Journal: Cell Death & Disease

Article Title: Hedgehog signalling is involved in acquired resistance to KRAS G12C inhibitors in lung cancer cells

doi: 10.1038/s41419-024-06436-9

Figure Lengend Snippet: A , B and F H23 and C , D and G H358 lung cancer cells were treated with 10 μM ARS-1620 for the indicated times. Time-dependent changes in ERK phosphorylation and accumulation of KRAS-GTP in H23 ( A , B ) and H358 cells ( C , D ) were determined by western blot analysis. Time-dependent changes in KRAS mRNA levels in H23 ( F ) and H358 cells ( G ) were determined by qRT-PCR. Fold-change in expression level was calculated relative to the values at time 0 for each cell. The graphs are mean ± standard deviation ( n = 3 independent experiments) (one-way ANOVA, ** P < 0.01; *** P < 0.001). E Representative plots of flow cytometry using propidium iodide staining for cell cycle analysis of H358 cells. Graphs represent the mean ± standard deviation ( n = 3 independent experiments).

Article Snippet: Human non–small cell lung carcinoma H358 (CRL-5807, KCLB 25807) and H23 (CRL-5800, KCLB 90023) cell lines harboring the KRAS G12C mutation were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Phospho-proteomics, Western Blot, Quantitative RT-PCR, Expressing, Standard Deviation, Flow Cytometry, Staining, Cell Cycle Assay

Journal: Cell Death & Disease

Article Title: Hedgehog signalling is involved in acquired resistance to KRAS G12C inhibitors in lung cancer cells

doi: 10.1038/s41419-024-06436-9

Figure Lengend Snippet: A – J The KRAS G12C inhibitor ARS-1620 enhances Hedgehog signaling in lung cancer cells. A – E H23 and F – J H358 cells were treated with 10 μM ARS-1620 for the indicated times. Time-dependent changes in GLI-1 ( A , F) , PTCH1 ( B , G) , IFT88 ( C , H) , and ARL6 ( D , I) mRNA levels were determined by qRT-PCR amplification. Fold change in expression levels was calculated relative to the values at time 0 for each cell. The graphs are mean ± standard deviation of three independent experiments (one-way ANOVA, ** P < 0.01; *** P < 0.001). Time-dependent changes in ERK phosphorylation in H23 ( E ) and H358 cells ( J ) were determined by western blot analysis. K – P The KRAS G12C inhibitor ARS-1620 induces primary cilia formation in lung cancer cells. K – M H23 and N – P H358 cells were treated with 10 μM ARS-1620 for 72 h and then stained for acetylated tubulin (Ac-Tu, red), Arl13B (green), and DAPI (blue). K , N Representative confocal microscopy images of H23 ( K ) and H358 ( N ) cells. L , O , M , P Percentages of ciliated H23 ( L ) and H358 cells ( O ) and the average length of cilia of H23 ( M ) and H358 cells ( P ) presented as the mean ± standard deviation ( n = 150 pooled from three independent experiments). Student’s t-test, * P < 0.05; ** P < 0.01; * * * P < 0.001.

Article Snippet: Human non–small cell lung carcinoma H358 (CRL-5807, KCLB 25807) and H23 (CRL-5800, KCLB 90023) cell lines harboring the KRAS G12C mutation were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Quantitative RT-PCR, Amplification, Expressing, Standard Deviation, Phospho-proteomics, Western Blot, Staining, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: Hedgehog signalling is involved in acquired resistance to KRAS G12C inhibitors in lung cancer cells

