AB033. Novel roles of mitochondrial outer membrane proteins in the maintenance of androgen receptor protein stability, resistance to anti-androgen receptor agents and progression of prostate cancer

Abstract

Background

Anti-androgen/androgen receptor (AR) agents, as the first-line hormone therapy, have been commonly administrated in prostate cancer (PCa) patients. However, the response of patients to these agents is temporary, the diseases inevitably developed to the androgen-refractory stage, and lost the response to these agents. In this current study, we investigated the novel roles of mitochondrial outer membrane (TOM) proteins in the maintenance of AR protein stability, and the development of resistance to anti-AR agents through mitochondria-mediated cell stress, and the activation of cell survival signaling pathways.

Methods

The co-localization of AR and TOM proteins was observed by confocal microscopy and biochemical assays. The physical interaction between AR and TOM proteins and the interactive domains were identified by the co-immunoprecipitation. TOM genes were interfered by RNAi, and AR protein stability was assessed by confocal microscopy and immunoblot, and the AR transcription activity was tested by RT-PCR and luciferase reporter assay. The mitochondrial functions and intracellular metabolism were evaluated by a Seahorse Bioscience XFe analyzer, and ROS was tested by flow cytometry. The phenotype transition was identified by the cell morphology and specific biomarkers. The sensitivity of anti-AR agents was tested by the MTS assay and cell colony assays.

Results

In AR-positive PCa cells, AR protein is located in mitochondria, and particularly interacts with TOM proteins through the LLXXL motifs at the C-terminal. Interfering one protein induced the degradation of another protein. The knockdown of TOM protein impaired the AR protein stability, nuclear translocation and the transcription activity. Alternatively, the knockdown of AR or anti-AR agent Casodex impaired the stability of TOM proteins. Furthermore, the interference of TOM proteins drastically altered intracellular metabolism, especially in direction to weaken cell respiration and enhance aerobic glycolysis, so called the “Warburg effect”. The consequence of the metabolic change contributed to the ROS elevation and the activation of PI3K/AKT pathway, cell survival and phenotype transition of PCa cells, and hence promoted cancer progression.

Conclusions

In addition to HSP proteins, we, at the first time, reported that TOM proteins are alternative novel machine to maintain AR protein stability. Importantly, anti-AR agents obtained the therapeutic effects on PCa by blocking androgen/AR, while activated alternative cell survival pathway through mitochondrial outer membrane proteins-mediated metabolism alternation and ROS elevation. These results implicated a novel mechanism in which PCa patients develop the resistance to anti-AR agents, and develop toward the androgen refractory stage.