Non-small Cell Lung Cancer Clinical Samples Research Articles

PIM1 kinase promotes EMT-associated osimertinib resistance via regulating GSK3β signaling pathway in EGFR-mutant non-small cell lung cancer

Acquired resistance is inevitable in the treatment of non-small cell lung cancer (NSCLC) with osimertinib, and one of the primary mechanisms responsible for this resistance is the epithelial-mesenchymal transition (EMT). We identify upregulation of the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and functional inactivation of glycogen synthase kinase 3β (GSK3β) as drivers of EMT-associated osimertinib resistance. Upregulation of PIM1 promotes the growth, invasion, and resistance of osimertinib-resistant cells and is significantly correlated with EMT molecules expression. Functionally, PIM1 suppresses the ubiquitin-proteasome degradation of snail family transcriptional repressor 1 (SNAIL) and snail family transcriptional repressor 2 (SLUG) by deactivating GSK3β through phosphorylation. The stability and accumulation of SNAIL and SLUG facilitate EMT and encourage osimertinib resistance. Furthermore, treatment with PIM1 inhibitors prevents EMT progression and re-sensitizes osimertinib-resistant NSCLC cells to osimertinib. PIM1/GSK3β signaling is activated in clinical samples of osimertinib-resistant NSCLC, and dual epidermal growth factor receptor (EGFR)/PIM1 blockade synergistically reverse osimertinib-resistant NSCLC in vivo. These data identify PIM1 as a driver of EMT-associated osimertinib-resistant NSCLC cells and predict that PIM1 inhibitors and osimertinib combination therapy will provide clinical benefit in patients with EGFR-mutant NSCLC.

METTL5: A Potential Biomarker for Nonsmall Cell Lung Cancer That Promotes Cancer Cell Proliferation by Interacting with IGF2BP3.

Objective: To examine if METTL5 promotes the proliferation of nonsmall cell lung cancer (NSCLC) cells by interacting with IGF2BP3. Methods: The expression patterns of METTL5 and IGF2BP3 in NSCLC tissues, their relationship with survival rate, and their correlation were analyzed using bioinformatics and clinical sample analyses. The effects of METTL5 overexpression and IGF2BP3 knockdown, as well as those of METTL5 knockdown and IGF2BP3 overexpression, on the proliferation of NSCLC cells were analyzed by transfecting appropriate constructs. The interaction between METTL5 and IGF2BP3 was verified using the co-immunoprecipitation (Co-IP) assay. The invivo effects of METTL5 and IGF2BP3 on NSCLC growth were analyzed using the tumor-bearing nude mouse model. Results: METTL5 and IGF2BP3 expression levels were positively correlated and were associated with poor clinical prognosis. The METTL5 and IGF2BP3 expression levels were upregulated in the clinical NSCLC samples. IGF2BP3 expression did not affect METTL5 expression but was regulated by METTL5. IGF2BP3 overexpression mitigated the METTL5 knockdown-induced impaired cell proliferation. Meanwhile, IGF2BP3 knockdown suppressed METTL5-mediated NSCLC cell proliferation. The Co-IP assay results revealed the interaction between METTL5 and IGF2BP3 in NSCLC cells. IGF2BP3 knockdown suppressed tumor growth, whereas IGF2BP3 overexpression enhanced tumor volume and quality. Conclusion: METTL5 induces NSCLC cell proliferation by interacting with IGF2BP3. Thus, METTL5 is a potential biomarker and a therapeutic target for NSCLC.

Circular RNA NFIX Functions as an Oncogene in Non-Small Cell Lung Cancer by Modulating the miR-214-3p/TRIAP1 Axis.

circRNA NFIX has been shown to exist as an oncogene in glioma. But its expression and role in NSCLC (non-small cell lung cancer) are still unclear. This research aimed to discover the expression and function of circRNA NFIX in NSCLC. In this research, qRT-PCR was utilized to investigate the expression levels of circRNA NFIX, miRNA-214-3p, and TRIAP1 in NSCLC tissues and cell lines. The binding sites between circRNA NFIX/TRIAP1 and miRNA-214-3p were predicted using the Starbase. These interactions were further validated using a double luciferase reporter assay. Cell proliferation and apoptosis were assessed through MTT and flow cytometry, respectively. The expression of apoptosis-related proteins was measured by western blot assay. miRNA-214-3p could link with circRNA NFIX. circRNA NFIX was upregulated, while miRNA-214-3p was downregulated in NSCLC cell lines and clinical samples. Besides, suppression of circRNA NFIX repressed cell proliferation and induced apoptosis in NSCLC cells by upregulating miRNA-214-3p expression. Besides, the data indicated that TRIAP1 was a target of miRNA-214-3p, and it was negatively regulated by miRNA-214-3p in NSCLC cells. The excessive expression of miRNA-214-3p suppressed NSCLC cell proliferation and increased apoptosis. In addition, overexpression of TRIAP1 significantly reversed the effects on NSCLC cells caused by miRNA-214-3p mimic. circRNA NFIX silencing repressed the proliferation of NSCLC cells and induced cell apoptosis by regulating the miR-214-3p/TRIAP1 axis, which was a potential diagnostic and therapeutic target for NSCLC.

