Dihydrotestosterone inhibits arylsulfatase B and Dickkopf Wnt signaling pathway inhibitor (DKK)‐3 leading to enhanced Wnt signaling in prostate epithelium in response to stromal Wnt3A

Abstract

Background:In tissue microarrays, immunostaining of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) was less in recurrent prostate cancers and in cancers with higher Gleason scores. In cultured prostate stem cells, decline in ARSB increased Wnt signaling through effects on Dickkopf Wnt Signaling Pathway Inhibitor (DKK)3. The effects of androgen exposure on ARSB and the impact of decline in ARSB on Wnt signaling in prostate tissue were unknown.Methods: Epithelial and stromal tissues from malignant and normal human prostate were obtained by laser capture microdissection. mRNA expression of ARSB, galactose-6-sulfate-sulfatase (GALNS) and Wnt-signaling targets was determined by QPCR.Non-malignant human epithelial and stromal prostate cells were grown in tissue culture, including two-cell layer cultures. ARSB was silenced by specific siRNA, and epithelial cells were treated with stromal spent media following treatment with IWP-2, an inhibitor of Wnt secretion, and by exogenous recombinant human Wnt3A.Promoter methylation was detected using specific DKK3 and ARSB promoter primers.The effects of DHT and of ARSB overexpression on DKK expression were determined. Cell proliferation was assessed by BrdU incorporation.Results: Normal stroma showed higher expression of vimentin, ARSB, and Wnt3A than epithelium. Normal epithelium had higher expression of E-cadherin, galactose 6sulfate-sulfatase (GALNS), and DKK3 than stroma. In malignant epithelium, expression of ARSB and DKK3 declined, and expression of GALNS and Wnt signaling targets increased. In cultured prostate epithelial cells, Wnt-mediated signaling was greatest when ARSB was silenced and cells were exposed to exogenous Wnt3A. Exposure to 5α-dihydrotestosterone (DHT) increased ARSB and DKK3 promoter rmethylation, and effects of DHT on DKK3 expression were reversed when ARSB was overexpressed. Conclusions: Androgen-induced declines in ARSB and DKK3 may contribute to prostate carcinogenesis by sustained activation of Wnt signaling in prostate epithelium in response to stromal Wnt3A. K E Y W O R D S arylsulfatase B, DHT, DKK3, methylation, Wnt3A 2 | BHATTACHARYYA ET AL. 690 1 | INTRODUCTION Chondroitin sulfates are present in significant amounts throughout mammalian tissues, including epithelium and stroma in all organs. With other glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, and hyaluronan, and by interactions with collagen, protein components of proteoglycans, and structural proteins, they form the framework that is necessary for proper tissue structure and function. The chondroitin sulfatase arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) removes 4-sulfates from the non-reducing end of chondroitin 4-sulfate (C4S) and is required for the subsequent degradation of C4S by glycosidases. 1-3 The chondroitin sulfatase galactose 6-sulfate-sulfatase (GALNS; N-acetylgalactosamine-6-sulfatase) removes 6-sulfate groups from chondroitin 6-sulfate (C6S) or chondroitin 4,6-disulfate (chondroitin sulfate E),...

Increased CHST15 follows decline in arylsulfatase B (ARSB) and disinhibition of non-canonical WNT signaling: potential impact on epithelial and mesenchymal identity

“…Consistent with these findings, marked increases in C4S, decline in C6S, and increased C4S: C6S ratio were shown in malignant prostate tissues and in human prostate stem cells [17,19]. In tissue obtained by laser capture microdissection, ARSB expression was markedly reduced in malignant prostate epithelium, but not in stroma, and expression of GALNS was greater in malignant epithelium than in normal epithelium and much greater in normal epithelium than in normal or malignant stroma [19]. In contrast, ARSB expression was significantly greater in normal or malignant prostate stromal tissue than in epithelium.…”

“…These effects were attributed to signaling mechanisms resulting from the increased sulfation of C4S when ARSB was reduced. Also, in malignant prostate tissues, ARSB activity and expression were reduced and activity and expression of GALNS (N-acetylgalactosamine-6-sulfatase) were increased [16][17][18][19]. Consistent with these findings, marked increases in C4S, decline in C6S, and increased C4S: C6S ratio were shown in malignant prostate tissues and in human prostate stem cells [17,19].…”

“…In our previous studies, we demonstrated functional effects due to the increase in chondroitin 4-sulfate (C4S), which follows decline in arylsulfatase B (ARSB, N-acetylgalactosamine-4-sulfatase) [16][17][18][19][20][21][22][23]. ARSB is the enzyme that removes 4-sulfate groups from N-acetylgalactosamine 4-sulfate, and is required for the degradation of C4S and dermatan sulfate (DS; [IdoA-GalNAc-4S] n ) [24][25][26].…”

“…These effects were attributed to the impact of increased C4S on binding with important mediators of cell functions, including galectin-3 and SHP2 (PTPN11), the ubiquitous non-receptor tyrosine phosphatase. When C4S was increased, due to inhibition of ARSB, binding of galectin-3 with C4S declined and binding of SHP2 with C4S increased [16][17][18][19][20][21][22][23], leading to significant effects on transcriptional and signaling events.…”

“…Also, in malignant prostate tissues, ARSB activity and expression were reduced and activity and expression of GALNS (N-acetylgalactosamine-6-sulfatase) were increased [16][17][18][19]. Consistent with these findings, marked increases in C4S, decline in C6S, and increased C4S: C6S ratio were shown in malignant prostate tissues and in human prostate stem cells [17,19]. In tissue obtained by laser capture microdissection, ARSB expression was markedly reduced in malignant prostate epithelium, but not in stroma, and expression of GALNS was greater in malignant epithelium than in normal epithelium and much greater in normal epithelium than in normal or malignant stroma [19].…”