Effect of HER3 dimer feedback upregulation via PI3K-AKT-mTOR pathway inhibition on androgen receptor (AR) stabilization in metastatic prostate cancer.

Abstract

226 Background: Metastatic castration resistant prostate cancer (CRPC) is dependent on persistent activation of the AR. In addition, the AR and PI3K-AKT-mTOR pathways have been shown to regulate each other through complex feedback mechanisms. Various early phase clinical studies are currently investigating the role of PI3K-AKT-mTOR pathway inhibitors in improving outcomes in metastatic CRPC. We hypothesise that the formation of a HER3 (human epidermal growth factor receptor) heterodimer, has an important role in regulating the AR and PI3K-AKT-mTOR feedback loop and that its detection can predict response to PI3K-AKT-mTOR pathway inhibition. Methods: LNCAP cells were stably lentiviral transduced with shRNA against HER3. The effect of PI3K-AKT-mTOR inhibition by DS7423, GDC0068, GDC0941 and Everolimus on AR and its direct target gene PSA was evaluated by western blot and real-time quantitative PCR. CWR22 and 22RVI mouse xenograft tumours were stained with fluorescently labelled antibodies for fluorescence lifetime imaging microscopy (FLIM) to assess HER2-HER3 dimerisation by FRET (fluorescence resonance energy transfer) using TRI2 software. Results: We show that there is a statistically significant increase in AR protein levels and PSA gene expression in LNCAP NTC vs HER3kd cells upon treatment with the dual PI3K-mTOR inhibitor DS7423. In addition, there is decreased inhibition of AKT phosphorylation in LNCAP NTC cells, whereas in the absence of HER3 there is complete abrogation of pAKT levels. The use of GDC0941 and Everolimus resulted in similar effect on AR in the LNCAP NTC vs HER3kd cells, however this is less prominent. The AKT inhibitor, GDC0068, has little effect on AR in the presence or absence of HER3. FLIM demonstrated increase in FRET as evidence of HER3 heterodimerisation in 22RVI compared to CWR22 xenograft tissue, showing that HER3 dimerisation emerges in metastatic prostate cancer that has progressed to a castrate resistant phenotype. Conclusions: Our results suggest that HER3 stabilises the AR upon PI3K-AKT- mTOR pathway inhibition and that detection of a HER3 heterodimer can be used as a predictive biomarker of response to these therapies.