Fig 2: ATF4 directly binds to the ABCC1 promoter region to activate transcription.A–C Correlation analysis between ACTA2, TGFB1, ATF4 expression, and ABCC1 expression using the gene expression profiling interactive analysis website. D Correlation analysis between ATF4 expression and ABCC1 expression in 48 PDAC tumor samples obtained in our institution. R = 0.3873, p = 0.0001. E, F qRT-PCR analysis revealed that silencing ATF4 expression inhibited ABCC1 expression but not ABCB1, ABCC2–5, or ABCG2 expression. G qRT-PCR analysis showed that TGF-β1 and CM-CAFs treatment upregulated ABCC1 expression, but SB431542 inhibited the promoting effect of CM-CAFs. H The luciferase assay indicated that the transcriptional activation of ABCC1 in PANC-1 cells was inhibited in ATF4-silenced cells and enhanced in TGF-β1- or CM-CAFs -treated cells with upregulated ATF4 expression. I Luciferase activity was detected in PANC-1 cells with mutations in the A1 site, A2 site, or both sites in the ABCC1 promoter region. Twenty-four hours after cotransfection with different human ABCC1 promoter-luciferase reporter constructs and pcDNA3.1-ATF4, all samples were harvested for luciferase activity experiments. The graph on the right side reveals the relative luciferase activity of the ABCC1 promoter. J ChIP assay was used to detect the directive binding of ATF4 to the ABCC1 promoter region. Specific antibodies against ATF4 were used for immunoprecipitation to harvest the protein-DNA complexes from PANC-1 cells. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the indicated primers and resolved in 2% agarose gels. The graphs shown are representative results of three repeated assays and are presented as the mean ± SD. ***p < 0.001.

Fig 3: Diagram of the crosstalk between CAFs and pancreatic cancer cells.CAFs secrete TGF-ß1 to facilitate SMAD2/3 phosphorylation followed by the upregulation of ATF4. Activation of ATF4 enhances proliferation, migration, and stemness in pancreatic cancer. Specifically, ATF4 directly binds to the ABCC1 promoter region and transactivates ABCC1, resulting in gemcitabine resistance.

Fig 4: CHL1 is downregulated in DDP and PTX-resistant A549 cells. (A) Cell survival of A549 and A549-resistant cells (A549/DDP and A549/PTX) treated with increasing concentrations of DDP and PTX, as assessed by MTT assay. (B) The IC50 values of DDP in A549/DDP and A549 cells, and the IC50 values of PTX in A549/PTX and A549 cells. *P<0.05 vs. A549 cells. (C) Western blotting demonstrated the expression of drug resistance-related proteins MDR1, MRP and LRP in A549 cells and A549-resistant cells (A549/DDP and A549/PTX). *P<0.05 vs. A549 cells. The protein and mRNA expression levels of CHL1 in A549 cells and A549-resistant cells (A549/DDP and A549/PTX) were analysed by (D) western blotting and (E) reverse transcription-quantitative PCR, respectively. *P<0.05 vs. A549 cells. (F) The mRNA expression of CHL1 in H460 and H460/DDP cells in the GSE21656 dataset. *P<0.05 vs. H460 cells. CHL1, close homolog of L1; DDP, cisplatin; PTX, paclitaxel; MDR1, multi-drug resistance gene 1; MRP, multidrug resistance-associated protein; LRP, low-density lipoprotein receptor-related protein; IC50, half maximal inhibitory concentration.