Human Non-small Cell Lung Cancer Research Articles

NF-κB pathway affects silica nanoparticle-induced fibrosis via inhibited inflammatory response and epithelial-mesenchymal transition in 3D co-culture

Long-term inhalation of silica nanoparticles (SiNPs) can induce pulmonary fibrosis (PF), nevertheless, the potential mechanisms remain elusive. Herein, we constructed a three-dimensional (3D) co-culture model by using Matrigel to investigate the interaction among different cells and potential regulatory mechanisms after SiNPs exposure. Methodologically, we dynamically observed the changes in cell morphology and migration after exposure to SiNPs by co-culturing mouse monocytic macrophages (RAW264.7), human non-small cell lung cancer cells (A549), and medical research council cell strain-5 (MRC-5) in Matrigel for 24 h. Subsequently, we detected the expression of nuclear factor kappa B (NF-κB), inflammatory factor and epithelial-mesenchymal transition (EMT) markers. The results showed that SiNPs produced toxic effects on cells. In the 3D co-culture state, the cell's movement velocity and displacement increased, and the cell migration ability was enhanced. Meanwhile, the expression of inflammatory factor tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) were upregulated, the epithelial marker E-cadherin (E-cad) was downregulated, the mesenchymal marker N-cadherin (N-cad) and myofibroblast marker alpha-smooth muscle actin (α-SMA) expression were upregulated, while NF-κB expression was also upregulated after SiNPs exposure. We further found that cells were more prone to transdifferentiate into myofibroblasts in the 3D co-culture state. Conversely, utilizing the NF-κB-specific inhibitor BAY 11–7082 effectively downregulated the expression of TNF-α, IL-6, interleukin-1β (IL-1β), N-cad, α-SMA, collagen-I (COL I), and fibronectin (FN), the expression of E-cad was upregulated. These findings suggest that NF-κB is involved in regulating SiNPs-induced inflammatory, EMT, and fibrosis in the 3D co-culture state.

Inhalable siRNA Nanoparticles for Enhanced Tumor-Targeting Treatment of KRAS-Mutant Non-Small-Cell Lung Cancer.

Kirsten rat sarcoma (KRAS) is the most commonly mutated oncogene in lung cancers. Gene therapy is emerging as a promising cancer treatment modality; however, the systemic administration of gene therapy has been limited by inefficient delivery to the lungs and systemic toxicity. Herein, we report a noninvasive aerosol inhalation nanoparticle (NP) system, termed "siKRAS@GCLPP NPs," to treat KRAS-mutant non-small-cell lung cancer (NSCLC). The self-assembled siKRAS@GCLPP NPs are capable of maintaining structural integrity during nebulization, with preferential distribution within the tumor-bearing lung. Inhalable siKRAS@GCLPP NPs show not only significant tumor-targeting capability but also enhanced antitumor activity in an orthotopic mouse model of human KRAS-mutant NSCLC. The nebulized delivery of siKRAS@GCLPP NPs demonstrates potent knockdown of mutated KRAS in tumor-bearing lungs without causing any observable adverse effects, exhibiting a better biosafety profile than the systemic delivery approach. The results present a promising inhaled gene therapy approach for the treatment of KRAS-mutant NSCLC and other respiratory diseases.

Phosphatidic Acid Stimulates Lung Cancer Cell Migration through Interaction with the LPA1 Receptor and Subsequent Activation of MAP Kinases and STAT3.

