TOX3 (TNRC9) Over Expression in Bladder Cancer Cells Decreases Cellular Proliferation and Triggers an Interferon-Like Response

Abstract

Background: Human TOX3 (TOX high mobility group box family member 3) regulates Ca 2+ dependent transcription in neurons and has been associated with breast cancer susceptibility. Aim of the study was to investigate the expression of TOX3 in bladder cancer tissue samples and to identify genes and pathways altered upon TOX3 dysregulation using a cell line model. Methods:We performed microarray transcript profiling of biopsies and validated the data with RT-qPCR. We used cell line models for Overexpression and siRNA mediated knockdown of TOX3. Pathway analysis was applied for target gene identification and immunoprecipitation studies were used for DNA binding studies.Results: Microarray transcript profiling of 89 bladder biopsies showed a significant up-regulation of TOX3 (p<10 -4 ) in non-muscle invasive (Ta-T1) bladder tumors compared to muscle-invasive (T2-T4) bladder tumors and normal urothelium. Microarray expression profiling of human bladder cancer cells over expressing TOX3 followed by Pathway analysis showed that TOX3 Overexpression mainly affected the Interferon Signaling Pathway. TOX3 up regulation induced the expression of several genes with a gamma interferon activation site (GAS), e.g. STAT1.In vitro functional studies showed that TOX3 was able to bind to the GAS-sequence located at the STAT1 promoter. siRNA mediated knockdown of TOX3 in RT4 bladder cancer cells decreased STAT1 expression suggesting a direct impact of TOX3 on STAT1. Immunoprecipitation of TOX3 over expressing cell extracts with an artificial "GAS"-DNA element resulted in an enrichment of the GAS containing DNA-sequence, providing evidence for a potential interaction of TOX3 with the GAS-sequence of STAT1.Conclusions: These results provide evidence for an alternative activation of the downstream interferon targets, independent of the initial interferon-receptor interaction, and consequently a biological role for TOX3.