Abstract
Supraphysiologic androgen (SPA) inhibits cell proliferation in prostate cancer (PCa) cells by transcriptional repression of DNA replication and cell-cycle genes. In this study, quantitative glycoprotein profiling identified androgen-regulated glycoprotein networks associated with SPA-mediated inhibition of PCa cell proliferation, and androgen-regulated glycoproteins in clinical prostate tissues. SPA-regulated glycoprotein networks were enriched for translation factors and ribosomal proteins, proteins that are known to be O-GlcNAcylated in response to various cellular stresses. Thus, androgen-regulated glycoproteins are likely to be targeted for O-GlcNAcylation. Comparative analysis of glycosylated proteins in PCa cells and clinical prostate tissue identified androgen-regulated glycoproteins that are differentially expressed prostate tissues at various stages of cancer. Notably, the enzyme ectonucleoside triphosphate diphosphohydrolase 5 was found to be an androgen-regulated glycoprotein in PCa cells, with higher expression in cancerous versus non-cancerous prostate tissue. Our glycoproteomics study provides an experimental framework for characterizing androgen-regulated proteins and glycoprotein networks, toward better understanding how this subproteome leads to physiologic and supraphysiologic proliferation responses in PCa cells, and their potential use as druggable biomarkers of dysregulated AR-dependent signaling in PCa cells.
Highlights
Supraphysiologic androgen (SPA) inhibits cell proliferation in prostate cancer (PCa) cells by transcriptional repression of DNA replication and cell-cycle genes
As shown p reviously[21,22,23], physiologic levels of androgen (i.e., 1 nM R1881) stimulated maximal proliferation of LNCaP cells, whereas SPA (i.e., 10 nM R1881) attenuated their proliferation (Supplemental Fig. 1A,B). These findings showed that androgenic responses in LNCaP cells are biphasic and dose-dependent, justifying their use as an experimental model for studying how the inhibition of SPA-mediated proliferation influences protein glycosylation in PCa cells
lectin weak affinity chromatography (LWAC) was performed using a mixture of wheat-germ agglutinin-(WGA) and concanavalin A-(ConA) conjugated sepharose beads, to capture both N-linked and O-linked glycoproteins (α-linked mannose, terminal glucose moieties) (Fig. 1A)
Summary
Supraphysiologic androgen (SPA) inhibits cell proliferation in prostate cancer (PCa) cells by transcriptional repression of DNA replication and cell-cycle genes. In the case of localized PCa, approximately 50% of patients of Caucasian/Eastern European-descended populations harbor gene rearrangements that place transcription factors of the oncogenic ETS family under the control of the androgen-regulated promoter element TMPRSS23 These TMPRSS2-ETS gene fusions promote ETS overexpression, increase tumor cell motility and invasiveness, and induce prostatic intraepithelial neoplasia in genetically engineered mice. CRPC involves the dysregulation of AR-dependent signaling by a variety of causes, including: AR mutations, AR amplification, AR variants, aberrant expression of AR coregulators, androgen synthesis by tumors[8,9], and amplification of upstream enhancers of AR10–12 These defects all enable the tumor cells to bypass the actions of ADT and second-generation AR pathway inhibitors (ARPIs) (i.e., enzalutamide and abiraterone)[13,14,15]. The glycoproteomic dataset represents a rich source of information about how androgenregulated protein glycosylation relates to androgen-mediated proliferation responses in PCa cells and, more generally, will be useful for interrogating the function of androgen-regulated glycoproteins in prostatic diseases
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