Phosphatidic acid (PA) is a key bioactive glycerophospholipid that is implicated in the regulation of vital cell functions such as cell growth, differentiation, and migration, and is involved in a variety of pathologic processes. However, the molecular mechanisms by which PA exerts its pathophysiological actions are incompletely understood. In the present work, we demonstrate that PA stimulates the migration of the human non-small cell lung cancer (NSCLC) A549 adenocarcinoma cells, as determined by the transwell migration assay. PA induced the rapid phosphorylation of mitogen-activated protein kinases (MAPKs) ERK1-2, p38, and JNK, and the pretreatment of cells with selective inhibitors of these kinases blocked the PA-stimulated migration of cancer cells. In addition, the chemotactic effect of PA was inhibited by preincubating the cells with pertussis toxin (PTX), a Gi protein inhibitor, suggesting the implication of a Gi protein-coupled receptor in this action. Noteworthy, a blockade of LPA receptor 1 (LPA1) with the specific LPA1 antagonist AM966, or with the selective LPA1 inhibitors Ki1645 or VPC32193, abolished PA-stimulated cell migration. Moreover, PA stimulated the phosphorylation of the transcription factor STAT3 downstream of JAK2, and inhibitors of either JAK2 or STAT3 blocked PA-stimulated cell migration. It can be concluded that PA stimulates lung adenocarcinoma cell migration through an interaction with the LPA1 receptor and subsequent activation of the MAPKs ERK1-2, p38, and JNK, and that the JAK2/STAT3 pathway is also important in this process. These findings suggest that targeting PA formation and/or the LPA1 receptor may provide new strategies to reduce malignancy in lung cancer.

Bioactivity-guided fractionation of Helicteres angustifolia L. extract and its molecular evidence for tumor suppression.

Helicteres angustifolia L. (Helicteres angustifolia) has been commonly used in folk medicine to treat cancer; however, its mechanisms of action remain obscure. In our earlier work, we reported that aqueous extract of H. angustifolia root (AQHAR) possesses attractive anticancer properties. In the present study, we isolated five ethanol fractions from AQHAR and investigated their therapeutic efficacy in human non-small cell lung cancer (NSCLC) cells. The results showed that among the five fractions, the 40% ethanol fraction (EF40) containing multiple bioactive compounds exhibited the best selective killing effect on NSCLC cells with no obvious toxicity to normal human fibroblasts. Mechanistically, EF40 reduced the expression of nuclear factor-E2-related factor 2 (Nrf2), which is constitutively expressed at high levels in many types of cancers. As a result, Nrf2-dependent cellular defense responses are suppressed, leading to the intracellular accumulation of reactive oxygen species (ROS). Extensive biochemical analyses revealed that EF40 caused cell cycle arrest and apoptosis through activation of the ROS-mediated DNA damage response. Furthermore, treatment with EF40 compromised NSCLC cell migration, as evidenced by the downregulation of matrix metalloproteinases (MMPs) and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). In vivo studies using A549 xenografts in nude mice also revealed significant suppression of tumor growth and lung metastasis in the treated group. We propose that EF40 may serve as a potential natural anti-NSCLC drug that warrants further mechanistic and clinical attention.

USP12 promotes nonsmall cell lung cancer progression through deubiquitinating and stabilizing RRM2.

RRM2 is the catalytic subunit of ribonucleotide reductase (RNR), which catalyzes de novo synthesis of deoxyribonucleotide triphosphates (dNTPs) and plays critical roles in cancer cell proliferation. RRM2 protein level is controlled by ubiquitination mediated protein degradation system; however, its deubiquitinase has not been identified yet. Here we showed that ubiquitin-specific peptidase 12 (USP12) directly interacts with and deubiquitinates RRM2 in non-small cell lung cancer (NSCLC) cells. Knockdown of USP12 causes DNA replication stress and retards tumor growth in vivo and in vitro. Meanwhile, USP12 protein levels were positively correlated to RRM2 protein levels in human NSCLC tissues. In addition, high expression of USP12 was associated with poor prognosis in NSCLC patients. Therefore, our study reveals that USP12 is a RRM2 regulator and targeting USP12 could be considered as a potential therapeutical strategy for NSCLC treatment.

Quantitative proteomics identifies tumour matrisome signatures in patients with non-small cell lung cancer.

The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry. We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and we defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC.

Hsa_circ_0003220 Drives Chemoresistance of Human NSCLC Cells by Modulating miR-489-3p/IGF1.

