Partial truncation of the C-terminal domain of PTCH1 in cancer promotes tumorigenesis by non-canonical activation of GLI transcriptional activity.

Loss of function mutations of the Hedgehog receptor PTCH1 are oncogenic drivers in some skin and brain cancers. We recently reported mutations in exons encoding the C-terminal tail of PTCH1 in colon cancer, which result in premature truncation but do not impair canonical Hedgehog signalling. In this study, we show that colon cancer cells engineered by CRISPR/Cas9 to express endogenous truncated PTCH1 have enhanced proliferation, colony formation, anchorage-independent growth and form larger tumours in vivo than isogenic cells expressing wild-type PTCH1. Analysis of the mechanisms underlying this growth advantage revealed profound transcriptional changes and unexpectedly, upregulation of GLI1 and GLI2 by a non-canonical route that proved to be necessary for the proliferative advantage. Furthermore, we found that truncation of PTCH1 C-tail upregulated several cancer-related pathways, including EGFR and several EGFR ligands and led to enhanced GLI-dependent PI3K activation, which exerted a positive feedback regulation on GLI expression and activity. Accordingly, PTCH1 mutant cells were highly sensitive to PI3K and GLI inhibitors and were only partially sensitive to EGFR and MEK inhibitors. Altogether, these findings reveal that PTCH1 C-tail truncating mutations promote colon cancer tumorigenesis through a non-canonical GLI-PI3K positive loop. Overall design: SW620 cells were engineered with by CRISPR/Cas9 to introduce indel mutations after D1222 of PTCH1. Two clones, C9 and C15, were isolated and sequenced, which had different frameshifts after D1222 and resulted in premature stop codons. Cells engineered with a scrambled sequence gRNA (SCR) were also selected as isogenic cells, and sequenced-verified to express WT PTCH1. RNA-seq from total RNA isolated from 3 consecutive passages of SCR, C9 and C15 cells growing in DMEM containing 10% FBS was performed.