A hormone-dependent feedback-loop controls androgen receptor levels by limiting MID1, a novel translation enhancer and promoter of oncogenic signaling

Abstract

BackgroundHigh androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR.MethodsAR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively.ResultsThe microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner.ConclusionPromotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.

The MID1 protein is a central player during development and in disease

“…At the same time AR regulates MID1 transcription: the MID1 gene contains several AR binding sites (8). In the presence of Androgen, AR suppresses MID1 transcription, whereas androgen withdrawal increases MID1 and concomitantly AR-protein levels (8). In line with these data MID1 is over expressed in a subtype of prostate cancer cells with a more aggressive phenotype (8), suggesting that MID1 is a tumour and metastasis promoting factor.…”

“…Recently MID1 was found to up regulate AR protein levels in several prostate cancer cell lines and its expression itself was negatively regulated by androgen signalling (see also section 6) (8). Like the AR gene MID1 is expressed in the GT in human embryos (46).…”

“…In this context it has been shown that the MID1 protein complex binds to two sites of the AR mRNA: a CAG repeat region and a GGY repeat region. Binding of the MID1 complex to the AR mRNA induces AR translation (8). Consequently, the knockdown of MID1 or its complex partners in prostate cancer cells leads to reduced AR levels.…”

“…Consequently, the knockdown of MID1 or its complex partners in prostate cancer cells leads to reduced AR levels. At the same time AR regulates MID1 transcription: the MID1 gene contains several AR binding sites (8). In the presence of Androgen, AR suppresses MID1 transcription, whereas androgen withdrawal increases MID1 and concomitantly AR-protein levels (8).…”

“…In prostate cancer cells MID1 binds to the androgen receptor (AR) mRNA and induces its translation. Also, MID1 is over expressed in a subtype of prostate cancer cells with a very aggressive phenotype (8) suggesting that MID1 is a tumour and metastasis-promoting factor.…”