Transcriptomic and functional analysis of ANGPTL4 overexpression in pancreatic cancer nominates targets that reverse chemoresistance

Figure 1. Expression of ANGPTL4 is associated with patient outcomes and modifies expression of resistance-associated genes. a) A Kaplan Meier curve for overall survival using TCGA-PAAD data (n=90). Top (red) and bottom (blue) 25% average ANGPTL4gene expression are significantly different. (p=0.043, log-rank test) b) Relative expression of ANGPTL4 transcript in MP2_ANGPTL4_KD (black circles), MP2 (pink squares), and MP2_ANGPTL4_OE (turquoise triangles) as measured by qPCR. P-values from unpaired, two-tailed parametric t-tests performed with 95% confidence intervals are displayed. Values are normalized with ACTB as the housekeeper. (P-values ≤ 0.0001=****) c) Heatmap displaying RNA-seq results from MP2_ANGPTL4_KD (black), MP2_NTC (pink), and MP2_ANGPTL4_OE (turquoise) cell lines. Displayed are 15 genes associated with both ANGPTL4 overexpression and gemcitabine resistance. Normalized expression for each of the 15 genes is plotted in the heatmap. Red (*) indicate ITGB4 used for later experiments.

Figure 2. Gemcitabine resistance signature in PDAC cells with high ANGPTL4 a) Cell viability was measured for MP2_ANGPTL4_KD (black), MP2_NTC (pink), and MP2_ANGPTL4_OE (turquoise) cells treated with 0, 5nM, or 12.5nM gemcitabine. A relative viability reflects normalization to cells not treated with gemcitabine. P-value asterisks: ≤0.01=**, ≤0.001=*** & ≤0.0001=****, unpaired, two tailed t-test b) Heatmap of gene expression measured by RNA-seq in MP2_ANGPTL4_KD (black/gray), MP2 (pink/light pink), and MP2_ANGPTL4_OE (turquoise/aqua) cell lines both untreated and treated with 1.5nM gemcitabine. Normalized expression of the 42 genes that resulted from an intersection of ANGPTL4 expression DEGs and gemcitabine treatment response DEGs are included in the heatmap. c) Kaplan-Meier curve for overall survival using TCGA-PAAD data (n=90) where patients were divided into quartiles with highest (red) and lowest (blue) using mean expression of the 42 genes in the intersection (40 genes were available in TCGA: KDM7aand STN1 were omitted). P-values were derived from a log-rank test.

Figure 3. ITGB4 and APOL1 inhibition sensitizes cells overexpressing ANGPTL4 to gemcitabine. a-b) Kaplan-Meier plot for overall survival using TCGA-PAAD data. Patients (n=90) with top (red) and bottom (blue) 25% average ANGPTL4gene expression and APOL1 or ITGB4 gene expression (p=0.01 and p=0.014, respectively, log-rank test). c)ANGPTL4 expression measured by qPCR normalized to MP2_ANGPTL4_OE+DsiRNA_NTC (turquoise) control: MP2 (pink), MP2_ANGPTL4_OE_ITGB4_KD (lavender), and MP2_ANGPTL4_OE_APOL1_KD (purple) compared to mean of MP2_ANGPTL4_OE+DsiRNA_NTC (MP2_ANGPTL4_OE cells transduced with non-targeting control siRNA). Expression is normalized to the housekeeping gene ACTB. P-values ≤ 0.0001=****; unpaired two-tailed parametric t-test d) Relative viability is plotted by normalizing all data to cells not treated with gemcitabine. MP2_ANGPTL4_KD (black), MP2_NTC (pink), MP2_ANGPTL4_OE+DsiRNA_NTC (turquoise), MP2_ANGPTL4_OE_ITGB4_KD (lavender), and MP2_ANGPTL4_OE_APOL1_KD (purple) cells treated with 0, 5nM, or 12.5nM gemcitabine. Sample size ranges from 4–11 per condition. P-value asterisks: ≤0.001=*** & ≤0.0001=****, unpaired, two-tailed parametric t-tests.

Figure 4. ANGPTL4 overexpression correlation with EMT. a) Pathway analysis of 1198 DEG revealed common cancer pathways. Red=log2FC >0.7 and Blue=log2FC <0.7. b) Wound healing assay results at 0 hrs (after initial wound) and 48 hours MP2_NTC (pink), MP2_ANGPTL4_OE+DsiRNA_NTC (turquoise), MP2_ANGPTL4_OE_ITGB4_KD (lavender), and MP2_ANGPTL4_OE_APOL1_KD (purple). Each set was performed in triplicate or quadruplicate. c) Time to close half the half the gap (t1/2 gap) for each of the four cell types is plotted. t1/2: MP2=57.7hrs, MP2_ANGPTL4_OE= 31.6 hrs., MP2_ANGPTL4_OE_ITGB4_KD= 192 hrs., and MP2_ANGPTL4_OE_APOL1_KD=52.96 hrs. P-values ≤0.01=**, unpaired parametric t-test