The β 2 -adrenergic receptor is a molecular switch for 1 neuroendocrine transdifferentiation of prostate cancer cells

36 The incidence of treatment-related neuroendocrine (NE) prostate cancer (t-NEPC) is rising as more 37 potent drugs targeting the androgen signaling axis are clinically implemented. Neuroendocrine 38 transdifferentiation (NEtD), an putative initial step in t-NEPC development, is induced by androgen 39 deprivation therapy (ADT) or anti-androgens, and by activation of the β 2 -adrenergic receptor 40 (ADRB2) in prostate cancer cell lines. Thus, understanding whether ADRB2 is involved in ADT-initiated 41 NEtD may assist in developing treatment strategies that can prevent or reverse t-NEPC emergence, 42 thereby prolonging therapeutic responses. 43 Here we found that in primary, treatment-naïve prostate cancers, ADRB2 mRNA was 44 positively correlated with expression of luminal differentiation markers, and ADRB2 protein levels 45 were inversely correlated with Gleason grade. ADRB2 mRNA was upregulated in metastatic prostate 46 cancer, and progressively downregulated during androgen deprivation therapy (ADT) and t-NEPC 47 emergence. In androgen-deprivated medium, high ADRB2 was required for LNCaP cells to undergo 48 NEtD, measured as increased neurite outgrowth and expression of neuron differentiation and NE 49 genes. ADRB2 overexpression induced an NE-like morphology in both AR positive and -negative 50 prostate cancer cell lines. ADRB2 downregulation in LNCaP cells increased canonical Wnt signaling, 51 and GSK3α/β inhibition reduced the expression of neuron differentiation and NE genes. In LNCaP 52 xenografts, more pronounced castration-induced NEtD was observed in tumors derived from high 53 than low-ADRB2 cells. 54 In conclusion, high ADRB2 expression is required for ADT-induced NEtD, characterized by 55 ADRB2 downregulation and t-NEPC emergence. 56 Implications: This data suggest a potential application of β-blockers to prevent cancer cells 57 committed to a neuroendocrine lineage from evolving into t-NEPC. The tissue microarray (TMA) includes radical prostatectomy tissue from 122 patients treated at 100 Skåne University Hospital between 1999 and 2002 (20). The TMA was immunohistochemically 101 stained as described previously (21) with an anti-ADRB2 primary antibody (MC2656; MBL Int., 102 Woburn, MA) in a 1:4000 dilution. Time of biochemical recurrence (BCR) was defined as the first date 103 of two consecutive rises in PSA above nadir (>0.2 ng/mL). Manual scoring of the ADRB2 antibody staining on a scale from 1-3 was performed by two 105 experienced uropathologists (WW, AS). The most profound Gleason grade in each spot was also 106 reported. In cases with more than one cancerous spot from an individual patient, the staining 107 intensity for the spot(s) with the highest Gleason grade was chosen for further analysis. All patients 108 included on the TMA provided informed consent. The study was approved by the Regional 109 Committees for Medical and Health Research Ethics (434-04153,S-04153c, 2009/373,09/00450-2/bso 110 2009/1028), Oslo University Research Support Unit (2011/3286) and by the Regional Ethical Review 111 Board at Lund University, Sweden (approval number DN. 445-07).