Exploring promoter silencing and re-expression of SH3GL2/endophilin A1 in urothelial cancer
Zucker, Isaac Jake
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INTRODUCTION: Bladder cancer (BC) is highly prevalent. It presents as either non-muscle invasive or muscle-invasive disease. The prognosis of muscle invasive disease is poor, with a 5-year survival rate of less than 50%. Treatment approaches for both types of BC have not advanced much in the last few years and new therapies are needed to overcome the large burden of BC. Recently, a large effort has been undertaken to classify BC into molecular subtypes. These analyses have revealed significant alterations in epigenetic modifiers in BC. A previous study from our group revealed that SH3GL2, a negative regulator of receptor tyrosine kinase (RTK) signaling, was lost with high frequency in BC, leading to increased growth of tumor cells in-vitro and in-vivo. Conversely, forced expression of SH3GL2 in BC cell lines attenuated oncogenic behaviors including growth and migration. In addition to genomic deletion, SH3GL2 is subject to methylation-induced silencing, a key epigenetic mechanism. OBJECTIVE: Epigenetic mechanisms of gene regulation are known to be perturbed in BC. The objectives of this study were to investigate methylation of the SH3GL2 promoter and to test whether agents that promote Deoxyribonucleic acid (DNA) demethylation could be used to re-express SH3GL2 thereby restoring regulation of RTK signaling. METHODS: Methylation of a specific CpG island in the SH3GL2 promoter was analyzed using methylation-specific Polymerase Chain Reaction (PCR) in a panel of BC cell lines with known SH3GL2 messenger Ribonucleic Acid (mRNA) status. Selected BC cell lines were treated with a variety of demethylating agents at different doses and for different times to evoke the re-expression of silenced SH3GL2. Demethylation inhibitors were combined with the histone deacetylase inhibitor, trichostatin A (TSA), to determine whether further re-expression could be achieved. RESULTS: The SH3GL2 promoter displayed differing extents of promoter methylation among cell lines examined. In RT4 cells, the only cell line with detectable expression of SH3GL2 mRNA and protein, the promoter was completely unmethylated. In contrast, T24 and 253J cells displayed significant promoter methylation with little to no SH3GL2 mRNA expressed, consistent with methylation-induced silencing. Treatment of T24 and 253J with 5-Aza-2’-deoxycytidine (5-Aza-dC, 20 M), a DNA methyltransferase (DNMT) inhibitor increased gene expression but this was not dose- or time-dependent. Two additional DNMT inhibitors, Zebularine and RG-108 were also tested. A much higher dosage of Zebularine was required to trigger activation (500 M) while RG-108 was unable to trigger gene reactivation at all. Combination treatment with 5-Aza-dC and TSA further increased SH3GL2 expression compared to either agent alone. These results suggest that DNA methyltransferase inhibition is an effective treatment to re-express SH3GL2 in cells with SH3GL2 promoter silencing. CONCLUSION: The present study shows silencing of SH3GL2 in a variety of BC cell lines as a consequence of DNA promoter hypermethylation. Treatment with demethylating agents was able to increase gene expression. Based on prior findings showing attenuation of tumor cell growth and migration with forced expression of SH3GL2, DNA methyltransferase inhibition represents an effective strategy to re-express SH3GL2 in BC and normalize tumor cell behavior.