Assessment of carcinoembryonic antigen-related cell adhesion molecule 5 expression by immunohistochemistry in real-world clinical samples of non-small cell lung cancer.

e20013 Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an emerging target of antibody-drug conjugate therapy for non-small cell lung cancer (NSCLC). High expression of CEACAM5 has been reported in approximately 25% of patients with lung adenocarcinoma (Lefebvre AM, et al. Lung Cancer. 2023; 184:107356). However, CEACAM5 expression has not been systematically examined in a real-world patient population with NSCLC. In this study, we evaluated our experience in the assessment of CEACAM5 expression by immunohistochemistry (IHC) in routinely diagnosed clinical samples. Methods: We assessed the expression of CEACAM5 by IHC on clinical biopsy/resection samples of consecutively diagnosed NSCLC from September 2022 to June 2023 at our institution, using the anti-CEACAM5 clone 769 antibody assay protocol developed for tusamitamab ravtansine clinical trials. Only cases adequate for PD-L1 22C3 and Oncomine Comprehensive Assay v3 NGS assay were included. CEACAM5 expression was scored independently by three pathologists based on the method proposed by Lefebvre et al. Expression levels were categorized as high (≥ 50% tumor cells at ≥ 2+ intensity), moderate (1–49% tumor cells at ≥ 2+ intensity), and negative (0 or 1+ intensity). Interrater reliability was assessed by Kendall’s coefficient of concordance (KCC) and Fleiss’ Kappa. Discordant classification was reviewed with the final classification achieved by consensus. Association with PD-L1 TPS and NGS results was determined by Chi-squared test. Results: This study cohort consisted of 150 consecutively diagnosed NSCLC, including 133 adenocarcinomas and 17 squamous cell carcinomas. The samples were derived from both primary (n = 138) and metastatic sites (n = 12). Both membranous and cytoplasmic CEACAM5 expression was observed in neoplastic cells, but was consistently absent in nonneoplastic cells. The prevalence of consensus high CEACAM5 membranous expression was 21% (n = 32) overall, 20% in primary (n = 28) and 33% (n = 4) in metastatic sites. The level of interrater agreement across all categories was moderate (KCC = 0.77). Interrater agreement between high CEACAM5 expression versus moderate/negative categories combined was also moderate (Kappa = 0.60). Challenging features included the determination of staining intensity and mixed membranous and cytoplasmic staining pattern, and cases near the cut-offs of the defined categories. CEACAM5 expression was not associated with PD-L1 TPS (p = 0.42) or EGFR mutations (p = 0.44). Conclusions: Our data showed that approximately 20% of routinely diagnosed clinical NSCLC samples had high CEACAM5 expression by IHC. The interrater reliability on the determination of high CEACAM5 expression was moderate among the three pathologists. We highlighted the challenging aspects in the evaluation of this biomarker.

Metabolic profile and therapeutic vulnerabilities of multi-omic characterization ofKRAS/STK11/KEAP1 co-mutant non–small cell lung cancer (NSCLC).

8576 Background: KRAS-mutant NSCLC with co-occurring loss-of-function mutations in STK11 and KEAP1(KSK) are remarkably aggressive and poorly responsive to chemo- and immunotherapy. Novel therapeutic strategies are urgently needed to improve outcomes for patients with KRAS/STK11/KEAP1co-mutant NSCLC (KSK). We interrogated the transcriptomic landscape using a large real-world dataset of NSCLC to identify therapeutic vulnerabilities that may help guide treatment selections in KSK. Methods: KRAS mutant NSCLC clinical samples (N=7210) were tested with NextGen Sequencing of DNA (592-gene panel or whole exome sequencing) and RNA (whole transcriptome sequencing) at Caris Life Sciences (Phoenix, AZ). Specimens were stratified into KRASMUT/STK11MUT/KEAP1MUT (KSK; N=698), KRASMUT/STK11MUT/KEAP1WT (KS; N=786), KRASMUT/STK11WT/KEAP1MUT (KK; N=466), and KRASMUT/STK11WT/KEAP1WT (K; N=4536). Real-world overall survival (OS) data was obtained from insurance claims and investigated using Kaplan-Meier estimates. Additionally, an in vitro CRISPR screen, bulk RNA sequencing, and phospho-kinase arrays were performed in KRAS/STK11/KEAP1 co-mutant, single mutant, and wild-type cell lines to better characterize these models. Results: KEAP1mutations (median OS: KK=7.83 months, KSK=7.23 months) were strongly associated with poor OS compared to STK11 mutations (median OS: KS=17.6months). Pathways significantly upregulated in KSK clinical samples included fatty acid metabolism and redox pathways. When compared to single mutant or wild-type conditions, KSK clinical samples had significant up-regulation of genes involved in ferroptosis evasion and metabolism like SLC7A11(KSK/KK=1.28, KSK/KS=4.82, KSK/K=10.24; all q<0.01) and SCD1(KSK/KS=1.19, KSK/K=1.24; both q<0.01). To confirm our clinical findings, CRISPR/Cas9-based genetic screening identified SCD1, and SLC7A11 master regulators of fatty acid metabolism and ferroptosis, as a potential therapeutic target in the KSK cell lines. SCD1 inhibition augmented the effect of ferroptosis inducer, erastin. Targeting SCD1 led to global metabolomic changes in KSK cells, including key pathways involved in lipid and glucose metabolismas compared to single mutants or wild-type cells. Finally, pharmacological inhibition of SCD1 caused significant tumor regression and augmented the anti-tumor response of SLC7A11 inhibitor, imidazole ketone erastin (IKE), in KSK syngeneic mouse models. Conclusions: Our study is the first to highlight the importance of the ferroptosis regulators, SCD1-SLC7A11,in regulating unique metabolic and ferroptosis evasion pathways in KSK patient tumor and preclinical models. The study data furthers the understanding of ferroptosis in NSCLCand the potential to translate SCD1 inhibitors and ferroptosis inducers in KRAS NSCLC clinical trials.

