Figure 4. The immuno-regulatory gene RORγ is a critical dependency of pancreatic cancer.
(A) Rorc expression in stem and non-stem REM2-KPf/fC tumor cells; representative of three biological replicates.
(B) Representative images of RORγ expression in normal adjacent human pancreas (left), PanINs (middle), and PDAC (right). RORγ (green), E-Cadherin (red), Dapi (blue), scale=50um.
(C) Frequency of RORγ+ cells within E-Cadherin+ epithelial fraction in patient samples quantified by immunofluorescence; Normal adjacent, n=3; pancreatitis, n=8; PanIN 1, n=10; PanIN 2, n=6; PDAC, n=8.
(D-E) Impact of shRNA-mediated RORγ inhibition on 3D growth of KPR172H/+C (D) and KPf/fC (E) cells, n=3 per shRNA.
(F) Impact of shRNA-mediated RORγ inhibition on Msi2-GFP stem cell content of in KPf/fC cells in 3D culture (H) n=3.
(G) Impact of shRNA-mediated RORγ inhibition on flank tumor growth of KPf/fC cells, n=4.
(H-I) Reduced tumor burden in Rorc−/−KP f/f C mice. Age-matched WT KP f/f C and Rorc−/−KP f/f C set shown displayed reduced tumor cell number (H), and reduced adenocarcinoma content (I); low-grade PanIN indicated with red arrow, PDAC indicated with black arrow, scale=100um; n=3 mice from 8–10 wks of age; representative plots and images from matched mice are displayed.
(J and K) Relative RNA expression of stem cell programs (J) and pro-tumorigenic factors (K) in KPf/fC cells transduced with shCtrl or shRorc. Red, over-represented; blue, under-represented; color denotes fold change. (L) Venn diagram of genes downregulated with RORγ loss (q<0.05, purple), Stem specific SE-associated genes (green), and genes associated with H3K27ac peaks with RORγ consensus binding sites (orange).
(M) Number of RORγ, CBFB, Sox9 binding sites found in stem cell SEs relative to random genomic background of equivalent base-pair coverage (p<0.05).
(N) Relative RNA expression of SE-associated oncogenes in KPf/fC cells transduced with shCtrl or shRorc. Red, over-represented; blue, under-represented; color denotes fold change from median values.
(O) H3K27ac ChIP-seq reads for genes marked by stem cell SEs and downregulated in RORγ-depleted KPf/fC cells. Data represented as mean +/− S.E.M. * p<0.05, ** p<0.01, *** p<0.001 by Student’s t-test or One-way ANOVA.