Switching mechanism from AR to EGFR signaling via 3-O-sulfated heparan sulfate in castration-resistant prostate cancer.

Androgen deprivation therapy is given to suppress prostate cancer growth; however, some cells continue to grow hormone-independently as castration-resistant prostate cancer (CRPC). Sulfated glycosaminoglycans promote ligand binding to receptors as co-receptors, but their role in CRPC remains unknown. Using the human prostate cancer cell line C4-2, which can proliferate in hormone-dependent and hormone-independent conditions, we found that epidermal growth factor (EGF)-activated EGFR-ERK1/2 signaling via 3-O-sulfated heparan sulfate (HS) produced by HS 3-O-sulfotransferase 1 (HS3ST1) is activated in C4-2 cells under hormone depletion. Knockdown of HS3ST1 in C4-2 cells suppressed hormone-independent growth, and inhibited both EGF binding to the cell surface and activation of EGFR-ERK1/2 signaling. Gefitinib, an EGFR inhibitor, significantly suppressed C4-2 cell proliferation and growth of a xenografted C4-2 tumor in castrated mouse. Collectively, our study has revealed a mechanism by which cancer cells switch to hormone-independent growth and identified the key regulator as 3-O-sulfated HS.

Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

Regulation of 3-O-Sulfation of Heparan Sulfate During Transition from the Naïve to the Primed State in Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (mESCs), which are established from the inner cell mass of pre-implantation mouse blastocysts, rapidly expand and form dome-shaped colonies. The pluripotent state of mESCs has been defined as the "naïve" state. On the other hand, characteristics of mouse epiblast stem cells (mEpiSCs), which are derived from the epiblast of mouse post-implantation blastocysts, has been described as the "primed" state. Human embryonic stem cells/induced pluripotent stem cells (hESCs/iPSCs) are also defined as primed state cells because their gene expression pattern and signal requirement are similar to those of mEpiSCs. Both mEpiSCs and hESCs/iPSCs proliferate slowly and form flat colonies. It is therefore difficult to genetically modify primed state cells and apply them to regenerative medicine. Therefore, stable methods of reversion from the primed to the naïve state are required. Clarifying the molecular mechanisms that underpin the primed-to-naïve transition is essential for the use of such cells in basic research and regenerative medicine applications. However, this is a challenging task, since the mechanisms involved in the transition from the naïve to the primed state are still unclear. Here, we induced mEpiSC-like cells (mEpiSCLCs) from mESCs. During induction of mEpiSCLCs, we suppressed expression of 3-O-sulfated heparan sulfate (HS), the HS4C3 epitope, by shRNA-mediated knockdown of HS 3-O-sulfotransferases-5 (3OST-5, formally Hs3st5). The reduction in the level of HS 3-O-sulfation was confirmed by immunostaining with an anti-HS4C3 antibody. This protocol provides an efficient method for stable gene knockdown in mESCs and for the differentiation of mESCs to mEpiSCLCs.

A defined glycosylation regulatory network modulates total glycome dynamics during pluripotency state transition.

Embryonic stem cells (ESCs) and epiblast-like cells (EpiLCs) recapitulate in vitro the epiblast first cell lineage decision, allowing characterization of the molecular mechanisms underlying pluripotent state transition. Here, we performed a comprehensive and comparative analysis of total glycomes of mouse ESCs and EpiLCs, revealing that overall glycosylation undergoes dramatic changes from early stages of development. Remarkably, we showed for the first time the presence of a developmentally regulated network orchestrating glycosylation changes and identified polycomb repressive complex 2 (PRC2) as a key component involved in this process. Collectively, our findings provide novel insights into the naïve-to-primed pluripotent state transition and advance the understanding of glycosylation complex regulation during early mouse embryonic development.

Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions.

Human induced pluripotent stem (hiPS) cells are attracting attention as a tool for regenerative medicine. However, several problems need to be overcome for their widespread and safe use, for example, the high cost of maintaining hiPS cells and the possibility of xenogeneic cell contamination in hiPS cell cultures. One of the main contributors to the high cost of maintaining hiPS cells is basic fibroblast growth factor (bFGF), which is essential for such cultures. Xenogeneic contamination can occur because of the use of mouse-derived feeder cells to culture hiPS cells. To overcome the problems of cell culture cost and xenogeneic contamination, we have developed a novel culture method in which the undifferentiated state and pluripotency of hiPS cells can be maintained under feeder-free and bFGF-free conditions. Our new approach involves the addition to the culture medium of highly sulfated hyaluronic acid (HA-HS), in which the hydroxyl groups of d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) are chemically sulfated. HA-HS promotes bFGF signaling and maintains the undifferentiated state and pluripotency of hiPS cells under feeder-free and bFGF-free conditions. By contrast, non-sulfated hyaluronic acid and low sulfated hyaluronic acid do not maintain the undifferentiated state and pluripotency of hiPS cells. These results indicate that the maintenance of hiPS cells under feeder-free and bFGF-free conditions is an HA-HS specific effect. This study is the first to demonstrate the effects of sulfated hyaluronic acid on mammalian pluripotent stem cells, and provides a novel method for maintaining hiPS cells using HA-HS.

Cell Profiling Based on Sugar-Chain-Cell Binding Interaction and Its Application to Typing and Quality Verification of Cells.

Developing methods to determine cell type and cell state has been a significant challenge in the field of cancer diagnosis as well as in typing and quality verification for cultured cells. Herein, we report a cell profiling method based on binding interactions between cell-surface sugar-chain-binding proteins and sugar-chain-immobilized fluorescent nanoparticles (SFNPs), together with a method for cell typing and cell quality verification. Binding profiles of cells against sugar chains were analyzed by performing flow cytometry analysis with SFNPs. Discrimination analysis based on binding profiles could classify cell type and evaluate the quality of cultured cells. By applying our method to differentiated cells originating from conventional cell lines and also to mouse embryotic stem cells, we could detect the cells before and after differentiation. Our method can be utilized not only for the biofunctional analysis of cells but also for diagnosis of cancer cells and quality verification of cultured cells.