CircNOX4 activates an inflammatory fibroblast niche to promote tumor growth and metastasis in NSCLC via FAP/IL-6 axis

BackgroundCancer-associated fibroblasts (CAFs) orchestrate a supportive niche that fuels cancer metastatic development in non-small cell lung cancer (NSCLC). Due to the heterogeneity and plasticity of CAFs, manipulating the activated phenotype of fibroblasts is a promising strategy for cancer therapy. However, the underlying mechanisms of fibroblast activation and phenotype switching that drive metastasis remain elusive.MethodsThe clinical implications of fibroblast activation protein (FAP)-positive CAFs (FAP+CAFs) were evaluated based on tumor specimens from NSCLC patients and bioinformatic analysis of online databases. CAF-specific circular RNAs (circRNAs) were screened by circRNA microarrays of primary human CAFs and matched normal fibroblasts (NFs). Survival analyses were performed to assess the prognostic value of circNOX4 in NSCLC clinical samples. The biological effects of circNOX4 were investigated by gain- and loss-of-function experiments in vitro and in vivo. Fluorescence in situ hybridization, luciferase reporter assays, RNA immunoprecipitation, and miRNA rescue experiments were conducted to elucidate the underlying mechanisms of fibroblast activation. Cytokine antibody array, transwell coculture system, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the downstream effectors that promote cancer metastasis.ResultsFAP+CAFs were significantly enriched in metastatic cancer samples, and their higher abundance was correlated with the worse overall survival in NSCLC patients. A novel CAF-specific circRNA, circNOX4 (hsa_circ_0023988), evoked the phenotypic transition from NFs into CAFs and promoted the migration and invasion of NSCLC in vitro and in vivo. Clinically, circNOX4 correlated with the poor prognosis of advanced NSCLC patients. Mechanistically, circNOX4 upregulated FAP by sponging miR-329-5p, which led to fibroblast activation. Furthermore, the circNOX4/miR-329-5p/FAP axis activated an inflammatory fibroblast niche by preferentially inducing interleukin-6 (IL-6) and eventually promoting NSCLC progression. Disruption of the intercellular circNOX4/IL-6 axis significantly suppressed tumor growth and metastatic colonization in vivo.ConclusionsOur study reveals a role of the circRNA-induced fibroblast niche in tumor metastasis and highlights that targeting the circNOX4/FAP/IL-6 axis is a promising strategy for the intervention of NSCLC metastasis.

Long noncoding RNA lnc-SNAPC5-3:4 inhibits malignancy by directly upregulating miR-224-3p in non-small cell lung cancer

