editing to replace the KRAS G12C mutation with other KRAS mutations found in B/AA patients, including KRAS G12V, KRAS G12A, and KRAS G13C. The lack of an appropriately diverse collection of lung cancer cell lines significantly limits racially and ethnically inclusive lung cancer research. This hinders the ability to develop inclusive models, screen comprehensively for therapeutic compounds, and pre-clinically test new drugs. Ultimately, this leads to an under-development of therapies that can increase the survival of minority and underserved patients. The noted lack of cell lines from underrepresented groups should mobilize researchers and clinicians to establish additional cell lines and ensure appropriate representation of all population groups in this critical pre-clinical research resource. Citation Format: Christopher Leon, Matthew Gladstone, Chunli Yan, Diana I. Romero, Yong Huang, Kweku Ofosu-Asante, Nazarius S. Lamango, Ite A. Offringa. Increasing racial diversity of lung adenocarcinoma cell lines to mitigate lung cancer health disparities [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C093.

Abstract C063: Frequency and spectrum of KRAS mutations in lung adenocarcinoma from Black/African American and White/European patients

Abstract Lung cancer is the most common cause of cancer death in the United States and a major source of cancer health disparity. Lung cancer incidence and mortality rates are higher in Black/African American (B/AA) men than in men from other racial/ethnic groups. Lung adenocarcinoma, arising in the alveoli or air sacs of the lung, is the most common subtype of lung cancer in all ethnic/racial groups. Genetic driver mutations can differ among racial/ethnic groups, in the identity of the mutated genes, their mutation frequencies, and the spectrum of mutations for each given gene. In addition, the same mutation could have different effects depending on an individual patient’s genetic background, resulting in differential responses to therapies. Therefore, it is important to identify the frequency and spectrum of driver mutations in different population groups. Protooncogenes from the RAS family, KRAS, NRAS, and HRAS, play a crucial role in cell growth regulation, cell proliferation, and survival. KRAS is frequently mutated in numerous cancer types, including lung adenocarcinoma, and has been reported to be the driver gene in ~30% of lung adenocarcinomas from White/European (W/E) patients. Due to the much lower number of lung cancer samples analyzed from B/AA patients, it has been unclear if the KRAS mutational frequency and spectrum in B/AA lung adenocarcinoma patients differs from that in W/E patients. Here we collected available existing data sets with mutational information on KRAS muations in lung adenocarcinoma from B/AA and W/E patients. We obtained data for 1555 B/AA and 13840 White lung adenocarcinoma patients. While B/AA lung adenocarcinoma patients had a lower prevalence of KRAS mutations (27% vs. 35%) than W/E patients, (χ2 p-value= 5.877e-10), KRAS was still the most frequently mutated cancer driver gene in both groups. The most common KRAS mutations in both B/AA and W/E patients were alterations of glycine 12 to cysteine (G12C), valine (G12V), aspartic acid (G12D), and alanine (G12A), as well as glutamine 61 to histidine (Q61H) and glycine 13 to cysteine (G13C). Given that a substantial proportion of lung adenocarcinoma patients carry a mutated KRAS gene, investigating new potential KRAS-targeting therapies remains especially urgent. To this end, we are investigating polyisoprenylated cysteinyl amide inhibitors (PCAIs), a group of potential cancer therapy agents designed to specifically disrupt and suppress hyperactive G-protein signaling, as caused by mutated RAS proteins. We are testing PCAI efficiacy on B/AA and W/E lung adenocarcinoma cell lines carrying various KRAS mutations. Citation Format: Daniel J. Mullen, Pablo E. Puente, Christopher Leon, Diana I. Romero, Kweku Ofosu-Asante, Yong Huang, Nazarius S. Lamango, Ite A. Offringa. Frequency and spectrum of KRAS mutations in lung adenocarcinoma from Black/African American and White/European patients [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C063.