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Ralaniten induction of expression of metallothionein isoforms is by a mechanism independent of androgen receptor

University of British Columbia

Background: Advanced prostate tumors that develop resistance to androgen deprivation therapy are incurable and uniformly fatal. Nonetheless, the manifestation of this lethal form, known as castration-resistant prostate cancer (CRPC), does not preclude most of these tumors from sustained dependence on the androgen receptor (AR) for growth and survival. To inhibit AR pathway signaling, the strategy of all currently approved drugs ultimately converge upon the C-terminus ligand-binding domain (AR-LBD) to target and disrupt AR activation. Ralaniten is a novel, first-in-class drug which binds the AR within its N-terminal domain (AR-NTD). Due to this unique mechanism of action, we predicted that Ralaniten would induce a distinct global response compared to alternative AR-inhibitors. This study initiates the characterization of Ralaniten specific gene expression profiles and unravels the mechanism of induction of an unexpected group of genes from the metallothionein (MT) gene family. Methods: In vitro experiments were performed in 4 human prostate cancer cell lines with experimentally useful genomic and phenotypic features. Preliminary gene expression data were generated by microarray. Pathway and statistical analyses revealed candidate genes for subsequent investigation. Transcriptional data were validated by qPCR and at the protein level by western blot. Reporter assays for gene activity were conducted after transient transfection of plasmids. Transient siRNA- mediated knockdown experiments assessed involvement of potentially relevant transcription factors. AR NTD inhibitors included Ralaniten, EPI-7170, SINT-1, and LPY26, whereas AR LBD inhibitors included the antiandrogens bicalutamide and enzalutamide. AR transactivation was mediated using the synthetic androgen R1881. Results: Microarray analyses revealed the MT family to be the most abundantly induced by Ralaniten in the absence of androgen. Induction was experimentally confirmed to be Ralaniten specific. Knockdown experiments implicate a central role for the transcription factor, MTF-1, in the induction of MT genes by Ralaniten, and have ruled out the requirement for the AR and the redox activated transcription factor, Nrf2, in this mechanism. Conclusions: Ralaniten induced the expression of MT genes by a mechanism independent of expression of AR and Nrf2. MT induction by Ralaniten is exquisitely dependent on the expression of the transcription factor, MTF-1.

Text

2021-01-06

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/76974

Pathology and Laboratory Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/