Molecular analysis of keratocystic odontogenic tumor cell lines derived from sporadic and basal cell nevus syndrome patients

Abstract

Keratocystic odontogenic tumor (KCOT) is a benign tumor often associated with basal cell nevus syndrome (BCNS). Mutations in Patched 1 (PTCH1), the Hedgehog (Hh) receptor, are responsible for BCNS. BCNS is distinguished by morphological anomalies and predisposition to benign and malignant tumors, including medulloblastoma, basal cell carcinoma, KCOT and ovarian fibromas. Among these tumors, KCOT is the least well studied because a suitable model system is not available for its investigation. To enable KCOT to be studied, we established two KCOT cell lines, one from a BCNS case (designated as iKCOT1) and one from a sporadic KCOT case (designated as sKCOT1). The BCNS‑derived KCOT cell line, iKCOT1, retained a germline-mutated PTCH1 allele and a wild-type PTCH1 allele. The sporadic KCOT-derived KCOT cell line, sKCOT1, had different loss-of-function PTCH1 mutations on both alleles. Both cell lines expressed stem cell markers (CD44, SOX2 and BMI1), mesenchymal cell markers (CDH2, VIM and SNAI2) and a neurogenic marker (NEFL). Culture of the cell lines in high calcium concentration media induced expression of epithelial cell and keratinocyte marker proteins (CDH1, CLDN1, KRT10 and IVL). Parakeratosis, which is characteristic for KCOTs, was observed in 2-D cultures. The similarities in protein expression patterns between the two cell lines suggested that common mechanisms underlie the development of both types of KCOT and a probable common origin of KCOT cells.

Decoding a gene expression program that accompanies the phenotype of sporadic and basal cell nevus syndrome‐associated odontogenic keratocyst

“…Our analysis revealed that sporadic and BCNS‐associated OKCs share a significantly common (85%) transcriptomics profile when compared with the DF. This is consistent with the results of our recent systematic review on the immunohistochemical profile of OKCs, 27 a study on the protein expression profile of OKC cell lines, 28 as well as an elegant proteomics study employing four sporadic and one BCNS‐associated OKCs, where no significant differences were found between the two subtypes 29 . Our data support the notion that sporadic and BCNS‐associated OKCs may share the same cell origin and their pathogenesis might be driven by common molecular mechanism(s) 1,28 .…”

Expression of stem cell markers in stroma of odontogenic cysts and tumors

“…In contrast, others reported expression of SOX2 in AMB 18,19 and suggested that since SOX2 expression was also positive in dental lamina of developing human primary molars and third molars, it probably acts in maintaining the progenitor state of ameloblastic epithelium and that AMB may originate from SOX2‐expressing dental lamina epithelium 18 . SOX2 has also been investigated in the epithelial component of sporadic and syndromic OKC, 14,15,19‐21 where it was found not only to be positive, but to be more expressed than in AMB 19,20 . Other odontogenic lesions with a positive epithelial expression of SOX2 were DC, 13 adenomatoid odontogenic tumor (AOT), 14 and odontoma 22 .…”

Establishment of a patient‑derived mucoepidermoid carcinoma cell line with the CRTC1‑MAML2 fusion gene

“…At the same time, the fibroblasts derived from the tumor tissue of the same patient, were obtained and grown in F-medium. Once cells reached confluence (80%), they were washed with PBS (Mg 2+ and Ca 2+ free) ( 23 ) and detached with 0.05% EDTA/trypsin for 5 min at 38˚C ( 24 ). After centrifugation at 167 x g for 5 min at 4˚C, the MEC cells were resuspended in F-medium, containing Y-27632 and seeded (0.3x10 6 cells) in 60 mm dishes.…”

“…Amplification of the GAPDH gene (forward, 5'-CAA TGA CCC CTT CAT TGA CC-3' and reverse, 5'-GAC AAG CTT CCC GTT CTC AG-3') was performed as a control. Successfully amplified RT-PCR products of the CRTC1-MAML2 fusion gene were purified and sequenced (24) using BigDye™ Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific, Inc.) and 2% agarose gel electrophoresis.…”