Abstract 4075: Regulation of NKX3.1 by RMND5 proteins.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Expression of the prostatic tumour suppressor, NKX3.1 is reduced or undetectable in a large proportion of advanced human prostate tumours. Although loss of heterozygosity (LOH) is common at the 8p21.2 locus which encompasses the NKX3.1 gene, mutations of the remaining NKX3.1 allele that would account for loss of NKX3.1 expression have not been identified. Indeed, the documented discordance between NKX3.1 mRNA and protein expression suggests that translational or posttranslational mechanisms contribute to the loss of NKX3.1 expression in prostate tumour tissues. We have identified two novel NKX3.1 binding proteins in LNCaP cells, RMND5A and RMND5B. The RMND5A gene locus at 2p11.1 is deleted or amplified in a variety of cancers, while the RMND5B locus at 5q35.3 undergoes frequent LOH in breast tumours from BRCA1 and BRCA2 mutation carriers and is located within an uncharacterised prostate cancer heritability locus. RMND5 proteins are widely expressed, multidomain proteins that each contain a putative RING domain, suggesting that they are able to function as E3 ubiquitin ligases. In in vitro ubiquitin assays, the RING domain of RMND5A was able to associate with UbcH2, UbcH5b and UbcH5c, while the RING domain of RMND5B interacted with UbcH5b and UbcH5c to mediate ubiquitin transfer. Both RMND5A and RMND5B were associated with ubiquitinated proteins in vivo and the increased expression of RMND5A or RMND5B augmented cellular levels of ubiquitinated proteins, providing further evidence of their E3 ubiquitin ligase activity. Overexpression of either RMND5A or RMND5B in LNCaP cells resulted in dose-dependent declines in endogenous NKX3.1 levels, which were attenuated following proteasome inhibition. In addition, RMND5A or RMND5B overexpression enhanced cellular levels of ubiquitinated NKX3.1. In LNCaP cells, RMND5 proteins were diffusely distributed in the cytoplasm and nucleus, with RMND5B occasionally exhibiting a punctate cytoplasmic distribution. When NKX3.1 and RMND5A or RMND5B were overexpressed, RMND5 proteins became predominantly nuclear, while the nuclear levels of NKX3.1 were markedly reduced. Together, these findings indicate that RMND5 proteins promote NKX3.1 ubiquitination and proteasome-dependent degradation and additionally modify the intracellular localisation of NKX3.1, potentially regulating its function as a transcription factor. Citation Format: Alison Louw, Marc A. Thomas, Jennet Harvey, Jacqueline M. Bentel. Regulation of NKX3.1 by RMND5 proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2013-4075