DNMT1-mediated epigenetic suppression of FBXO32 expression promoting cyclin dependent kinase 9 (CDK9) survival and esophageal cancer cell growth

Esophageal cancer (EC) is a common and serious form of cancer, and while DNA methyltransferase-1 (DNMT1) promotes DNA methylation and carcinogenesis, the role of F-box protein 32 (FBXO32) in EC and its regulation by DNMT1-mediated methylation is still unclear. FBXO32 expression was examined in EC cells with high DNMT1 expression using GSE163735 dataset. RT-qPCR assessed FBXO32 expression in normal and EC cells, and impact of higher FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with EMT-related proteins. The xenograft model established by injecting EC cells transfected with FBXO32 was used to evaluate tumor growth, apoptosis, and tumor cells proliferation and metastasis. Chromatin immunoprecipitation (ChIP) assay was employed to study the interaction between DNMT1 and FBXO32. HitPredict, co-immunoprecipitation (Co-IP), and Glutathione-S-transferase (GST) pulldown assay analyzed the interaction between FBXO32 and cyclin dependent kinase 9 (CDK9). Finally, the ubiquitination assay identified CDK9 ubiquitination, and its half-life was measured using cycloheximide and confirmed through western blotting. DNMT1 negatively correlated with FBXO32 expression in esophageal cells. High FBXO32 expression was associated with better overall survival in patients. Knockdown of DNMT1 in EC cells increased FBXO32 expression and suppressed malignant phenotypes. FBXO32 repressed EC tumor growth and metastasis in mice. Enrichment of DNMT1 in FBXO32 promoter region led to increased DNA methylation and reduced transcription. Mechanistically, FBXO32 degraded CDK9 through promoting its ubiquitination.</p

The quantitative expression of N-cadherin and Cx43 cultured for 3, 7 and 14 days.

<p>(<b>A, B, D, E</b>) N-Cadherin, PKP2 and PG significantly decreased at day 7 but significantly increased at day 14. (<b>A, C</b>) Cx43 increased a little at day 7 and then dramatically decreased at day 14. **<i>p</i><0.01; *<i>p</i><0.05.</p

Immunofluorescent staining and quantification of ID-related proteins of the EHTs cultured for 3, 7 and 14 days.

<p>(<b>A–D</b>) At day 3, the cell membranes were positively stained with N-cadherin, Cx43, PKP2 and PG. (<b>E–H</b>) At day 7, N-cadherin (<b>E</b>), PKP2 (<b>G</b>) and PG (<b>H</b>) staining were positive and mainly localized to the contact sites of adjacent cells (arrow). Whereas the Cx43 positive staining was distributed along the whole plasma membrane in a punctuate pattern (<b>F</b>). (<b>I–L</b>) At day 14, ID-related proteins concentrated at the polar ends (arrow) of cardiomyocytes. (<b>M</b>) The FI of N-Cadherin, PKP2 and PG decreased from day 3 to 7 but increased at day 14. Cx43 was expressed the opposite. **<i>p</i><0.01; *<i>p</i><0.05.</p

Immunofluorescent staining and quantification of ID-related proteins in primary cultured neonatal rat cardiomyocytes at different time point, adult and neonatal rat heart.

<p>(<b>A</b>) ID-related proteins were progressively changed their localization from the cells periphery to cell-cell contact sites. (<b>B</b>) ID-related proteins significantly increased at day 7 but slightly decreased (N-Cadherin, PKP2 and PG) or kept unchanged (Cx43) at day 14. **<i>p</i><0.01; *<i>p</i><0.05. (<b>C–D</b>) ID-related proteins accumulated in the contact sites of adjoining cells in neonatal and adult rat hearts. RC: rat cardiomyocytes; RH: rat heart.</p

Ultra-structures of the nascent ID in the EHTs cultured for 3 and 7 days.

<p>(<b>A</b>) Closely apposed cell membranes at day 3 with scarce plaque-like structure (black arrow), subplasmalemmal vesicles (dotted-line area) and fibril-like structure (arrowhead). (<b>B</b>) Adherens junctions (white arrow) and desmosomes (black arrow) dispersed among the ID at day 7. New intercellular junctions were forming (black-line area) and a single gap junction profile appeared (arrowhead). Note an adhesion region with a dense plaque of adherens junction (star) anchoring actin filaments and a desmosome (black arrow). Myo: myofibril; Z: Z-line; Nc: nucleus.</p