The mounting body of evidence demonstrates the growing importance of long noncoding RNAs in the advancement of tumors. This study aimed to investigate the molecular mechanism of lnc-SNAPC5-3:4 in non-small cell lung cancer (NSCLC). We investigated the expression of miR-224-3p and lnc-SNAPC5-3:4 in clinical NSCLC samples and NSCLC cell lines using reverse transcription polymerase chain reaction (RT-PCR). In vitro studies, A549 cell growth was estimated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EDU), and flow cytometry assays. In vivo studies, NSCLC tumorigenesis was determined using xenograft tumor mouse models, tumor growth was evaluated using antigen Kiel 67 (Ki67) staining, and tumor apoptosis was detected through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The relationship between lnc-SNAPC5-3:4 and miR-224-3p was determined by luciferase reporter gene assay. Results indicated that the expression of lnc-SNAPC5-3:4 was observed to be downregulated in NSCLC tissues and cell lines. After overexpression of lnc-SNAPC5-3:4 in cultured A549 cells, proliferation decreased and apoptosis increased. Furthermore, the expression of miR-224-3p was targeted and negatively regulated by lnc-SNAPC5-3:4. The lnc-SNAPC5-3:4 upregulation inhibited cell proliferation and promoted apoptosis, which was partially blocked by miR-224-3p overexpression in A549 cells. In addition, we constructed a subcutaneous inoculation model using BALB/c nude mice, and the results indicated that lnc-SNAPC5-3:4 overexpression restrained the growth of subcutaneous tumors, decreased Ki67 expression levels, and increased apoptosis, as indicated by TUNEL staining in nude mice. However, miR-224-3p transfection resulted in the reversal of the inhibitory effect of lnc-SNAPC5-3:4 on tumor growth. In conclusion, our study revealed that lnc-SNAPC5-3:4 inhibits tumor progression in NSCLC by targeting miR-224-3p. This study provides a potential therapeutic target for inhibiting NSCLC progression.

TRIM3 facilitates ferroptosis in non-small cell lung cancer through promoting SLC7A11/xCT K11-linked ubiquitination and degradation.

Ferroptosis, a unique form of regulated necrotic cell death, is caused by excessive iron-dependent lipid peroxidation. However, the underlying mechanisms driving ferroptosis in human cancers remain elusive. In this study, we identified TRIM3, an E3 ubiquitin-protein ligase, as a key regulator of ferroptosis. TRIM3 is downregulated in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), two major types of non-small cell lung cancer (NSCLC). Forced expression of TRIM3 promotes cell death by enhancing the cellular level of ROS and lipid peroxidation. Moreover, our in vivo study determined that TRIM3 overexpression diminishes the tumorigenicity of NSCLC cells, indicating that TRIM3 functions as a tumor suppressor in NSCLC. Mechanistically, TRIM3 directly interacts with SLC7A11/xCT through its NHL domain, leading to SCL7A11 K11-linked ubiquitination at K37, which promotes SLC7A11 proteasome-mediated degradation. Importantly, TRIM3 expression exhibits a negative correlation with SCL7A11 expression in clinical NSCLC samples, and low TRIM3 expression is associated with a worse prognosis. This study reveals that TRIM3 functions as a tumor suppressor that can impede the tumorigenesis of NSCLC by degrading SLC7A11, suggesting a novel therapeutic strategy against NSCLC.

LncRNA KCNQ1OT1/miR-496/HMGB1 Signaling Axis Promotes Invasion and Migration of Non-small Cell Lung Cancer Cells.

Enhanced invasion and migration of non-small cell lung cancer (NSCLC) cells is the major cause of metastasis and poor prognosis in NSCLC. This study was conducted to investigate the role and mechanism of lncRNA KCNQ1OT1 in the proliferation, invasion, and migration of NSCLC cells. The expression of KCNQ1OT1 in NSCLC was analyzed in the StarBase database, and the target miRNA of KCNQ1OT1 as well as the target genes of the miRNA was predicted. Then, the mRNA expression levels of KCNQ1OT1, miR-496, and HMGB1 were detected in clinical tissue samples and cells by qRT-PCR assay. Besides, the protein levels of HMGB1 were detected by Western blot. MTT assay, transwell assay, and scratch assay were used to determine the proliferation, invasion, and migration ability of NSCLC cells, respectively. Correlation analysis was performed to assess the correlation between the expression of KCNQ1OT1, miR-496, and HMGB1 in clinical NSCLC samples. Dual-luciferase reporter gene assay was conducted to analyze the interaction between KCNQ1OT1 and miR-496 and between miR-496 and HMGB1. The database results showed that KCNQ1OT1 was highly expressed in NSCLC. Similarly, we found that the expression level of KCNQ1OT1 was significantly higher in NSCLC tissues and cells than that in the corresponding normal tissues and cells. The results of MTT assay, transwell assay, and scratch assay demonstrated that KCNQ1OT1 significantly enhanced the proliferation, invasion, and migration of NSCLC cells. Further mechanism exploration revealed that KCNQ1OT1 could sponge miR-496, and miR-496 directly targeted and regulated the expression of HMGB1. The expression of miR-496 and either KCNQ1OT1 or HMGB1 were negatively correlated in NSCLC, while the expression of KCNQ1OT1 and HMGB1 were positively correlated. Compared with normal paracancer tissues, miR-496 was much lower and HMGB1 was much higher expressed in NSCLC tissues. The results of cotransfection also further demonstrated that miR-496 inhibitor or sh-HMGB1 cotransfected with sh-KCNQ1OT1 could significantly decrease or increase the ability of sh-KCNQ1OT1 to inhibit the proliferation, invasion, and migration of H1299 cells, respectively. In conclusion, lncRNA KCNQ1OT1 promotes the invasion and migration of NSCLC cells through miR-496/HMGB1 signaling axis.