Circular RNAs (circRNAs) have been shown to have critical roles in developing cancer and treatment resistance in an increasing body of research. The aim was to look into the functions and processes of hsa_circ_0003220 in the non-small cell lung cancer (NSCLC) chemoresistance. The NSCLC cell lines H460 and A549 were employed in present work. hsa_circ_0003220, miR-489-3p, and insulin-like growth factors (IGF1) mRNA levels were assessed with a quantitative real time polymerase chain reaction (qRT-PCR). The cisplatin, docetaxel, and paclitaxel (PTX) resistances were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the enzyme linked immunosorbent assay (ELISA) test measured IGF1 expression. In order to corroborate the miR-489-3p relation with hsa_circ_0003220 or IGF1, a dual-luciferase reporter method was applied. The level of hsa_circ_0003220 was raised in cells and tissues from PTX-resistant (PR) NSCLC. In PR NSCLC cells, hsa_circ_0003220 knockdown reduced chemoresistance. For the purpose of the mechanism study, hsa_circ_0003220 knockdown substantially reduced IGF1 expression via miR-489-3p sponging, reducing chemoresistance in PR NSCLC cells. By controlling the miR-489-3p/IGF1 axis, hsa_circ_0003220 knockdown helped NSCLC overcome chemoresistance, suggesting a potential circRNA-targeted therapy for the disease.

Dual receptor specific nanoparticles targeting EGFR and PD-L1 for enhanced delivery of docetaxel in cancer therapy

Dual-receptor targeted (DRT) nanoparticles which contain two distinct targeting agents may exhibit higher cell selectivity, cellular uptake, and cytotoxicity toward cancer cells than single-ligand targeted nanoparticle systems without additional functionality. The purpose of this study is to prepare DRT poly(lactic-co-glycolic acid) (PLGA) nanoparticles for targeting the delivery of docetaxel (DTX) to the EGFR and PD-L1 receptor positive cancer cells such as human glioblastoma multiform (U87-MG) and human non-small cell lung cancer (A549) cell lines. Anti-EGFR and anti-PD-L1 antibody were decorated on DTX loaded PLGA nanoparticles to prepare DRT-DTX-PLGA via. single emulsion solvent evaporation method. Physicochemical characterizations of DRT-DTX-PLGA, such as particle size, zeta-potential, morphology, and in vitro DTX release were also evaluated. The average particle size of DRT-DTX-PLGA was 124.2 ± 1.1 nm with spherical and smooth morphology. In the cellular uptake study, the DRT-DTX-PLGA endocytosed by the U87-MG and A549 cells was single ligand targeting nanoparticle. From the in vitro cell cytotoxicity, and apoptosis studies, we reported that DRT-DTX-PLGA exhibited high cytotoxicity and enhanced the apoptotic cell compared to the single ligand-targeted nanoparticle. The dual receptor mediated endocytosis of DRT-DTX-PLGA showed a high binding affinity effect that leads to high intracellular DTX concentration and exhibited high cytotoxic properties. Thus, DRT nanoparticles have the potential to improve cancer therapy by providing selectivity over single-ligand-targeted nanoparticles.

Tumor-associated fibrosis impairs immune surveillance and response to immune checkpoint blockade in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. Immune checkpoint blockade has improved survival for many patients with NSCLC, but most fail to obtain long-term benefit. Understanding the factors leading to reduced immune surveillance in NSCLC is critical in improving patient outcomes. Here, we show that human NSCLC harbors large amounts of fibrosis that correlates with reduced T cell infiltration. In murine NSCLC models, the induction of fibrosis led to increased lung cancer progression, impaired T cell immune surveillance, and failure of immune checkpoint blockade efficacy. Associated with these changes, we observed that fibrosis leads to numerically and functionally impaired dendritic cells and altered macrophage phenotypes that likely contribute to immunosuppression. Within cancer-associated fibroblasts, distinct changes within the Col13a1-expressing population suggest that these cells produce chemokines to recruit macrophages and regulatory T cells while limiting recruitment of dendritic cells and T cells. Targeting fibrosis through transforming growth factor-β receptor signaling overcame the effects of fibrosis to enhance T cell responses and improved the efficacy of immune checkpoint blockade but only in the context of chemotherapy. Together, these data suggest that fibrosis in NSCLC leads to reduced immune surveillance and poor responsiveness to checkpoint blockade and highlight antifibrotic therapies as a candidate strategy to overcome immunotherapeutic resistance.

