Fig 2: GLI expression and activity correlates with EMT and the basal-like subtype of PDA.(A) Human PDA cell lines segregate into two groups based on protein expression of EMT associated markers as indicated. Mesenchymal markers (ZEB1, VIM, N-Cadherin), epithelial markers (E-Cadherin, ESRP1). (B,C) GLI activity (B) as measured by GLI luciferase assay and SHH protein levels (see Figure 1) (C) correlated to EMT score. Linear regression line is shown in red along with corresponding statistics. Epithelial cell lines are indicated in blue, and mesenchymal cell lines are indicated in red. Intermediate lines are indicated in grey. (D) mRNA expression of the classical subtype gene set (see Materials and methods) across 14 human PDA cell lines. Epithelial cell lines (blue) express higher levels of the classical gene program. (E) Expression of Hh pathway components correlates with PDA subtypes. GLI2 mRNA expression is higher in TCGA PDA samples (n = 149) classified as basal-like (n = 65), while SHH and IHH expression is higher in samples classified as classical (n = 84). Bar represents average normalized z-score. (F) Table outlining the relationship between GLI and Hh expression to PDA subtypes in PDA human cell lines.10.7554/eLife.45313.007Figure 2—source data 1.GLI expression and activity correlates with EMT and the basal-like subtype of PDA.

Fig 3: Expression and activity of GLI proteins is anti-correlated with Hh ligand levels in PDA.(A) Immunoblots showing expression of the indicated proteins in 14 human PDA cell lines. Cell lines denoted with an asterisk represent GLIhi/Hhlo lines. (B) The indicated cell lines were transfected with the 8 × 3’Gli-binding site luciferase plasmid and luciferase activity was measured 48 hr post-transfection. For each box-and-whisker plot, center asterisk indicates the mean of n = 3 experiments with a total of 10 independently transfected cultures for each cell line. (C) GLI activity as measured by GLI luciferase assay (B) is anti-correlated with SHH protein levels (A). Linear regression line is shown in red along with corresponding statistics. (D,E) High expression of GLI2 predicts shorter overall (D: left; GLI2 high n = 147, GLI2 low n = 38) and disease-free (D: right; GLI2 high n = 114, GLI2 low n = 27) survival while high SHH expression predicts extended disease-free survival (E: SHH high n = 11, SHH low n = 130) for patients with PDA from The Cancer Genome Atlas (TCGA). Data from 185 patients. p-Value calculated by Log-rank test.10.7554/eLife.45313.005Figure 1—source data 1.GLI proteins are expressed in basal-like PDA.

Fig 4: ROS1-regulated Gli1 level is required for cell proliferation. (a) Western blotting for quantification/comparison of Gli1 level in OC3 and OC3-IV2 cells. (b) The mRNA levels for the indicated genes in OC3-IV2 cells were measured by Q-PCR and normalized to those in OC3 cells. (c) ChIP assays were performed to compare modifications of H3K4me3 and H3K27me3 or EZH2 occupancy of the GLI1 promoter in OC3 and OC3-IV2 cells. (d) Comparison of ChIP assay results for STAT1 occupancy of the GLI1 gene region for OC3 cells, OC3-IV2 cells, and OC3-IV2 cells treated with 20 μM U0126 or LY294002 for 24 h. (e) Q-PCR analysis showing relative GLI1 mRNA expression between OC3-IV2-Scr and OC3-IV2-shROS1#1 cells. (f) STAT1 ChIP assays at the GLI1 gene region were performed using lysates from OC3-IV2-Scr and OC3-IV2-shROS1#1. (g) Relative mRNA level of GLI1 was compared among OC3 and OC3-IV2 cells treated with 20 μM U0126 or LY294002 for 24 h by Q-PCR. (h) Left: Relative mRNA expression from GLI1 between OC3-IV2-Scr, OC3-IV2-shGli1#1, and #2 cells. *Compared with the relative mRNA expression in OC3-IV2-Scr cells. Right: The Boyden chamber assay was used to assess migration and invasion of OC3-IV2-Scr, OC3-IV2-shGli1#1, and #2 cells. (i) Proliferation of OC3-IV2-Scr, OC3-IV2-shGli1#1, and #2 cells was assessed by MTT and colony formation assays. *Compared with OC3-IV2-Scr cells at the indicated time point. Data from at least three independent experiments are presented as mean±SEM (*P<0.05).

Fig 5: GLI2 is required to maintain the basal-like state in PDA.(A) Immunoblot shows the effect of shRNA-mediated GLI2 and GLI1 combined knockdown on expression of the indicated proteins in KP4 cells. (B,C) Graph shows the effect of siRNA mediated GLI2 and GLI1 combined knockdown on EMT associated gene expression (B) and basal-like subtype gene expression (C) determined by qRT-PCR, displayed as fold change normalized to a scrambled siRNA control in KP4 cells (n = 2). (D) Brightfield images of Panc0327 and KP4 cells grown in 2D monolayer show loss of a mesenchymal phenotype upon GLI2 knockout (GLI2 KO) compared to Cas9 control cells (CTRL). Scale bar, 100 μm. (E) Immunofluorescence staining (left) and quantification (right) of percentage ESRP1 nuclear localization in KP4 control (shGFP) and shRNA mediated GLI2 knockdown cells. Data represent 10 fields from two independent experiments. Solid arrowheads indicate absence of nuclear ESRP1; open arrowheads indicate nuclear ESRP1. Scale bar, 50 μm. (F) Quantification of in vitro growth rate of KP4 Cas9 CTRL and GLI2 KO cells. (G) Quantification of in vivo growth of KP4 CTRL and GLI2 KO subcutaneous xenografts. Error bars represent s.e.m. (H) Immunohistochemistry of KP4 xenografts show downregulation of basal-like marker S100A2 (top) and upregulation of GATA6 (bottom) in GLI2 KO cells compared to CTRL, consistent with loss of the basal-like state. Scale bars, 200 μm. p-Values were calculated by two-tailed unpaired t test. n.s. = not significant; *p<0.05; **p<0.01; ***p<0.001.10.7554/eLife.45313.013Figure 4—source data 1.GLI2 is required to maintain a basal-like state in PDA.