Abstract 5504: A tumor-promoting senescent secretome triggered by platinum chemotherapy exploits a targetable TGFβR1/Akt-mTOR axis in lung cancer

Abstract Background Platinum-based chemotherapy is commonly used for the treatment of non-small cell lung cancer (NSCLC), yet clinical outcomes and survival rates remain very poor and continues to be a cancer of unmet need. Recent evidence points to cellular senescence, a response to oncogenic- and therapy-induced genotoxic stress, and its associated proinflammatroy secretory phenotype (SASP) as an emerging hallmark of cancer. Hence, targeting senescence and its associated tumor-promoting activities is emerging as a novel and promising therapeutic strategy but largely unexplored in lung cancer. Experimental Procedures Our study includes a variety of methodologies, including: - Functional in vitro analyses: proliferation, colony formation, tumor spheres, migration assays. High throughput unbiased analyses: RNAseq, proteomics and microenvironment microarrays (MEMA). - In vivo models of lung cancer: xenografts, orthotopic and genetically engineered mouse models. Longitudinal tumor burden by IVIS and microCT and mouse survival. - Clinical samples: Histological and in silico analyses. Results Here we show that cisplatin-derived SASP enhances the malignant phenotype of lung cancer cells. Using xenograft, orthotopic and KrasG12V-driven murine NSCLC models, we demonstrate that cisplatin-induced senescent cells strongly promote tumor progression. Mechanistically, we find that a TGF-β-enriched SASP drives pro-proliferative effects through TGFβR1 and Akt/mTOR pathway activation. We validate the translational relevance of chemotherapy-induced SASP using clinical NSCLC samples from a trial with patients who received neoadjuvant platinum-based chemotherapy. Importantly, TGFβR1 inhibition with galunisertib or senolytic treatment significantly reduces tumor promotion driven by cisplatin-induced senescence. Finally, we demonstrate, using distinct murine NSCLC models, that addition of TGFBR1 inhibitors to platinum-based chemotherapy reduces tumor burden and improves survival, providing pre-clinical proof-of-concept for future trial designs on combination therapies. Conclusions and Impact We regard this work as a major conceptual dissection of tumor-promoting activities of therapy-induced senescence, and a preclinical advance in the management of lung cancer with potential wide therapeutic applications in precision medicine. Of note, we expect our findings to have implications for multiple cancer types, including ovarian, breast, mesothelioma, oesophageal, head and neck, bladder and brain cancers, where platinum-based therapies remain important standard-of-care treatments. Citation Format: Estela González-Gualda, David Macias, Samir Morsli, José Ezequiel Martín, Hui-Ling Ou, Mary Denholm, Ioana Olan, Reuben Hoffmann, Mark Dane, Dimitris Veroutis, Guillermo Medrano, Francisca Mulero, Carla P. Martins, Mariano Barbacid, Vassilis Gorgoulis, James E. Korkola, Doris M. Rassl, Gary J. Doherty, Robert C. Rintoul, Masashi Narita, Daniel Muñoz-Espín. A tumor-promoting senescent secretome triggered by platinum chemotherapy exploits a targetable TGFβR1/Akt-mTOR axis in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5504.

Universal and highly accurate detection of circulating tumor DNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system

Circulating tumor DNA (ctDNA) is a promising biomarker for real-time, minimally invasive diagnostics and monitoring in patients with non-small cell lung cancer (NSCLC), especially when representative tissue biopsies are not available. However, the current methods for ctDNA detection are cumbersome and expensive. While advanced CRISPR/Cas-based assays offer advantages of simplicity, low cost and high sensitivity, their application for ctDNA detection is restricted by the requirement of a protospacer adjacent motif (PAM) near the mutation site and off-target cleavages (i.e., false-positive results) due to the extreme similarities between the mutant and wild-type sequences, especially single nucleotide variants. Herein, we propose a novel strategy comprising recombinase polymerase amplification (RPA) and CRISPR/Cas12a to detect ctDNA with high universality and accuracy. The use of artificially inserted PAMs by modified RPA primers or suboptimal PAMs unlocks the PAM restriction; introducing single- or double-base mismatches in CRISPR RNA effectively reduces the off-target effects and improves the specificity to single-base resolution. Under optimized conditions, this method detected ctDNA mutations with a limit of detection at 100 aM and identified mutations down to 0.02% variant allele frequency in 50 min, requiring only isothermal control. We successfully applied this method to multiple clinical samples of NSCLC and the results were validated using real-time polymerase chain reaction analysis. In summary, we established a rapid, sensitive, universal and highly accurate method for ctDNA detection that has great potential application in the early diagnosis, therapy guidance and prognosis prediction of NSCLC.

