Journal: Translational Lung Cancer Research

Article Title: Targeting of arachidonic acid-modulated autophagy to enhance the sensitivity of ROS1 + or ALK + non-small cell lung cancer to crizotinib therapy

doi: 10.21037/tlcr-2025-105

Figure Lengend Snippet: Downregulation of MAPK/p-STAT3/PTGS2 drove metabolic reprogramming in crizotinib-resistant cells. (A) UMAP plots based on the top 5 principal components of all single-cell transcriptomes after quality control, color-coded by treatment group (ALK TKI-sensitive or TKI-resistant) or by subsets identified through unsupervised dimensionality reduction and clustering. The analysis revealed two TKI-sensitive clusters (SensC1 and SensC2) and six TKI-resistant clusters (ResiC1–ResiC6). (B) Differentially expressed genes in each subset, with the top 5 genes per subset being shown (see A for color codes). (C) Mean pathway activity scores for different cell subsets. (D) Human phospho-kinase array analysis was performed to evaluate signaling pathways in HCC78 and HCC78CR cells treated with 2 µM of crizotinib for 24 hours. The green arrow indicates the puncta of p-STAT3 (S727). (E) HCC78 and HCC78CR cells were treated with the 1 µM crizotinib for 24 hours. The protein levels of p-P38MAPK (T180/T182) and p-STAT3 (S727) were detected via western blotting. β-actin was used as the loading control. The gray value ratios of phosphorylated to total proteins are shown on the right. (F) Volcano plot of RNA-sequencing analysis comparing gene expression profiles between parental and resistant HCC78 cells. (G) KEGG pathway enrichment analysis of differentially expressed genes between parental and resistant HCC78 cells. (H) CNET mapping of four differential signaling pathways (lL-17 signaling pathway, cytokine-cytokine receptor interaction, NF-κB signaling pathway, and regulation of lipolysis in adipocytes). (I) UMAP and dot plots showing the expression levels of EPCAM and PTGS2 in each subset in ALK-TKI-sensitive and TKI-resistant tumors. (J) Unsupervised transcriptional trajectory analysis of different cell subsets generated using R package Monocle2, colored by pseudotime, cell subsets, and PTGS2 expression levels, respectively, in ALK TKI-sensitive and TKI-resistant tumors. (K) The levels of PTGS2 mRNA in HCC78CR and H3122CR cells, as compared to their corresponding parental cells, were measured using RT-qPCR. Data are presented as the mean ± SD. (E) Brown-Forsythe and Welch ANOVA tests and (K) Unpaired two-sided Student’s t -test. ns: no significance. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ANOVA, analysis of variance; ALK, anaplastic lymphoma kinase; Cri, crizotinib; CNET, computational network; CR, crizotinib resistance; EPCAM, epithelial cell adhesion molecule; KEGG, Kyoto Encyclopedia of Genes and Genomes; p38MAPK, mitogen-activated protein kinase; p-p38MAPK(T180/182), phosphorylation of mitogen-activated protein kinase on T180 and T182; STAT3, signal transducer and activator of transcription 3; p-STAT3(S727), phosphorylation of signal transducer and activator of transcription 3 on S727; no-diff, no difference; PTGS2, prostaglandin endoperoxide synthase 2; SD, standard deviation; TKI, tyrosine kinase inhibitor; UMAP, uniform manifold approximation and projection; WT, wild type.

Article Snippet: The primary antibodies used were LAMP1 [cat. no. 9091; Cell Signaling Technology (CST), Danvers, MA, USA], LC3B (cat. no. 83506; CST), p-p38MAPK (Thr180/Tyr182) (cat. no. 4511; CST), p38 MAPK (cat. no. 8690; CST), p-STAT3 (S727) (cat. no. 9134; CST), STAT3 (cat. no. 9139; CST), p-IRE1 (S724) (cat no. AP1442; ABclonal, Woburn, MA, USA), IRE1 (cat no. A17940; ABclonal), sXBP1 (cat no. A17007; ABclonal), p-PERK (T982) (cat no. AP0886; ABclonal), and PERK (cat. no. A18196; ABclonal). β-actin, HRP-conjugated goat anti-rabbit, and goat anti-mouse secondary antibodies were obtained from Boster Bio (Pleasanton, CA, USA).

Techniques: Control, Activity Assay, Protein-Protein interactions, Western Blot, RNA Sequencing, Gene Expression, Expressing, Generated, Quantitative RT-PCR, Phospho-proteomics, Standard Deviation