Panel A shows a scatter plot comparing EPHA2 expression in normal pancreas tissue and PDAC tissue. The x-axis represents the tissue type (normal and tumor), and the y-axis represents EPHA2 expression levels. Panel B is a bar graph showing the mutational frequency of common oncodrivers in PDAC, comparing low and high EPHA2 expression tumors. The x-axis lists the oncodrivers, and the y-axis shows the mutational frequency. Panel C is a scatter plot comparing EPHA2 mRNA abundance in KRAS wild-type and mutant PDAC tumors. The x-axis represents the KRAS status, and the y-axis shows EPHA2 mRNA levels. Panel D is a scatter plot comparing EPHA2 promoter methylation levels in KRAS wild-type and mutant PDAC tumors. The x-axis represents the KRAS status, and the y-axis shows the methylation levels. Panel E is a box plot comparing EPHA2 mRNA abundance in KRAS wild-type and mutant PDAC tumors using microarray data. The x-axis represents the KRAS status, and the y-axis shows EPHA2 mRNA levels. Panel F is a box plot comparing EPHA2 mRNA abundance in KRAS wild-type and mutant PDAC tumors using RNAseq data. The x-axis represents the KRAS status, and the y-axis shows EPHA2 mRNA levels. Panel G is a survival plot showing the probability of overall survival in PDAC patients with low and high EPHA2 expression. The x-axis represents the overall survival in months, and the y-axis shows the probability of survival. Panel H is a box plot comparing EPHA2 mRNA expression across different PDAC subtypes. The x-axis lists the PDAC subtypes, and the y-axis shows EPHA2 mRNA levels. Panel I is a line graph showing Kras mRNA expression after treatment with cET-ASONT in wild-type and Epha2 knockout KPC cells. The x-axis represents the concentration of cET-ASONT, and the y-axis shows Kras mRNA expression. Panel J shows Western blots comparing the effect of cET-ASONT and cET-ASOKras on MEK1/2 and ERK1/2 phosphorylation and KRAS expression in KPC cells. Panel K shows Western blotting of KPC cell lysates comparing EPHA2 expression in non-targeting and Epha2-targeting sgRNA transduced cells. Panel L is a bar graph showing Kras mRNA expression in non-targeting and Epha2CRISPR-KO KPC cells after treatment with cET-ASOKras. The x-axis represents the cell type, and the y-axis shows Kras mRNA expression. Panel M shows Western blotting of non-targeting and Epha2CRISPR-KO KPC cells after treatment with cET-ASOKras, showing the effect on MEK1/2 and ERK1/2 phosphorylation and KRAS expression. Panel N shows Western blotting of H1299 cell lysates comparing EPHA2 expression in non-targeting and EPHA2-targeting sgRNA transduced cells. Panel O is a bar graph showing KRAS mRNA expression in non-targeting and EPHA2CRISPR-KO H1299 cells after treatment with cET-ASOKras. The x-axis represents the cell type, and the y-axis shows KRAS mRNA expression. Panel P shows Western blotting of non-targeting and EPHA2CRISPR-KO H1299 cells after treatment with cET-ASOKras, showing the effect on MEK1/2 and ERK1/2 phosphorylation and KRAS expression. Panel Q shows density plots of the number of ASO-positive vesicles in cells treated with vehicle or chloroquine in combination with vehicle or ASO for 4 or 16 hours. The x-axis represents the log10-transformed number of ASO-positive vesicles, and the y-axis shows the density.