CRISPR-Based Fluorescent Reporter (CBFR) Assay for Sensitive, Specific, Inexpensive, and Visual Detection of a Specific EGFR Exon 19 Deletion in NSCLC.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Mutations in the EGFR gene, including deletions in exon 19 and the mutation L858R, induce responsiveness of non-small cell lung cancer (NSCLC) to a group of drugs known as tyrosine kinase inhibitors. Here, we report the development of the CRISPR-based fluorescent reporter (CBFR) assay including a two-step strategy combining PCR amplification and Cas12a-driven cleavage to detect the delE746_A750 subtype of EGFR exon 19 deletions. Sensitivity and specificity of the CBFR assay were analyzed with different concentrations of fluorescence reporter and different amounts of PCR product. The results demonstrated that increasing the fluorescent reporter to 4μM and the PCR product to 5μl enhanced sensitivity. The CBFR assay could detect EGFR exon 19 deletion even with a frequency of 1% in samples. In clinical NSCLC samples, optimized CBFR assay enabled visual detection of the delE746_A750 subtype in less than 1h. The CBFR assay provides a sensitive, specific, and simple strategy designed based on a straightforward and inexpensive process. We suggest that the CBFR assay could serve as a diagnostic approach to detect mutations, deletions, and pathogens in underequipped laboratories and promote personalized therapeutic approaches.

A SERS microfluidic chip based on hpDNA-functioned Au-Ag nanobowl array for efficient simultaneous detection of non-small cell lung cancer-related microRNAs

In this paper, a novel surface-enhanced Raman scattering (SERS) microfluidic chip is proposed for the simultaneous and efficient detection of miR-92a and miR-339-3p, two microRNAs (miRNAs) related to non-small cell lung cancer (NSCLC). The sample solution can flow along the microchannel into the reaction zone during the assay, driven by the capillary force generated by the comb-like structured channel. A one-step paired hybridization reaction with the corresponding hairpin DNA (hpDNA) on the hpDNA-functioned Au-Ag nanobowl (Au-AgNBs) array takes place when the target strand is present in the sample solution, amplifying the SERS signal. The detection limits of miR-92a and miR-339-3p in serum were 44.36 aM and 63.58 aM, respectively, using this method, with a detection and analysis time of only 8 min. The microfluidic chip showed good specificity, reproducibility, and stability. Finally, the method was used to detect miR-92a and miR-339-3p in the serum of NSCLC clinical samples, and the results were consistent with the quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, this new SERS microfluidic chip is a very promising tool for detecting miRNA expression levels, which can help in the early diagnosis and prognosis of NSCLC.

Clinical significance of ALKBH4 expression in non-small cell lung cancer.

BackgroundGene methylation is deeply involved in epigenetics and affects both the development and maintenance of homeostasis and carcinogenesis. ALKBH4 is a member of the AlkB homolog (ALKBH) family that controls demethylation of DNA and RNA.MethodsThis study enrolled 160 patients with non-small cell lung cancer (NSCLC) who underwent complete resection. The expression of ALKBH4 in cancer tissue was evaluated by immunohistochemistry. The correlation among the expression of ALKBH4, clinicopathological factors, and prognostic outcome was evaluated.ResultsIn the NSCLC clinical samples, the expression of ALKBH4 was identified not only in cell membranes but also in the cytoplasm of cancer cells. In 140 of 160 cases, ALKBH4 was more highly expressed in the cancerous tissue than in the surrounding normal tissue. The proportion of cancer cells expressing ALKBH4 was higher in adenocarcinoma than in other histological types. In addition, the expression intensity of ALKBH4 in each cancer cell was also stronger in adenocarcinoma than in squamous cell carcinoma. The expression of ALKBH4 was not associated with clinicopathological factors, except for histological type. In adenocarcinoma, the recurrence-free survival (RFS) and overall survival (OS) rates were significantly lower in the ALKBH4-positive group than in the ALKBH4-negative group (P=0.008, 0.031, respectively). A multivariate logistic regression analysis indicated that the ALKBH4 expression was an independent prognostic factor for RFS (P=0.003) and OS (P=0.013). The expression of ALKBH4 was observed in all four patients with adenocarcinoma in situ.ConclusionsThe ALKBH4 expression may be a useful predictor of the postoperative outcomes of lung adenocarcinoma (LUAD) patients.