Design, synthesis, and anticancer activity of novel 4,6-dimorpholinyl-1,3,5-triazine compounds

Abstract A series of novel 4,6-dimorpholinyl-1,3,5-triazine derivatives 6a–6r were obtained through N-substitution and Claisen-Schmidt condensation. 1H NMR, 13C NMR, and mass spectrometry were used to characterize the molecular structures of the derivatives. The in vitro antiproliferation activity of derivatives was evaluated using the MTT assay against SW620 (human colon cancer cells), A549 (human nonsmall cell lung cancer cells), HeLa (human cervical cancer cells), and MCF-7 (human breast cancer cells). Compound 6o bearing a pyridyl group exhibited good cytotoxicity against four cancer cells, with IC50 values of 8.71, 9.55, 15.67, and 21.77 μM, sequentially. In addition, compound 6a showed some selectivity against SW620.

Association of cachexia with loss of function mutations in STK11/LKB1 for NSCLC.

e15099 Background: Cancer cachexia (CC), a wasting syndrome of muscle and adipose tissue resulting in weight loss, occurs in 50% of non-small cell lung cancer (NSCLC) patients. Management of CC is limited by the absence of biomarkers and knowledge of molecules that drive its phenotype. The evolving landscape of increased genomic testing of human tumors and derived cell lines has further given us an opportunity to leverage these findings to identify a genetic biomarker that regulates CC development through a novel in vivo human NSCLC cachexia screen. Methods: To identify such molecules, we injected 54 human non-small cell lung cancer (NSCLC) lines into immunodeficient mice, 17 of which produced an unambiguous phenotype of cachexia (n = 10) or non-cachexia (n = 7). Whole exome sequencing of tumors was conducted to identify candidate cachexia-associated genes and gene variant analysis was conducted to identify gene variants that associated with NSCLC-associated CC. CRISPR/Cas9 targeted gene silencing in human NSCLC and mouse cancer lines verified if target genes conferred the cachexia phenotype. Circulating tumor DNA analysis of a 246 NSCLC patient cohort was used to confirm candidate gene variants association with cancer-related weight loss. Results: Whole exome sequencing revealed that 8 of 10 cachexia lines, but none of the non-cachexia lines, possessed mutations in STK11/LKB1 (p = 2x10-12), a regulator of nutrient sensor AMP kinase. Silencing of STK11/LKB1 in human NSCLC and murine colorectal carcinoma lines conferred a cachexia phenotype after cell transplantation into immunodeficient and immunocompetent models, respectively. This host wasting was associated with an alteration in the immune cell repertoire of the tumor microenvironments that led to increases in local mRNA expression and serum levels of CC-associated cytokines resulting in host adipose and muscle tissue wasting. Mutational analysis of circulating tumor DNA from 246 NSCLC patients identified 89% concordance between STK11/LKB1 mutations and weight loss at cancer diagnosis (OR = 17 ± 1, p = 0.004). Conclusions: The current data provides evidence that tumor STK11/LKB1 loss of function is a driver of CC in NSCLC, simultaneously serving as the first genetic biomarker for this wasting syndrome.

Advance nanotherapeutic approach for systemic co-delivery of mitoxantrone loaded chitosan coated PLGA nanoparticles to improve the chemotherapy against human non-small cell lung cancer

Poly(lactide-co-glycolide) nanoparticles (PLGA NPs) are a type of polymeric nanoparticles that offer benefits over metallic NPs, such as the capacity to sustain therapeutic drug levels for extended periods. In the current study, in order to enhance the solubility, stability, and anti-cancer effect of the mitoxantrone drug (Mtx), mitoxantrone-loaded chitosan-coated PLGA NPs (Mtx-loaded Cht-PLGA NPs) were synthesized. Mtx-loaded Cht-PLGA NPs were coated with Cht to improve mucoadhesiveness and colloidal stability. The synthesized NPs were checked for their size, morphology, zeta potential, encapsulation efficiency, FTIR and in vitro release studies. The anti-cancer potential of Mtx-loaded Cht-PLGA NPs were assessed in the A-549 lung cancer cell lines. Additionally, compared to pure Mtx, the viability of A-549 cells markedly decline with increasing concentrations of Mtx-loaded Cht-PLGA NPs. The cellular uptake study also confirms that chitosan coating greatly enhanced the uptake of the nanoparticles. Further, the ability to stimulate the reactive oxygen species (ROS) generation at the IC50 concentration of Mtx-loaded Cht-PLGA NPs was confirmed by using carboxy-2,7-dichloro-dihydro-fluorescein diacetate. Further, the western blotting revealed the down-regulation of TGF-β, vimentin, and upregulation of p53. This study reveals that the developed mitoxantrone-loaded cationically charged polymeric nano vehicle would be effectively less cytotoxic and have improved efficacy against lung cancer.