doi: 10.1038/s41419-024-06436-9

Figure Lengend Snippet: A – D ARS-1620 treatment downregulates the expression of AURKA in lung cancer cells. H23 ( A , B) and H358 ( C , D) cells were treated with 10 μM ARS-1620 for the indicated times. Time-dependent changes in mRNA ( A , C) and protein levels of AURKA ( B , D) were determined by qRT-PCR and western blot analysis, respectively. Fold change in expression levels was calculated relative to the values at time 0 for each cell. The graphs are mean ± standard deviation of three independent experiments (one-way ANOVA, ** P < 0.01; *** P < 0.001). E – K Inhibition of AURKA using siRNA ( E– H ) or an inhibitor (Tozasertib) ( I– K ) induced Hedgehog signaling and accumulation of KRAS-GTP. H358 cells were transfected with control scRNA or siRNA against AURKA (siAURKA) or treated with a 10 μM inhibitor. Expression levels of AURKA ( E , H , and K ), GLI-1 ( F , H , I , and K ), and KRAS ( G , H , J , and K ) were determined by qRT-PCR amplification and Western blot analysis. L – N Ectopic expression of AURKA attenuates the induction of Hedgehog signals and re-expression of KRAS in ARS-1620-treated cells. H358 cells were transfected with p-Aurora A GFP-AURKA-GFP expression vector or empty vector as a control, followed by treatment with 10 μM ARS-1620 for the indicated times. L Expression of AURKA was confirmed by qRT-PCR amplification, as were time-dependent changes in M GLI-1, and N KRAS mRNAs. Fold-change in expression levels was calculated relative to the values at time 0 for each cell. The graphs are mean ± standard deviation of three independent experiments (one-way ANOVA, ** P < 0.01; *** P < 0.001). O Ectopic expression of AURKA blocks ARS-1620–mediated activation of KRAS promoter. H358 cells were co-transfected with the luciferase reporter vector pEZX-PG04.1/KRAS promoter and p-Aurora A GFP-AURKA-GFP expression vector or empty vector served as a control, followed by treatment with 10 μM ARS-1620 for 48 h. The same volume of dimethylsulfoxide was added to the cells as controls. The fold change in luciferase activity was calculated relative to that of empty vector-transfected dimethylsulfoxide control. The graphs are the mean ± standard deviation of three independent experiments (one-way ANOVA, *** P < 0.001).

Article Snippet: Human non–small cell lung carcinoma H358 (CRL-5807, KCLB 25807) and H23 (CRL-5800, KCLB 90023) cell lines harboring the KRAS G12C mutation were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Inhibition, Transfection, Control, Amplification, Plasmid Preparation, Activation Assay, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: Hedgehog signalling is involved in acquired resistance to KRAS G12C inhibitors in lung cancer cells

doi: 10.1038/s41419-024-06436-9

Figure Lengend Snippet: H358 cells were co-treated with 10 μM ARS-1620 and 10 μM Smo inhibitor sonidegib for the indicated times. A Transcriptome profiles were analyzed by RNA-seq as described in Fig. legends. Gene set enrichment plot of KRAS signaling negatively enriched in cells co-treated with ARS-1620 and the Hedgehog signal inhibitor sonidegib for 48 h versus cells treated with ARS-1620 for 48 h. B – I The Smo inhibitor sonidegib suppressed ARS-1620-induced Hedgehog signaling in both H23 ( B – E ) and H358 cells ( F – I ). Time-dependent changes in IFT88 ( B , F ), ARL6 ( C , G ), and GLI-1 mRNA levels ( D , H ) were determined by qRT-PCR amplification. Fold change in expression level was calculated relative to the values at time 0 for each cell. The graphs are mean ± standard deviation of three independent experiments (one-way ANOVA, ** P < 0.01; *** P < 0.001). Time-dependent changes in IFT88, ARL6 and GLI-1 shown in H23 ( E ) and H358 cells ( I ) were determined by western blot analysis. J – O The Smo inhibitor sonidegib suppresses ARS-1620-induced primary cilia formation in both H23 ( J – L ) and H358 cells ( M – O ). Representative confocal microscopy images of H23 ( J ) and H358 cells ( M ) stained for acetylated tubulin (Ac-Tu, red), Arl13B (green), and DAPI (blue). K , L , N , O Graphs depict the percentages of ciliated H23 ( K ) and H358 cells ( N ) and average length of cilia of H23 ( L ) and H358 cells ( O ) and are presented as the mean ± standard deviation ( n = 150 pooled from three independent experiments). Student’s t-tests, * P < 0.05; ** P < 0.01; *** P < 0.001. P – S The Smo inhibitor sonidegib suppresses ARS-1620–induced re-expression and reactivation of ERK signal in lung cancer cells. Time-dependent changes in KRAS mRNA levels in H23 ( P ) and H358 cells ( R ) were determined by qRT-PCR amplification. Fold change in expression level was relative to the values at time 0 for each cell. The graphs are mean ± standard deviation of three independent experiments (one-way ANOVA, ** P < 0.01; *** P < 0.001). Time-dependent changes in ERK phosphorylation in H23 ( Q ) and H358 cells ( S ) were determined by western blot analysis ( T , U ). The Smo inhibitor sonidegib suppressed the generation of cells resistant to KRAS G12C inhibitors. At the indicated times, H23 ( T ) and H358 cells ( U ) were stained with crystal violet and photographed. Three independent experiments were performed and representative images are shown.

Article Snippet: Human non–small cell lung carcinoma H358 (CRL-5807, KCLB 25807) and H23 (CRL-5800, KCLB 90023) cell lines harboring the KRAS G12C mutation were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: RNA Sequencing, Quantitative RT-PCR, Amplification, Expressing, Standard Deviation, Western Blot, Confocal Microscopy, Staining, Phospho-proteomics