LINC00514 facilitates cell proliferation, migration, invasion, and epithelial-mesenchymal transition in non-small cell lung cancer by acting on the Wnt/β-catenin signaling pathway

ABSTRACT The long non-coding RNA (lncRNA) LINC00514 was identified to play an essential oncogenic function in different human cancers, but its effects in non-small cell lung cancer (NSCLC) are yet to be elucidated. In this study, we evaluated the function of LINC00514 in NSCLC. LINC00514 expression and prognosis in NSCLC were analyzed using qRT-PCR and online bioinformatic tools. The bioeffects of LINC0514 in NSCLC cells were examined using cell counting kit-8, colony formation, and transwell assays. Western blotting was used to measure the expression of the target proteins. The LINC00514 regulation of the Wnt/β-catenin signaling pathway was assessed using a specific agonist (LiCl) and luciferase reporter assay. We found that LINC00514 expression was elevated in NSCLC cells and clinical samples and that increased LINC00514 expression predicted poorer patient prognosis. Silencing LINC00514 suppresses proliferation, migration, and invasion of NSCLC cells. Downregulation of LINC00514 inhibited Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT). Moreover, suppression of the biological phenotypes of NSCLC cells induced by LINC00514 gene silencing was restored after LiCl treatment. Finally, we found that silencing LINC00514 attenuated the growth of xenograft tumors in vivo. Altogether, this study provides the latest convincing evidence that LINC00514 facilitates the malignant biological behavior of NSCLC cells through activation of the Wnt/β-catenin pathway, which might offer a beneficial approach for the treatment of NSCLC.

Targeting SNORA38B attenuates tumorigenesis and sensitizes immune checkpoint blockade in non-small cell lung cancer by remodeling the tumor microenvironment via regulation of GAB2/AKT/mTOR signaling pathway

BackgroundNon-coding RNAs (ncRNAs), including small nucleolar RNAs (snoRNAs), are widely involved in the physiological and pathological processes of human beings. While up to date, although considerable progress has been achieved...

Inhibin βA is an independent prognostic factor that promotes invasion via Hippo signaling in non‑small cell lung cancer.

Inhibin βA (INHBA) serves a prognostic and tumor-promoting role in numerous types of cancer. The present study aimed to determine the clinical significance of INHBA in non-small cell lung cancer (NSCLC) and the mechanisms underlying its potential tumor-promoting effect. INHBA expression was detected in clinical NSCLC samples using immunohistochemistry. In vivo loss- and gain-of-function studies were performed to determine the effects of INHBA on NSCLC invasion. In addition, protein and mRNA expression levels of INHBA, yes-associated protein (YAP), large tumor suppressor 1/2 kinase (LATS1/2), connective tissue growth factor, cysteine rich angiogenic inducer 61 and Merlin were assessed using western blotting and reverse transcription-quantitative PCR, respectively, to investigate the mechanism by which INHBA may affect the invasion of NSCLC. The present study revealed that INHBA was significantly upregulated in 238 clinical NSCLC samples compared with its expression levels in paired adjacent non-cancerous tissues, and in metastatic nodules compared with in primary tumors. Notably, high INHBA expression was statistically associated with clinicopathological features, including poor differentiation and advanced tumor stage. INHBA positivity was statistically related to decreased 5-year overall survival, for which INHBA was an independent prognostic factor. Furthermore, INHBA promoted NSCLC invasion in vitro. In NSCLC, INHBA expression was associated with the nuclear levels of YAP and INHBA overexpression enhanced the invasive abilities of NSCLC cells via inhibiting the Hippo pathway. Mechanistically, INHBA inhibited l LATS1/2 phosphorylation and induced YAP nuclear translocation by downregulating the protein expression levels of Merlin. In conclusion, INHBA may negatively regulate the Hippo pathway to act as a tumor promotor, and could represent a marker of prognosis in NSCLC.

LINC00852 is associated with poor prognosis in non-small cell lung cancer patients and its inhibition suppresses cancer cell proliferation and chemoresistance via the hsa-miR-145-5p/KLF4 axis.

Emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in human cancer. In the present study, we examined the expression, prognostic implications and functional roles of a lncRNA, LINC00852 in non-small cell lung cancer (NSCLC). LINC00852 expression was examined by quantitative real-time PCR (qRT-PCR) in both NSCLC clinical samples and in vitro NSCLC cell lines. In patients with NSCLC, postoperative overall survival was estimated according to endogenous LINC00852 expression in their cancerous lung tissues. In NSCLC cell line SW900 and H441 cells, LINC00852 was down-regulated to examine its effects on cancer proliferation, cisplatin chemoresistance and cell-cycle transition in vitro, as well as tumorigenicity in vivo. The potential downstream target of LINC00852, the axis of human microRNA-145-5p (hsa-miR-145-5p) and Kruppel-like factor 4 (KLF4) gene, was investigated in NSCLC, by dual-luciferase assay, qRT-PCR and genetic knockdown functional assays. LINC00852 is up-regulated in both NSCLC tumors and NSCLC cell lines. High LINC00852 expression was significantly correlated with NSCLC patients' short overall survival. In NSCLC cell lines, LINC00852 down-regulation had anti-cancer effects by suppressing cancer cell proliferation, cisplatin chemoresistance and cell-cycle transition in vitro, as well as explant growth in vivo. Moreover, the hsa-miR-145-5p/KLF4 axis was demonstrated to be directly regulated by LINC00852 in NSCLC. Inhibiting hsa-miR-145-5p or overexpressing KLF4 could reverse the LINC00852-down-regulation-induced anti-cancer effects on NSCLC cancer cell proliferation and chemoresistance. LINC00852 may be a prognostic biomarker for NSCLC. The epigenetic signaling pathway of LINC00852/hsa-miR-145-5p/KLF4 may be considered as a novel molecular target for fighting NSCLC.