Comparison on the effect of MART-10 and calcitriol on the treatmentof non-small cell lung cancer by determining which one is a morepotent drug in treating non-small cell lung cancer

Non-small cell lung cancer is one of the most common types of lung cancer, with a high mortality rate worldwide.The current treatment methods have apparent side effects and inevitable drawbacks. A compelling new drug is badlyin need of development. A previous study has reported that calcitriol and MART-10 can effectively block anaplasticthyroid cancer (ATC) spread. MART -10 is more potent than calcitriol in inhibiting migration and invasion of ATC.This study investigates the effect of NSCLC using MART-10 and calcitriol and finds out which is more potent, both invitro and in vivo. The experiments will use known human non-small cell lung cancer cell lines (A549 cell line) and twovitamin D compounds: MART-10 and calcitriol and Xenograft mouse system. MTT measures cell proliferation. FACSmeasures cell apoptosis for AnnexinV and PI. Measure development in tumor size by mouse xengraft system. A positivecontrol is taxol, and a negative control is PBS. There are three most possible results: (1) MART-10 and calcitriol cankill A549 lung cancer cells, and MART-10 reduce the number of A549 lung cancer cells at a lower concentration thanwith calcitriol. (2) MART-10 and calcitriol can kill A549 lung cancer cells, and calcitriol reduces the number of A549lung cancer cells at a lower concentration than with MART-10. (3) MART-10 and calcitriol do not significantly killA549 lung cancer cells. The result of the study will provide critical information for the future clinical trial of MART10 and calcitriol in treating NSCLC. It would increase our understanding of the more effective treatment of NSCLC andimprove human health. Future studies should focus on combinations of MART-10 or calcitriol with various anti-cancerchemotherapy drugs.

Light, pH, and Temperature Triple-Responsive Magnetic Composites for Highly Efficient Phosphopeptide Enrichment.

Smart materials can dynamically and reversibly change their structures and functions in response to external stimuli. In this study, we designed a smart magnetic composite (MNP-pSPA-b-pNIPAm) with a triple response to ultraviolet (UV) light, pH, and temperature. Relying on the response of spiropyranyl acrylate (SPA) and N-isopropylacrylamide (NIPAm) to external stimuli (light, pH, and temperature), MNP-pSPA-b-pNIPAm was used for the controlled capture and release of phosphopeptides. The established phosphopeptide enrichment platform exhibits high sensitivity (detection limit of 0.04 fmol), high selectivity (BSA/β-casein, 1000:1), and good reusability (6 cycles). In addition, the method was also applied to the enrichment of phosphopeptides in real samples (skim milk, human saliva, and serum), demonstrating the feasibility of this method for phosphoproteomic analysis. After enriching from human nonsmall cell lung cancer cell (A549) lysates with MNP-pSPA-b-pNIPAm, 2595 phosphopeptides corresponding to 2281 phosphoproteins were identified. The novel responsive enrichment probe is highly specific for phosphoproteomic analysis and provides an effective method for studying the significance of protein phosphorylation in complex biological samples.

A phase 1 trial of umbilical cord blood–derived tumor-reactive PD-L1+ natural killer cells engineered to express soluble IL-15 (TRACK-NK) in patients with non–small-cell lung cancer (NSCLC) refractory to PD-1/PD-L1 inhibitors.

