Fig 2: Enhancement of β-catenin is critical for ARL4C to reduce TKI Erlotinib resistance. (A) Western blot showed β-catenin protein expression in HCC827-ARL4C-SH, HCC827-Vector, and HCC827 (control) cells. (B) The expression of β-catenin-target genes Axin, CD44, Ccnd1, Lgr-5, and MMP7 in HCC827-ARL4C-SH, HCC827-Vector, and HCC827 (control) cells detected by real-time PCR. (C) Detection of β-catenin protein expression in HCC827/ER-ARL4C-OE, HCC827/ER-Vector, and HCC827/ER (control) cells by Western blot. (D) The expression of β-catenin-target genes Axin2, CD44, Ccnd1, Lgr-5, and MMP7 in HCC827/ER-ARL4C-OE, HCC827/ER-Vector, and HCC827/ER (control) cells measured by real-time PCR. (E,F) β-catenin (CTNNB1) expression in HCC827/ER/ARL4C-OE, HCC827/ER/ARL4C-OE–vector, and HCC827/ER/ARL4C-OE-CTNNB1-OE in the rescue assay analyzed by real-time PCR and Western blot. (G) Real-time PCR quantified the expression of β-catenin-target genes, Axin2, CD44, Ccnd1, Lgr-5, and MMP7 in HCC827/ER/ARL4C-OE, HCC827/ER/ARL4C-OE–vector, and HCC827/ER/ARL4C-OE-CTNNB1-OE. (H,I) Colony formation of HCC827/ER/ARL4C-OE, HCC827/ER/ARL4C-OE–vector, and HCC827/ER/ARL4C-OE-CTNNB1-OE. (J) Erlotinib IC50 for HCC82/ER/ARL4C-OE, HCC827/ER/ARL4C-OE–vector, and HCC827/ER/ARL4C-OE-CTNNB1-OE measured by MTS assay. (K) Transwell assays on the migration of HCC827/ER/ARL4C-OE, HCC827/ER/ARL4C-OE–vector, and HCC827/ER/ARL4C-OE-CTNNB1-OE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: Differential Gene-Expression Analysis between Treatment Time Points Reveals Treatment Stage-Specific Transcriptional Signatures(A) Boxplots showing the expression level of the alveolar signature across treatment time points as well as non-cancerous AT2 cells from our cohort. ***p < 0.001.(B) Fold-change expression of NKX2-1 as quantified by RT-PCR in EGFR mutant PC9 cells after specified treatment duration (see STAR Methods), ***p < 0.001.(C) Representative IHC images of TN, RD, and PD tumor tissue sections stained for AQP4 demonstrating increased expression at the RD time point. Red arrows indicate cancer cells of interest. Scale bars correspond to 50 µm.(D) Kaplan-Meier plot of the relationship between the alveolar signature and patient OS within the TGCA dataset. Patients were stratified by signature expression quartile (Q1 = 128, Q2 = 127, Q3 = 128, Q4 = 127), where Q1 is the lowest expression and Q4 is the highest expression.(E and F) Representative IHC images of TN, RD, and PD tumor tissue sections stained for SUSD2 (E) and CTNNB1 (F) demonstrating increased expression at the RD time point. Red arrows indicate example regions of interest. Scale bars correspond to 50 µm.(G–J) Treatment response upon inhibition of ß-catenin activity in EGFR mutant PC9 cells and ALK fusion-positive H3122 cells. Relative viability is shown as percent confluency compared to DMSO control. PC9 cells were treated with XAV-939 (G) or PRI-724 (H) with or without the combination of 50nM osimertinib. H3122 were treated with XAV-939 (I) or PRI-724 (J) with or without the combination of 50 nM alectinib. p values were calculated for all end points (day 6) values compared to single agent TKI. Error bars represent mean ± standard error of the mean (SEM), n = 4 technical replicates. *p < 0.05, **p < 0.01.(K) Boxplots showing the expression levels of the kynurenine signature expression across different treatment time points. ***p < 0.001.(L) Fold-change expression of QPRT as quantified by RT-PCR in PC9 cells after treatment with osimertinib as in (B) (see STAR Methods) (AR), *p < 0.05.(M) Kaplan-Meier plot of the relationship between the kynurenine signature and patient OS within the TGCA dataset. Patients were stratified by signature expression quartile (Q1 = 128, Q2 = 127, Q3 = 128, Q4 = 127), where Q1 is the lowest expression and Q4 is the highest expression.(N) Boxplot showing the expression levels of the plasminogen activation pathway signature across different treatment time points.(O) Boxplot showing the expression levels of the SERPINE1 across different treatment time points.(P) Kaplan-Meier plot of the relationship between the plasminogen activating pathway signature and patient OS within the TGCA dataset, respectively. Patients were stratified by signature expression quartile (Q1 = 128, Q2 = 127, Q3 = 128, Q4 = 127), where Q1 is the lowest expression and Q4 is the highest expression.(Q) Kaplan-Meier plot of the relationship between SERPINE1 expression and patient OS within the TGCA dataset, respectively. Patients were stratified by signature expression quartile (Q1 = 128, Q2 = 127, Q3 = 128, Q4 = 127), where Q1 is the lowest expression and Q4 is the highest expression.(R) Boxplot showing the expression levels of the gap-junction signature across treatment time points.(S) Kaplan-Meier plots of relationships between the gap-junction signature and patient OS within the TGCA dataset. Patients were stratified by signature expression quartile (Q1 = 128, Q2 = 127, Q3 = 128, Q4 = 127), where Q1 is the lowest expression and Q4 is the highest expression.See also Figures S3, S4, and S5 and Tables S2, S4, S5, and S6.

Fig 4: β-catenin is a direct target of miRNA-142-3p. (A) The two predicted binding sites and the mutations that disrupt the associations between miRNA-142-3p onto the β-catenin (CTNNB1) mRNA 3′-UTR. (B) The relative luciferase activity (FL/RL) was measured in HT29 cells after co-transfection of the β-catenin 3′-UTR or β-catenin 3′-UTR mutant luciferase constructs with either miRNA-142-3p mimics, NC, or inhibitors (100 nmol/L). All data are shown as means ± SD. *** represents significant difference with p < 0.001, while † represents p > 0.05. ** represents significant difference with p < 0.05.

Fig 5: Transforming growth factor‐β (TGF‐β) restores the serum deprivation response (SDPR)‐induced MET phenotype. (a) Cellular morphology of the SDPR‐overexpressed MDA‐MB‐231 with or without TGF‐β1 treatment. (b) RT‐qPCR analysis of mRNA expression of the mesenchymal markers Vimentin and N‐cadherin (CDH2), and epithelial markers E‐cadherin (CDH1) and β‐catenin (CTNNB1) in the SDPR‐overexpressed MDA‐MB‐231 with or without TGF‐β1 treatment. (c) Western blot analysis of protein expression of the mesenchymal markers Vimentin and N‐cadherin, and epithelial markers E‐cadherin and β‐catenin in the SDPR‐overexpressed MDA‐MB‐231 with or without TGF‐β treatment.