Figures and data
FAK downregulation in serrated tumors.
a Summary of FAK IHC staining in 11 human BRAFV600E-mutant CRC samples. N represents normal colon; P represents polyp; C represents carcinoma; NA, not applicable; ↔ represents no change; ↑ represents an increase. ↓ represents a decrease. b Representative IHC staining of BRAFV600E-mutant patient SSA/P, serrated colorectal adenoma, and adjacent normal tissues. c IHC staining of Fak in small intestine tumors in a 12-month-old BC mouse. d Representative IHC staining of Fak in colon tumor in 12-month-old BC mice. Scale bars in b: 1 mm (upper panel) and 100 μm (lower panel). Scale bars in c and d: 100 μm.
Fak loss enhances BRAFV600E-driven cecal tumorigenesis in mice.
a Representative hematoxylin and eosin (H&E) staining of the small intestine, cecum, and colon from indicated 6-week-old mice. b Summary of tumor incidence at small intestine and colon in indicted mice at the indicated age. c Summary of tumor incidence and tumor stage at cecum in indicated mice at the indicated age. d H&E staining of the cecum in BC mice and cecal serrated adenoma/polyp and carcinoma in FBC mice at the indicated age. e Representative IHC staining of Fak in cecal tumors in FBC mice. Scale bars: 100 μm.
Molecular characterization of cecal tumors in FBC mice.
a GSEA plot showing enrichment of human SSA/Ps signature genes (upregulated genes in SSA/Ps) in FBC cecal tumors vs normal cecal mucosa of B mice. b GSEA plot showing that downregulated genes in human SSA/Ps were also reduced in FBC cecal tumors. c Microsatellite instability status of FBC mice cecal mucosa and cecal carcinomas. Each column represents one sample.
BRAFV600E mutation and Fak loss-mediated changes in signaling pathways.
GSEA analysis showing upregulation of MAPK signature (a), intestinal WNT signaling (b), YAP/TAZ target gene signature (c) and intestinal differentiation signature (d), and downregulation of intestinal stem cell signature (e) in the cecum of BC mice vs B mice (n=4 per group). GSEA plots revealed no significant change in MAPK signature (f), intestinal WNT signaling (g), YAP/TAZ target gene signature (h), and intestinal differentiation signature (i) in the cecum of FBC mice vs BC mice, but enrichment of stem cell signature in FBC mice (j) (n=4 per group). k GSEA analysis showing upregulation of upregulated fetal spheroid markers in the cecum of BC mice vs B mice, and further enrichment in the cecum of FBC mice vs BC mice (n=4 per group). l Immunoblotting analysis of LGR4 in the cecum from indicated 6-week-old mice. m Representative in situ hybridization (ISH) staining of tumor sections from ApcMin/+, BC, and FBC mice using Lgr4 and Lgr5 probes. Scale bars: 100 μm.
Fak loss inhibits ERK phosphorylation and upregulates Lgr4.
a and b Immunoblotting analysis of intestinal mucosa lysates from indicated bowel subsites in indicated 6-week-old mice. c Immunoblotting analysis of cecum lysates from 6-week-old BC mice treated with vehicle or EGFR inhibitor erlotinib for 4 hours. Each lane represented a single mouse. d Immunoblotting analysis of cecum lysates from 6-week-old BC mice treated with vehicle or FAK inhibitor PF-562271 for 4 hours. Each lane represented a single mouse. e Immunoblotting analysis of lysates from freshly isolated cecal crypts and cecal organoids treated with DMSO, MEK inhibitor PD0325901, or erlotinib, respectively as described in Method. f qRT-PCR of Lgr4 using lysates from HT-29 cells treated with the vehicle and MEKi for 4 hours. Data presented as mean ± SD (***P<0.001; Student’s t-test, two-tailed). g qRT-PCR of Lgr4 using cecum lysates from BC mice treated with vehicle or MEKi for 6 hours. Data presented as mean ± SD (**P<0.01; Student’s t-test, two-tailed). Abrogation of ERK phosphorylation at T202/Y204 in the cecum was confirmed by western blot. h qRT-PCR of Lgr4 in cecum from BC and FBC mice (n=3 per group). Data presented as mean ± SD (P value calculated using two-tailed Student’s t-test). i Immunoblotting analysis of the lysates from HT-29 cells treated with cycloheximide (100 μg/ml) and/or MEK inhibitor PD0325901 (10 μM) as indicated.
ERK activation is FAK/EGFR-independent in KC mice.
Representative hematoxylin and eosin (H&E) staining of the small intestine, cecum, and colon from indicated 9-month-old mice. b Immunoblotting analysis of intestinal mucosa lysates from indicated bowel subsites in indicated 6-week-old mice. c The cecal mucosa lysates from 6-week-old KC and BC mice were used for immunoprecipitation and immunoblotting with the indicated antibodies. d Immunoblotting analysis of cecum lysates from 6-week-old KC mice treated with vehicle or EGFR inhibitor erlotinib for 4 hours. e and f Comparison of FAK expression levels between CRCs with indicated mutations by analysis of TCGA RNA-sequencing dataset. Data were analyzed for statistical significance using a Student t-test. g Diagram of the “just-right” MAPK signaling model in the serrated pathway.
