Abstract
Odontogenic keratocysts (OKCs) are common cystic lesions of odontogenic epithelial origin that can occur sporadically or in association with naevoid basal cell carcinoma syndrome (NBCCS). OKCs are locally aggressive, cause marked destruction of the jaw bones and have a propensity to recur. PTCH1 mutations (at ∼80%) are frequently detected in the epithelia of both NBCCS-related and sporadic OKCs, suggesting that PTCH1 inactivation might constitutively activate sonic hedgehog (SHH) signalling and play a major role in disease pathogenesis. Thus, small molecule inhibitors of SHH signalling might represent a new treatment strategy for OKCs. However, studies on the molecular mechanisms associated with OKCs have been hampered by limited epithelial cell yields during OKC explant culture. Here, we constructed an isogenic PTCH1R135X/+ cellular model of PTCH1 inactivation by introducing a heterozygous mutation, namely, c.403C>T (p.R135X), which has been identified in OKC patients, into a human embryonic stem cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. This was followed by the induction of epithelial differentiation. Using this in vitro isogenic cellular model, we verified that the PTCH1R135X/+ heterozygous mutation causes ligand-independent activation of SHH signalling due to PTCH1 haploinsufficiency. This activation was found to be downregulated in a dose-dependent manner by the SHH pathway inhibitor GDC-0449. In addition, through inhibition of activated SHH signalling, the enhanced proliferation observed in these induced cells was suppressed, suggesting that GDC-0449 might represent an effective inhibitor of the SHH pathway for use during OKC treatment.
Highlights
Odontogenic keratocysts (OKCs), known as keratocystic odontogenic tumours (KCOTs), are common odontogenic lesions that can occur sporadically or in association with naevoid basal cell carcinoma syndrome (NBCCS)
Binding of sonic hedgehog protein (SHH) to PTCH1 or mutational inactivation of PTCH1 relieves the inhibition of SMO, a G protein-coupled receptor-like protein, which might result in SHH pathway activation in a liganddependent or ligand-independent manner, respectively
More than 200 PTCH1 mutations have been detected in syndromic and sporadic OKCs, most of which were the predicted truncationcausing mutations resulting in partial loss of PTCH1 expression
Summary
Jiemei Zhai[1,2], Heyu Zhang[3], Jianyun Zhang[1], Ran Zhang[1], Yingying Hong[4], Jiafei Qu1, Feng Chen[3] and Tiejun Li1. We constructed an isogenic PTCH1R135X/+ cellular model of PTCH1 inactivation by introducing a heterozygous mutation, namely, c.403C>T (p.R135X), which has been identified in OKC patients, into a human embryonic stem cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. This was followed by the induction of epithelial differentiation. Using this in vitro isogenic cellular model, we verified that the PTCH1R135X/+ heterozygous mutation causes ligand-independent activation of SHH signalling due to PTCH1 haploinsufficiency.
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