Abstract A34: Decline in Arylsulfatase B activates Wnt signaling by effects on DKK-3 methylation

Abstract

Abstract Immunostaining and activity of the enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) were reduced in prostatic malignancies, and decline in ARSB was associated with higher Gleason score and biochemical recurrence in two prostate cancer tissue microarrays. Since the only known function of ARSB is to remove 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate, a mechanism for this association of ARSB with more aggressive prostate cancer was unknown. Subsequent studies showed transcriptional effects of ARSB, mediated by reduced binding of galectin-3 to the more highly sulfated C4S present when ARSB was reduced. Decline in ARSB induced increased expression of the extracellular matrix proteoglycan versican, which has chondroitin 4-sulfate attachments and has been shown to be increased in more aggressive prostate cancers. The increase in versican was mediated by increased nuclear galectin-3 and AP-1 effects on the versican promoter. Other experiments following decline in ARSB showed increased expression of Wnt9A, mediated by increased nuclear galectin-3 and Sp1. These findings suggested that ARSB acted as a tumor suppressor gene and decline in ARSB enhanced tumor progression. Experiments were performed to address transcriptional mechanisms by which decline in ARSB and the accompanying increase in chondroitin 4-sulfation could activate Wnt signaling and lead to more aggressive prostate cancers. The impact of ARSB on Wnt signaling was evaluated in prostate tissue from the ARSB-null mouse, and human prostate stromal (WPMY-1, ATCC), epithelial (RWPE-1, ATCC), and stem (WPE-stem, ATCC) cell lines. The effects of ARSB on Wnt signaling were assessed by ELISA, Western blot, silencing by siRNA, ARSB overexpression, DNA methyltransferase (DNMT) activity assays, QRT-PCR, and measurements of promoter methylation. Decline in ARSB increased Wnt signaling, manifested by increases in nuclear beta-catenin, DNA-bound TCF-LEF, nuclear c-myc and cyclin D1. To determine if increased Wnt signaling was attributable to reduction of the inhibitory effect of Dickkopf (DKK), an established negative regulator of Wnt signaling and tumor suppressor, the expression of DKK 1-4 was determined by QRT-PCR. DKK-3 expression in the prostate cells following ARSB silencing and in the ARSB-null tissue was significantly reduced. ARSB overexpression increased the DKK-3 expression. DNA methyltransferase (DNMT) activity was significantly increased when ARSB was silenced, and reduced when ARSB was overexpressed. Expression of DNMT 1 and 3a was increased in the ARSB-null mouse prostate tissue and prostate cells, consistent with the decline in DKK-3 expression. Determinations of DKK-3 promoter methylation showed increased methylation following ARSB silencing and in the malignant prostate tissue. Hence, decline in ARSB activated Wnt signaling by increased methylation of DKK-3, leading to disinhibition of Wnt signaling, attributable to reduced binding of DKK-3 with the Frizzled - LRP5/6 receptor complex. These findings demonstrate an epigenetic mechanism whereby decline in ARSB increases Wnt signaling in prostate tissue and can lead to more aggressive prostate malignancy. Also, the role of ARSB as a tumor suppressor gene is supported by this demonstration of inverse effects of decline and overexpression of ARSB on Wnt signaling and on DNMT expression and activity. Experiments are ongoing to clarify the intermediates in the pathway by which ARSB affects DNMT expression. Note: This abstract was not presented at the conference. Citation Format: Sumit Bhattacharyya, Leo Feferman, Joanne Kramer Tobacman. Decline in Arylsulfatase B activates Wnt signaling by effects on DKK-3 methylation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A34.