Nuclear localization of β-catenin and expression of target genes are associated with increased Wnt secretion in oral dysplasia

• Published in: Oral oncology 2019-07, Vol.94, p.58-67
• Main Authors: Reyes, Montserrat, Peña-Oyarzun, Daniel, Maturana, Andrea, Torres, Vicente A.
• Format: Article
• Language: English
• Subjects: beta Catenin - metabolism; Carcinoma, Squamous Cell - physiopathology; Cell Line, Tumor; Cell signaling; Humans; Keratinocyte; Keratinocytes - metabolism; Mouth Neoplasms - physiopathology; Oral cancer; Oral dysplasia; Signal Transduction; Survivin; Wnt; Wnt1 Protein - metabolism; β-Catenin
• ISSN: 1368-8375; 1879-0593
• DOI: 10.1016/j.oraloncology.2019.05.010

[Display omitted] •Dysplastic oral keratinocytes have increased levels of nuclear β-catenin.•Wnt3a is required for nuclear localization of β-catenin in oral dysplastic cells.•Expression of β-catenin target genes depend of Wnt3a in oral dysplastic cells.•Wnt3a and nuclear β-catenin are simultaneously increased in oral dysplasia biopsies.•Targeting Wnt3a represents a potential therapeutic target in oral dysplasia. To evaluate the localization of β-catenin in oral dysplastic cells, the expression of target genes upregulated in oral dysplasia, and the role of Wnt ligands in these events. Subcellular localization of total and non-phosphorylated (transcriptionally active) β-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 β-catenin were evaluated in biopsies by tissue immunofluorescence. Immunofluorescence and fractionation experiments showed augmented nuclear β-catenin (total and transcriptionally active) in DOK, when compared with OKF6 and CAL27 cells. Intriguingly, conditioned medium from DOK promoted nuclear accumulation of β-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 β-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 β-catenin in oral dysplasia, but not in healthy mucosa biopsies. These data indicate that secretion of Wnt ligands is critical for β-catenin nuclear localization and expression of target genes in oral dysplasia.