Journal
ORAL ONCOLOGY
Volume 94, Issue -, Pages 58-67Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.oraloncology.2019.05.010
Keywords
Oral dysplasia; beta-Catenin; Keratinocyte; Wnt; Survivin; Oral cancer; Cell signaling
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Funding
- National Fund for Scientific and Technological Development FONDECYT [1180495]
- Advanced Center for Chronic Diseases, FONDAP-ACCDiS [15130011]
- National Commission for Scientific and Technological Research (CONICYT) Fellowship [21170490]
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Objectives: To evaluate the localization of beta-catenin in oral dysplastic cells, the expression of target genes up-regulated in oral dysplasia, and the role of Wnt ligands in these events. Materials and methods: Subcellular localization of total and non-phosphorylated (transcriptionally active) beta-catenin was evaluated by immunofluorescence and biochemical fractionation in dysplastic oral keratinocytes (DOK), non-dysplastic oral keratinocytes (OKF6), oral squamous carcinoma cells (CAL27) and primary oral keratinocytes. Tcf/Lef-dependent transcription was measured by luciferase reporter assays. Expression of target genes, survivin and cyclin D1, was evaluated by RT-qPCR and Western blotting. Wnt secretion was inhibited with the inhibitor of porcupine, C59. Wnt3a and beta-catenin were evaluated in biopsies by tissue immunofluorescence. Results: Immunofluorescence and fractionation experiments showed augmented nuclear beta-catenin (total and transcriptionally active) in DOK, when compared with OKF6 and CAL27 cells. Intriguingly, conditioned medium from DOK promoted nuclear accumulation of beta-catenin and Tcf/Lef-dependent transcription in OKF6 and primary oral keratinocytes, suggesting the participation of secreted factors. Treatment of DOK with C59 decreased Wnt3a secretion, nuclear beta-catenin and the expression of survivin and cyclin D1 at both mRNA and protein levels. Accordingly, DOK secreted higher Wnt3a levels than OKF6, and inhibition of Wnt3a secretion prevented DOK-induced Tcf/Lef-dependent transcription in OKF6. These observations were confirmed in clinical samples, since tissue immunofluorescence analysis showed simultaneous expression of Wnt3a and nuclear beta-catenin in oral dysplasia, but not in healthy mucosa biopsies. Conclusion: These data indicate that secretion of Wnt ligands is critical for beta-catenin nuclear localization and expression of target genes in oral dysplasia.
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