Supplementary data related to Figs. 1 and 2. (A) EPHA2 expression in normal pancreas (TCGA and GTEx, n = 171) compared with expression in PDAC (TCGA, n = 179). (B) Analysis of the mutational frequency in the most common oncodrivers in PDAC (TCGA), comparing low EPHA2 expression tumors (blue, n = 88) with high EPHA2 expression tumors (magenta, n = 89). Two-sided Fisher exact test. (C) Comparison of the abundance of EPHA2 mRNA in either KRAS WT (blue, n = 67) or KRAS mutant (magenta, n = 117) PDAC tumors (TCGA). Student’s t test. (D) Comparison of the promoter methylation levels on the EPHA2 gene in either KRAS WT (blue, n = 67) or KRAS mutant (magenta, n = 117) PDAC tumors (TCGA). Student’s t test. (E) Comparison of the abundance of EPHA2 mRNA in either KRAS WT (blue) or KRAS mutant (magenta) PDAC tumors (microarray data, PDAC, and intraductal papillary mucinous neoplasm—IPNM). Kras WT n = 24, Kras mutant n = 207. Student’s t test. (F) Comparison of the abundance of EPHA2 mRNA in either KRAS WT (blue) or KRAS mutant (magenta) PDAC tumors (RNA-seq data30). Kras WT n = 11, Kras mutant n = 84. Student’s t test. (G) Survival plot; probability of overall survival of PDAC patients comparing patients with low EPHA2 expression (blue, n = 88) and those with high EPHA2 expression (magenta, n = 89). Log-rank test. (H)EPHA2 mRNA expression comparison across the different PDAC subtypes as defined by Bailey et al. (2016). ADEX n = 16, immunogenic n = 25, progenitor n = 30, and squamous n = 25. Kruskal–Wallis. (I)Kras mRNA expression after 72 h of treatment with the indicated concentrations of cET-ASONT in WT (Epha2+/+, blue line) or Epha2 knockout (Epha2−/−, magenta line) KPC cells. Two-way ANOVA, Sidak multiple comparison test, n = 3 individual experiments. (J) Western blots comparing the effect of cET-ASONT and cET-ASOKras on MEK1/2 and ERK1/2 phosphorylation and KRAS expression in KPC cells. Vinculin was used as a loading control. (K) Western blotting of KPC cell lysates obtained from either KPC cells transduced with a nontargeting sgRNA (n.t.) or an Epha2-targeting sgRNA (Epha2CRISPR-KO), showing the depletion of EPHA2 expression in the latter. GAPDH was used as a loading control. (L)Kras mRNA expression in either nontargeting or 60 Epha2CRISPR-KO KPC cells after treatment with 0.5 μM cET-ASOKras for 72 h. Data are mean ± SEM, n = 6 independent experiments, Student’s t test. (M) Western blotting of either nontargeting or Epha2CRISPR-KO KPC cells after treatment with 0.5 μM cET-ASOKras for 72 h, showing the effect of the ASO on MEK1/2 and ERK1/2 phosphorylation and KRAS expression. Vinculin was used as a loading control. (N) Western blotting of H1299 (non-small cell lung carcinoma) cell lysates obtained from either H1299 cells transduced with either a nontargeting sgRNA (n.t.) or an EPHA2-targeting sgRNA (EPHA2CRISPR-KO), showing the depletion of EPHA2 expression in the latter. β-actin was used as a loading control. (O)KRAS mRNA expression in either nontargeting or EPHA2CRISPR-KO H1299 cells after treatment with 0.5 μM cET-ASOKras for 72 h. Data are mean ± SEM, n = 4 independent experiments, Student’s t test. (P) Western blotting of either nontargeting or EPHA2CRISPR-KO H1299 cells after treatment with 0.5 μM cET-ASOKras for 72 h, showing the effect of the ASO on MEK1/2 and ERK1/2 phosphorylation and KRAS expression. Vinculin was used as a loading control. (Q) Density plots of the number of ASO-positive vesicles (number per area of cytoplasm) in cells treated either with vehicle or the indicated concentrations of chloroquine in combination with either vehicle or ASO (5 μM) for either 4 or 16 h. Data are expressed as log10-transformed, n = 3 independent experiments; all conditions are compared with vehicle-ASO–treated cells, ANOVA, and Tukey post hoc test. Source data are available for this figure: SourceData FS1.