TPS2665 Background: The mechanism by which patients with PD-L1 – tumors can respond favorably to anti-PD-L1 monoclonal antibody therapy has been shown to involve the PD-L1+ NK cell in the tumor microenvironment (TME). PD-L1 is induced on the cell surface when NK cells recognize tumors, leading to enhanced NK-cell function without exhaustion. The PD-L1 + NK cell is capable of trafficking to the lung TME, recognizing and killing tumor cells as well as inducing activation of endogenous T cells via potent cytokine secretion, all without the expression of a chimeric antigen receptor (CAR). TRACK-NK cells are PD-L1+ NK cells derived from cord blood (CB) and engineered to express soluble IL-15 (sIL-15). The TRACK-NK cells express high levels of tumor reactive receptors that recognize "tumor stress" including DNAM-1, NKp30 and NKG2D. TRACK-NK cells induced significant reduction in tumor volume in mice injected with human A549 NSCLC cells compared to mock-transduced NK cells, or NK cells expressing sIL-15 (sIL-15-NK) but without ex vivo activation. Survival was also prolonged in the H460 NSCLC model compared to vehicle alone. We hypothesized that TRACK-NK cells can be safely administered, and will provide meaningful clinical benefit in patients with NSCLC. Methods: NCT05334329 is a first-in-human, phase 1 dose finding study of allogeneic, off-the shelf frozen and thawed TRACK-NK cells in patients with NSCLC progressing on treatment with PD-1/PD-L1 inhibitors. TRACK-NK cells (CB NK cells engineered with retroviral transduction to express sIL-15 and ex vivo expanded and activated to express PD-L1) are administered in four weekly intravenous infusions per cycle following lymphocyte depletion (Fludarabine/Cyclophosphamide x 3 days) in the outpatient setting. Patients not experiencing disease progression (per RECIST v1.1) following cycle 1 may receive a second cycle. Primary endpoints include: 1. Toxicity/adverse events as per NCI-CTCA version 5.0, and ASTCT Consensus Grading for Cytokine Release Syndrome (CRS) and Neurotoxicity; and 2. Magnitude/duration of TRACK-NK cell persistence in peripheral blood via droplet digital PCR. We are employing a Utility Based-Bayesian Optimal Interval design to direct optimal dose determination. Dose limiting toxicity is defined as any ≥ grade 3 non-hematologic toxicity; any grade 3 CRS that does not resolve to ≤ grade 1 within 7 days; any ≥ grade 4 CRS; any ≥ grade 2 neurotoxicity that does not resolve to grade 1 within 72 hours; any ≥ grade 3 autoimmune, hypersensitivity, and acute graft-versus-host reactions. Correlatives also include cytokines levels, quantity and activation of CD4 and CD8 cells in peripheral blood, and NK and T cell trafficking and TME changes in paired tumor biopsies using quantitative immunofluorescence and gene expression analysis. Clinical trial information: NCT05334329 .

Epithelial-mesenchymal transition is associated with osteopontin-induced EGFR‑TKI resistance in EGFR mutant non-small cell lung cancer.

Resistance restricts the long-term therapeutic efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of non-small cell lung cancer (NSCLC) with positive EGFR mutations. The present study sought to identify the potential protein osteopontin (OPN) involved in EGFR-TKI resistance and examine its therapeutic mechanism in NSCLC. The expression of OPN in NSCLC tissues was evaluated by immunohistochemistry (IHC). Western blot (WB), quantitative real‑time polymerase chain reaction (qRT-PCR), and immunofluorescence staining were used to analyze OPN and epithelial-mesenchymal transition (EMT)-related protein expression in the PC9 and PC9 gefitinib resistance (PC9GR) cells. Enzyme-linked immunosorbent assays (ELISAs) were used to detect the secreted OPN. Cell Counting Kit-8 (CCK-8) assays and flow cytometry were used to examine the effect of OPN on the gefitinib-induced growth and death of PC9 or PC9GR cells. OPN was upregulated in the human NSCLC tissues and cells resistant to EGFR-TKIs. The overexpression of OPN inhibited EGFR-TKI-induced apoptosis and was associated with the formation of EMT. By activating the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)-EMT pathway, OPN contributed to the development of EGFR-TKI resistance. Reducing OPN expression and inhibiting PI3K/AKT signaling improved EGFR-TKI sensitivity significantly more than the use of either agent alone. This study showed that OPN increased EGFR-TKI resistance in NSCLC through the OPN-PI3K/AKT-EMT pathway. Our findings may provide a possible therapeutic target for overcoming EGFR-TKI resistance in this pathway.