Targeting DNAJC19 overcomes tumor growth and lung metastasis in NSCLC by regulating PI3K/AKT signaling

BackgroundSome driver oncogenes are still unknown in non-small-cell lung cancer (NSCLC). DNAJC19, a major component of the translocation machinery of mitochondrial membranes, is a disease-associated protein. Herein, we report the role of DNAJC19 in NSCLC cell growth and metastasis.MethodsImmunohistochemistry (IHC) was performed to investigate DNAJC19 expression in NSCLC clinical samples. For knockdown or overexpression assays in A549 or NCI-H1299 lung cancer cells, lentiviral vectors were used. After assessment of cell functions, DNAJC19-knockdown A549 cells were further applied to establish mouse xenograft and metastasis tumor models. Assessments based on the RNA-seq data, western blotting, PCR and IHC were performed for the mechanistic study.ResultsExpression of DNAJC19 was higher in tumors than in noncancerous adjacent tissues. Survival analysis indicated that low DNAJC19 levels were correlated with an increased progression-free survival rate. ShRNA-mediated knockdown of DNAJC19 markedly inhibited cell growth, viability, migration and invasion. Moreover, RNA-seq analysis revealed that the PI3K/AKT signaling pathway was involved in molecular events when A549 cells were treated with shDNAJC19. In addition, DNAJC19 knockdown decreased PI3Kp85a, AKT and p-AKT expression in A549 cells, and cellular functions were greatly rescued in DNAJC19-knockdown A549 cells by ectopic overexpression of AKT. Furthermore, tumor xenograft growth and lung metastasis were markedly repressed in the shDNAJC19 group compared to the control group. As expected, the expression levels of DNAJC19, PI3K and AKT in xenograft mouse samples were also lower in the shDNAJC19 group than in the shCtrl group.ConclusionsDNAJC19 greatly promotes NSCLC cell growth and lung metastasis by regulating PI3K/AKT signaling, providing a novel therapeutic target for treating NSCLC patients.

Programmed Death Ligand 1 (PD-L1) Expression and its Association with Clinicopathologic Profile in Patients with Non-Small Cell Lung Cancer in a Philippine Tertiary Medical Center

Introduction. The current management of advanced non-small cell lung cancer (NSCLC) includes the characterization of Programmed Death Ligand-1 (PD-L1) expression for potential immune checkpoint inhibitor treatment. There is currently no available data regarding the patterns of PD-L1 expression in NSCLC, as well as their association with clinicopathologic profile in Filipino patients. Methodology. Clinicopathologic characteristics of 187 consecutive NSCLC clinical samples with PD-L1 testing using the clone 22C3 pharmaDx kit were collected. The presence of stromal tumor-infiltrating lymphocytes (TILs) were assessed in hematoxylin and eosin-stained slides. PD-L1 expression on tumor cells (TC) and stromal TILs were evaluated. Results. Of the 187 cases, there were 112 males and 75 females. The mean age at diagnosis was 66.4 years old (32-92 years old). It is composed of 131 cases of Adenocarcinoma, 15 Squamous cell carcinoma, 4 Adenosquamous carcinoma, 32 Non-small cell carcinoma, not otherwise specified, 3 poorly differentiated malignancy, 1 Large cell carcinoma, and 1 Mucinous carcinoma. Specimen types included 17 pleural fluid cell blocks, 60 tumor cell block samples, and 110 tissue biopsies. Tumor cell PD-L1 expression was identified in 59.1% of the 110 tissue biopsies. PD-L1 TPS for histologic specimens are as follows: TPS >50%, TPS 1-49%, and TPS <1% were observed in 23.6%, 35.5%, and 40.9% in our lung cancer cohort, respectively. Of the 77 cytology specimens, 50.6% presented with TC PD-L1 expression. TPS for this subgroup include: 49.4% with no PD-L1 expression, 35.1% with low PD-L1 expression, and 15.6% showing high PD-L1 expression. PD-L1 expression on TC did not correlate with age, sex, or histology for both specimen type subgroups. Stromal tumor-infiltrating lymphocytes were noted in 74.5% of tissue biopsies. Tumor cell block samples did not demonstrate stromal TILs. For tissue biopsies, female gender and TPS 1-49% were more likely to have <50% PD-L1 expression on TILs. Conclusion. Overall TC PD-L1 expression was observed in more than half (55.6%) of NSCLC patients in our cohort. The prognostic value of PD-L1 and clinical response to immune checkpoint inhibitors in the Filipino population needs to be further investigated.