Fig 2: KLF4 bound to the promoter of BIK. a Schematic illustration of pGL3-based reported constructs were used in luciferase assays to examine the transcriptional activity of BIK. b Parts of the promoter of BIK, named P1, P2 and P3, were individually transfected into PC3 cells with or without 20 µM cisplatin treatment. Luciferase activity was measured. Data represent the mean ± SD of three independent experiments. **p < 0.01, *** p < 0.001 vs. control. c The promoter of BIK, named P1, was transfected into PC3 and DU145 cells with or without KLF4 KO and then the cells were treated with 20 µM cisplatin at the indicated times. Luciferase activity was measured. Data represent the mean ± SD of three independent experiments. ***p < 0.001 vs. control. d The potential KLF4 binding sites were inspected by JASPAR. Schematic illustration of KLF4 wild type binding site (BS) and the matching mutant (BSM) that were used in luciferase assays. e-f The wild type promoter (BS) or the matching mutant (BSM) were individually transfected into PC3 cells with or without KLF4 knockout and the cells then were treated with 20 µM cisplatin for the indicated times. Luciferase activity was measured. Data represent the mean ± SD of three independent experiments. *** p < 0.001 vs. control. g-h ChIP analysis showing the binding of KLF4 to the promoter of BIK in KLF4 WT or KO PC3 cells in response to 20 µM cisplatin treatment. An isotype-matched IgG was used as a negative control

Fig 3: KLF4 enhanced cisplatin-induced apoptosis in prostate cancer cells. a PC3 and DU145 cells were treated with 20 µM cisplatin (CDDP) at the indicated times. The protein levels of KLF4 were analysed by western blotting. b-e PC3 and DU145 cells with or without KLF4 knockout (KO) were treated with 20 µM cisplatin at the indicated times. Cell apoptosis was detected by western blotting. Cell viability was measured by a CCK8 assay. Data represent the mean ± SD of three independent experiments. ***p < 0.001 vs. control. f-i PC3 and DU145 cells with or without KLF4 knockdown were treated with 20 µM cisplatin at the indicated times. Cell apoptosis was detected by western blotting and cell viability was measured by a CCK8 assay. Data represent the mean ± SD of three independent experiments. **p < 0.01 vs. control. j-m PC3 cells with or without KLF4 overexpression were treated with 20 µM cisplatin as indicated. Cell apoptosis was analysed by flow cytometer and western blotting. Cell viability was detected by a CCK8 assay. Data represent the mean ± SD of three independent experiments. **p < 0.01 vs. control

Fig 4: MUC1 associates with MYC and decreased survival in the TCGA-PAAD dataset. (A) GSEA of the 163 PDAC tumors in the TCGA-PAAD/PDAC dataset for associations of MUC1 expression with the indicated HALLMARK gene signatures. (B–D) Enrichment plots for the HALLMARK INTERFERON ALPHA RESPONSE (B) HALLMARK MYC TARGETS V1 (C) and HALLMARK NOTCH SIGNALING (D) pathways, comparing MUC1-high to MUC1-low PDAC tumors in the TCGA-PAAD/PDAC dataset. (E) Probability of survival comparing MUC1-high, MYC-high (blue curve) to MUC1-low, MYC-high (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. (F) Probability of survival comparing MUC1-high, MYC-low (blue curve) to MUC1-low, MYC-low (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. (G) Schematic representation of MUC1-C in driving PDAC NE lineage specification. MUC1-C induces STAT1 and the type I and II IFN pathways, which are associated with KRAS mutant PDAC tumors and poor patient outcomes (9). MUC1-C also activates MYC, which is required for oncogenic mutant KRAS signaling in PDACs. Further studies are needed, at least in part, to determine whether MUC1-C independently activates STAT1 and MYC. The present data further indicate that MUC1-C integrates MYC activation with induction of the (i) OCT4, SOX2 and KLF4 pluripotency factors, (ii) BRN2, ASCL1 and AURKA NE markers, and (iii) NOTCH1/2 stemness TFs. In this way, MUC1-C promotes NE dedifferentiation, self-renewal capacity and tumorigenicity in PDAC progression.

Fig 5: KLF4 is positively regulated by MALAT1 and negatively regulated by miR-145 in A549 cells. (A) miR-145 mimics markedly increased miR-145 levels in A549 cells. (B) MALAT1 levels were increased in A549 cells transfected with pcDNA3-MALAT1. (C) The KLF4 mRNA level was increased in response to transfection of pcDNA3-MALAT1 and was decreased following transfection of miR-145 mimics in A549 cells. (D) The protein level of KLF4 was increased in response to transfection of pcDNA3-MALAT1 and was decreased following transfection of miR-145 mimics in A549 cells. **P<0.001 and ***P<0.0001. KLF4, Kruppel-like factor 4; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription quantitative polymerase chain reaction; NC, negative control.