MiR-183-5p promotes the progression of non-small cell lung cancer through targeted regulation of FOXO1

Abstract Objective To investigate miR-183-5p targeting to forkhead box protein O1 (FOXO1) and its corresponding effect on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection. Several cell lines were cultured in vitro, including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549, SPCA-1, PC-9, and 95-D. miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR; the corresponding correlations in NSCLC tissues were analyzed using the Pearson test, and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed. The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase, RNA-binding protein immunoprecipitation (RIP) assay, and pull-down experiments. A549 cells were divided into control, anti-miR-NC, anti-miR-183-5p, miR-NC, miR-183-5p, miR-183-5p+pcDNA3.1, and miR-183-5p+pcDNA3.1-FOXO1 groups. Cell proliferation, invasion, migration, apoptosis, and cell cycle distribution were detected using an MTT assay, clone formation assay, Transwell assay, scratch test, and flow cytometry, respectively. The expression of EMT-related proteins in the cells was analyzed by western blotting. The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice. Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues, whereas FOXO1 mRNA expression was significantly down-regulated. There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues (P < 0.05). Additionally, the expression of miR-183-5p was significantly correlated with tumor size, tumor differentiation, and tumor-node-metastasis stage in patients with NSCLC (P < 0.05). miR-183-5p targeted and inhibited FOXO1 expression. Compared to the anti-miR-NC group, the cell proliferation, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group, whereas the protein expression of E-cadherin and α-catenin and the proportion of G0/G1 phase cells were significantly higher; additionally, the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group (P < 0.05). Compared to the miR-NC group, the cell proliferation, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells in the miR-183-5p group were significantly higher, whereas the E-cadherin and α-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower; furthermore, the frequency of colony formation and invasion were significantly higher in the miR-183-5p group (P < 0.05). Compared with the miR-183-5p+pcDNA3.1 group, the OD value, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group, whereas E-cadherin and α-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher; additionally, the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group (P < 0.05). Overall, silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression. Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation, migration, invasion, and EMT, of NSCLC cells by down-regulating FOXO1 expression.

PiRNA-14633 underexpression as an independent prognostic marker in non-small cell lung cancer.

e21223 Background: To determine the expression and function of piRNA-14633 in non-small cell lung cancer (NSCLC) samples were taken from cancer lesions (n = 30) and adjacent normal tissue (n = 30) in NSCLC patients. Methods: piRNA-14633 expression was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The effect of piRNA-14633 overexpression was examined in vitro utilizing a human NSCLC cell line H1650 stably transfected with a recombinant lentivirus and compared to empty vector-transfected controls. piRNA-14633 overexpression was verified by RT-PCR. The expression of proteins involved in cell cycle regulation (CDK4) and apoptosis (Bcl-6) and cell migration (MMP-9) in cells was analyzed by Western blot. The effect of piRNA-14633 overexpression on cell viability and proliferation was assessed by an MTT assay cells. Flow cytometric analyses were used to evaluate the effect of piRNA-14633 expression on cell cycle distribution and apoptosis. The migration and invasion potential of piRNA-14633 cells was examined by plating cells in Matrigel-coated chambers. Results: The level of piRNA-14633 expression was found to be significantly lower in NSCLC tissue than normal tissues (P < 0.05). Decreased expression of piRNA-14633 was significantly correlated with lymph node metastasis, clinical stage, and histological grade of patients with NSCLC (P < 0.05). Meanwhile, loss of piRNA-14633 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P < 0.05). The result of biological function shown that H1650 cell-transfected piRNA-14633 had a lower survival fraction, higher percentage of the G0/G1 phases, higher cell apoptosis, significant decrease in migration and invasion, and lower CDK4, Bcl-6, and MMP-9 expression compared with H1650 cell without piRNA-14633 (P < 0.05). Conclusions: Reduced piRNA-14633 expression is associated with increased disease severity, suggesting it is a negative regulator of NSCLC and can serve as a prognostic indicator.

