Abstract 697: Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines

The proteoglycan CSPG4 [chondroitin sulfate proteoglycan 4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP) or neuron-glial antigen 2 (NG2)] has been associated with progression of melanoma, and intervention to reduce CSPG4 has been considered as treatment of melanoma. In recent work, we have reported that decline in the enzyme arylsulfatase B (ARSB, N-acetylgalactosamine 4-sulfatase) is associated with malignancy, including prostate, mammary, and colon. ARSB is the enzyme that removes 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate (DS), and thereby regulates the degradation of the sulfated glycosaminoglycan (GAG) chain. Hence, the absence of ARSB leads to the accumulation of more highly sulfated C4S or DS. In prior published work, decline in ARSB and the associated increase in C4S led to transcriptional events, mediated through a decline in galectin-3 binding to the more highly sulfated C4S present. Transcription factors activator protein 1 (AP-1) and Specificity protein 1 (Sp1) interacted with galectin-3 to mediate the increased transcription of versican and Wnt9A. In this study, the mechanism by which decline in ARSB increased expression of CSPG4 and. increased the invasiveness of melanoma cell lines was addressed. Techniques included: cell culture of normal melanocytes (ATCC PCS 200-013) and melanoma cell lines obtained from radial or vertical growth phase [WM1552C, WM1552C/mock (mock CSPG4 transfection), WM1552C/MCSP (CSPG4 transfected), WM1552C/MCSPACD (transfected with CSPG4 without the cytoplasmic domain), WM35] and from metastatic melanoma (1205 Lu); QPCR of CSPG4; ARSB activity assay using the exogenous substrate 4-methylumbelliferylsulfate; silencing of ARSB by siRNA; invasion assay; measurements of total sulfated glycosaminoglycans and C4S using 1,9-dimethylmethylene blue dye; and measurements of matrix metalloproteinase (MMP) activity. The results show that ARSB activity was reduced in the metastatic melanoma cells to 51% of the level in the normal melanocytes. Silencing of ARSB was associated with increase to 2.8 times the baseline level in the mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4) in the normal melanocytes. Invasiveness of the melanoma cells increased by 70% when ARSB was silenced, with corresponding increase in MMP activity. Since CSPG4 has C4S attachments, the effects of ARSB on CSPG4 expression may affect the availability of C4S GAG chains to interact with extracellular matrix components and affect invasiveness. The findings indicate that decline in ARSB and the associated increase in C4S have profound effects on the invasive potential of melanoma and that increased attention to ARSB and C4S may yield new therapeutic interventions. Citation Format: Sumit Bhattacharyya, Leo Feferman, Kaoru Terai, Arkadiusz A. Dudek, Joanne K. Tobacman. Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 697.