WNT SIGNALLING REGULATES SELF-RENEWAL AND DIFFERENTIATION OF PROSTATE CANCER CELLS WITH STEM CELL CHARACTERISTICS Running Title WNT regulates prostate cancer stem cell attributes Key words prostate cancer, stem cell, WNT, androgen receptor, LNCaP, prostasphere Authors

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in nonadherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the affect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen independent self renewal of cells with stem cell characteristics and the androgen dependent proliferation of transit amplifying cells. As the canonical WNT signalling effector β-catenin can also associate with the androgen receptor. We propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. The inhibition of WNT signalling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve therapeutic outcome.