Preclinical evidence for synergistic effects of autophagy inhibitor and alpelisib in PI3KCA mutated non-small cell lung cancer: Implications for future clinical trials.

e21022 Background: The PI3K pathway is frequently activated in a variety of cancers including non-small cell lung cancer (NSCLC). PI3K mutation is associated with worse response to chemotherapy and consequently decrease the effectiveness of single administration of anticancer drugs. In this study, we checked the therapeutic efficacy of alpelisib either alone or in combination with an autophagy inhibitor for the treatment of NSCLC. Methods: We treated alpelisib and autophagy inhibitors in human NSCLC cell lines and measured the cell viability and protein expression of autophagy indicators, p62 and LC3B. Co-treatment of alpelisib and autophagy inhibitors significantly decrease the cell viability and proliferation compared to alpelisib alone. Based on the results of the combination experiment of alpelisib and autophagy inhibitors, Chloroquine (CQ) was selected for further study. Co-treatment of alpelisib and CQ significantly increase apoptosis and decrease tumor-related protein expression in in vitro and in vivo. Results: Our data demonstrates that alpelisib and autophagy inhibitor have a synergistic efficacy against NSCLC in vitro and in vivo. Conclusions: Based on these results, a Phase 1 clinical trial is currently being planned.

Exploration of biomarkers in potentially resectable stage IIIB NSCLC patients receiving neoadjuvant chemoimmunotherapy.

e20591 Background: Non–small cell lung cancer (NSCLC) is one of the main leading causes of cancer mortality in the world. NSCLC is incurable in most patients with potentially resectable locally advanced stage IIIB disease. Numerous studies suggest that advantageous chemoimmunotherapy is a promising treatment for resectable NSCLC. The determination of PD-L1, T-cell receptor (TCR) repertoire and tumor mutational burden (TMB) have been reported to predict the response to immunotherapy. However, the value of pathologic response determination in the context of neoadjuvant chemoimmunotherapy in resectable stage IIIB NSCLC is limited. Therefore, it is necessary to identify biomarkers for the prediction of the benefit from neoadjuvant chemoimmunotherapy in patients with potentially resectable stage IIIB NSCLC. Methods: A total of 15 patients with potentially resectable clinical stage IIIB NSCLC were enrolled into the study receiving neoadjuvant sintilimab (200mg IV Q3W) plus chemotherapy therapies for 4-6 weeks followed by surgery. All peripheral blood and tissue samples were collected and detected by using TCR repertoire sequencing before and after chemoimmunotherapy. The patients were divided into MPR group (n=5) and non-MPR group (n=10) according to MPR (<10% viable tumour at resection) as an index. Furthermore, TMB spectra of 1021-gene panels and PD-L1 immunohistochemical staining were performed in paraffin-embedded human NSCLC tissue before neoadjuvant chemoimmunotherapy. The Pearson correlation coefficient method was used to analyze correlation. The ROC analysis were performed using “pROC” R package. Results: In baseline tissue samples, the levels of TMB and NAD were higher in MPR compared with non-MPR groups (P=0.0328, P=0.015, respectively). The negative correlation was existed between pretreatment tissue TCR richness and TMB in non-MPR group (P=0.028, R=-0.66). In MPR group, TCR clones and evenness showed significant difference (P=0.00237, P=0.04658, respectively). In non-MPR group, the TCR repertoire of tissue in posttreatment showed significant difference compared with those of pretreatment, including clones (P=1.04x10-6), shannon’s diversity (P=0.00142), evenness (P=0.00433), richness (P=0.00591). The clinical data showed that MPR patients with DDR mutation had longer DFS after sintilimab plus chemotherapy therapies (18.4±5.41 months, X-squared=9.8, P=0.02034). DDR mutation, TMB and TCR richness revealed better sensitivity and specificity to classify patients achieving MPR after neoadjuvant chemoimmunotherapy (AUC=1.000, AUC=0.796, AUC=0.773, respectively). Conclusions: This study confirmed the superiority of neoadjuvant chemoimmunotherapy in patients with potentially resectable stage IIIB NSCLC in terms of MPR. TCR repertoire, TMB and DDR mutation were associated with pathologic response to neoadjuvant chemoimmunotherapy as biomarkers for effective anti-tumor immunity. Clinical trial information: ChiCTR2000040673 .