Fig 2: Knockdown of MELK inhibits the metastasis of ESCC cells in vivo. (A) KYSE150 cells with MELK knockdown were intravenously injected into nude mice (n=7 per group) through the tail vein. Representative images of excised lungs after 5 weeks of injection are shown (arrows indicate the metastatic foci). (B) The number of tumor nodules on lung surfaces. (C) Lung metastases in each mouse were stained with H&E. Arrows indicate the metastatic colonization of tumor cells in the lung tissues. Scale bars, 1 mm (left), 200 µm (right). (D) Schematic illustrating the role of MELK in stimulating metastasis of ESCC cells. *P<0.05. MELK, maternal embryonic leucine zipper kinase; ESCC, esophageal squamous cell carcinoma; H&E, hematoxylin and eosin; sh, short hairpin RNA; NC, negative control; IKK, IκB kinase; NF-κB, nuclear factor-κB; ZEB2, zinc finger E-box-binding homeobox 2; E-cadherin, epithelial cadherin; ZO-1, zonula occludens-1; N-caherin, neural cadherin; MMPs, matrix metalloproteinases; EMT, epithelial-mesenchymal transition.

Fig 3: GSDME-dependent pyroptosis in mesenchymal cells upon drug treatment. (A) Parental and TGFβ-stimulated HCC827 cell lines were treated with erlotinib (4 μM) or salinomycin (4 μM) at a time course manner and analyzed by Western blot for GSDME protein cleavage. GSDME-FL, full-length GSDME; GSDME-N, GSDME N-terminal domain. (B) Parental and TGFβ-stimulated NCI-H1975 cell lines were treated with osimertinib (4 μM) or salinomycin (4 μM) at a time course manner and analyzed by Western blot for GSDME protein cleavage. (C) GSDME was knocked out in HCC827 cells using the CRISPR-Cas9 system. Cells were stimulated with TGFβ (5 ng/μl) to induce EMT, treated with erlotinib (4 μM) or salinomycin (4 μM), and analyzed by Western blot for GSDME protein cleavage. (D) GSDME was knocked out in HCC827 cells using the CRISPR-Cas9 system. Cells were transfected with exogenous ZEB2 to induce EMT, treated with erlotinib (4 μM) or salinomycin (4 μM), and analyzed by Western blot for GSDME protein cleavage. (E) GSDME was knocked out in HCC827 cells using the CRISPR-Cas9 system. Cells were stimulated with TGFβ (5 ng/μl) to induce EMT, and LDH release in the presence of DMSO or indicated inhibitors was measured. Data are means ± SEM pooled from three independent experiments. (F) GSDME was knocked out in HCC827 cells using the CRISPR-Cas9 system. Cells were transfected with exogenous ZEB2 to induce EMT, and LDH release in the presence of DMSO or indicated inhibitors was measured. Data are means ± SEM pooled from three independent experiments.

Fig 4: HES1 upregulates Slug in TNBC. A) Protein and B) mRNA levels of EMT-inducing transcription factors (Twist, Snail, Slug, ZEB1, ZEB2) was detected in HES1-knockdown TNBC cell lines (MDA-MB-231 and HCC-1937).

Fig 5: Mutant p53 activates CPT1C expression through the miR-200c-ZEB2 axis. (A) miR-200c-deficiency leads to enhanced expression of CPT1C in mammary epithelial cells. The expression of CPT1C and Actin in control (Ctrl) and miR-200c-KO MCF12A cells (MCF12A-Sg1 and MCF12A-Sg2) were examined by immunoblotting. (B) The box and whisker plot of CPT1C expression in p53 wide-type breast cancer patients (n = 657) from TCGA with high (n = 328) or low miR-200c expression (n = 329). The statistical significance of differential CPT1C expression in miR-200c high expression and low expression groups was determined by Wilcoxon rank-sum test. p: corresponding p-value. (C, D) Restoring the expression of miR-200c compromises the enhanced CPT1C expression caused by mutant p53. The expression of CPT1C in control (MCF12A-Ctrl), p53 mutant cells (MCF12A-p53R249S, MCF12A-p53R273H and MCF12A-p53R280K) and miR-200c-overexpressing p53 mutant cells (MCF12A-p53R249S-miR-200c, MCF12A-p53R273H -miR-200c and MCF12A-p53R280K-miR-200c) were examined by Q-PCR (C) or immunoblotting (D). (E) The box and whisker plot of CPT1C expression in breast cancer patients (n = 290) harboring p53 missense mutations with high (n = 145) or low miR-200c expression (n = 145). Statistic method: Wilcoxon rank-sum test. p: corresponding p-value. (F) The box and whisker plot of CPT1C expression in total breast cancer patients (n = 1074) from TCGA with high (n = 537) or low miR-200c expression (n = 537). Statistic method: Wilcoxon rank-sum test. p: corresponding p-value. (G) The Spearman correlation plot for CPT1C versus miR-200c in total breast cancer patients from the TCGA database. R: correlation coefficient value; P: corresponding p-value. (H–J) Knockdown of ZEB2 interferes with the enhanced expression of CPT1C in miR-200c-KO (H). MCF12A-miR-200c-Sg2), p53 mutant-overexpressing (I), MCF12A-p53 R273H), and BT549 (J) cells. (K) The Pearson’s pairwise correlation plot for CPT1C versus ZEB2 in total breast cancer patients from TCGA. R: correlation coefficient value; P: corresponding p-value. The result is derived from bc-GeneExMiner v4.5 (http://bcgenex.ico.unicancer.fr).