Robert Kleszcz1, Anna Szymańska1, Violetta Krajka-Kuźniak1, Wanda Baer-Dubowska1, Jarosław Paluszczak2. 1. Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, ul. Święcickiego 4, 60-781, Poznań, Poland. 2. Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, ul. Święcickiego 4, 60-781, Poznań, Poland. paluszcz@ump.edu.pl.
Abstract
PURPOSE: Activation of the Wnt pathway contributes to the development of head and neck squamous cell carcinomas (HNSCC) and its inhibition has recently emerged as a promising therapeutic strategy. Here, we aimed at identifying suitable molecular targets for down-regulation of canonical Wnt signaling in HNSCC cells. METHODS: Candidate target genes (PORCN, WNT3A, FZD2, FZD5, LRP5, DVL1, CIP2A, SET, KDM1A, KDM4C, KDM6A, CBP, CARM1, KMT2A, TCF7, LEF1, PYGO1, XIAP) were silenced using siRNA and selected targets were subsequently blocked using small molecule inhibitors. The effect of this treatment on the expression of β-catenin-dependent genes was assessed by qRT-PCR. The effect of the inhibitors on cell viability was evaluated using a resazurin assay in HNSCC-derived cell lines. A luciferase reporter assay was used for confirmation of the inhibition of Wnt-dependent gene expression. Cell migration was evaluated using a scratch wound healing assay. Cytometric analysis of propidium iodide stained cells was used for cell cycle distribution evaluation, whereas cytometric analysis of caspase 3/7 activity was used for apoptosis induction evaluation. RESULTS: We found that inhibition of Porcupine and CBP/β-catenin interaction by IWP-2 and PRI-724, respectively, most strongly affected β-catenin-dependent gene expression in HNSCC cells. These inhibitors also induced apoptosis and affected HNSCC cell migration. CONCLUSIONS: Targeting Porcupine or the CBP/β-catenin interaction seems to be an effective strategy for the inhibition of canonical Wnt signaling in HNSCC cells. Further studies are required to confirm the possible therapeutic effect of IWP-2 and PRI-724 in HNSCC.
PURPOSE: Activation of the Wnt pathway contributes to the development of head and neck squamous cell carcinomas (HNSCC) and its inhibition has recently emerged as a promising therapeutic strategy. Here, we aimed at identifying suitable molecular targets for down-regulation of canonical Wnt signaling in HNSCC cells. METHODS: Candidate target genes (PORCN, WNT3A, FZD2, FZD5, LRP5, DVL1, CIP2A, SET, KDM1A, KDM4C, KDM6A, CBP, CARM1, KMT2A, TCF7, LEF1, PYGO1, XIAP) were silenced using siRNA and selected targets were subsequently blocked using small molecule inhibitors. The effect of this treatment on the expression of β-catenin-dependent genes was assessed by qRT-PCR. The effect of the inhibitors on cell viability was evaluated using a resazurin assay in HNSCC-derived cell lines. A luciferase reporter assay was used for confirmation of the inhibition of Wnt-dependent gene expression. Cell migration was evaluated using a scratch wound healing assay. Cytometric analysis of propidium iodide stained cells was used for cell cycle distribution evaluation, whereas cytometric analysis of caspase 3/7 activity was used for apoptosis induction evaluation. RESULTS: We found that inhibition of Porcupine and CBP/β-catenin interaction by IWP-2 and PRI-724, respectively, most strongly affected β-catenin-dependent gene expression in HNSCC cells. These inhibitors also induced apoptosis and affected HNSCC cell migration. CONCLUSIONS: Targeting Porcupine or the CBP/β-catenin interaction seems to be an effective strategy for the inhibition of canonical Wnt signaling in HNSCC cells. Further studies are required to confirm the possible therapeutic effect of IWP-2 and PRI-724 in HNSCC.
Entities:
Keywords:
CBP; Head and neck cancer; IWP-2; PRI-724; Porcupine; Wnt pathway