N-linked glycosylation supports cross-talk between receptor tyrosine kinases and androgen receptor

PLoS One. 2013 May 28;8(5):e65016. doi: 10.1371/journal.pone.0065016. Print 2013.

Abstract

Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs). In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP) than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R) ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an important challenge in the future.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosynthetic Pathways
  • Cell Line, Tumor
  • Gene Expression Regulation, Neoplastic
  • Glycosylation
  • Hexosamines / biosynthesis
  • Humans
  • Male
  • Prostatic Neoplasms / enzymology
  • Prostatic Neoplasms / genetics
  • Protein Processing, Post-Translational
  • Protein Transport
  • Receptor Cross-Talk*
  • Receptor, IGF Type 1 / metabolism*
  • Receptors, Androgen / metabolism*
  • Up-Regulation

Substances

  • AR protein, human
  • Hexosamines
  • Receptors, Androgen
  • Receptor, IGF Type 1

Grants and funding

HMI is funded by an Early Stage Researcher fellowship as part of the EU FP7 Marie Curie Integrated Training Network, PRO-NEST (Prostate Research Organizations – Network Early Stage Training). IGM is supported by funding from the Norwegian Research Council, Helse Sor-Ost and the University of Oslo through the Centre for Molecular Medicine (Norway), which is part of the Nordic EMBL (European Molecular Biology Laboratory) partnership. IGM is also supported by the Norwegian Cancer Society and by EU FP7 funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.