Heparan sulfate promotes TRAIL-induced tumor cell apoptosis

Reviewer #1 (Public Review):

Summary: Yin Luo and colleagues describe a new regulatory mode of TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand) -induced apoptosis of tumor cells. The work is timely because high expectations existed on the possibility to induce tumor-specific apoptosis by activation of TRAIL-receptors DR4 and DR5. So far, however, attempted TRAIL-based anti-tumor therapies failed, and several tumor types were found to resist TRAIL-induced apoptosis. The work is also important because it aims at a better mechanistic understanding of TRAIL-induced apoptosis and TRAIL resistance of some tumor types.

Strengths: The major novel finding of this study is that extracellular heparan sulfate (HS) acts as a positive regulator of TRAIL-induced tumor cell apoptosis, and that HS expression of different tumor cell lines correlates with their capacity to induce cell death. The authors first show by affinity chromatography and SPR that murine and human TRAIL bind strongly to heparin (heparin is a highly sulfated, and thus strongly negatively charged form of HS that is derived from connective tissue type mast cells), and identify three basic amino acids on the TRAIL N-terminus that are required for the interaction. Size exclusion chromatography (SEC) and multiangle light scattering (MALS) revealed that TRAIL exists as a trimer that requires a minimum heparin length of 8 sugar residues for binding, and small angle X-ray scattering (SAXS) showed that TRAIL interaction with longer oligosaccharides induced higher order multimerization of TRAIL. Consistent with these biochemical and biophysical analyses, HS on tumor cells contributes to TRAIL-binding to their cell surface and subsequent apoptosis. Minor novel findings include domain swapping observed by TRAIL trimer crystallization and different degrees of HS core protein and DR receptor expression in different tumor cell types. These findings are well supported and together with the advanced and established methodology used by the authors are the strengths of this paper. The paper will be of great interest to medical biologists studying TRAIL-resistance of tumors, to biologists interested in DR4 and DR5 receptor function and the effects of receptor internalization, and to glycobiologists aiming to understand the multiple important roles that HS plays in development and disease. The authors also raise the important point (and support it well) that routine heparin treatment of cancer patients potentially interferes with TRAIL-based therapies, providing one possible reason for their failure.

Weaknesses: Despite the importance and the clear strengths of the paper, some of its aspects could have been developed further to better support the authors claims and their hypothesis that HS downregulation may represent one strategy to explain tumor resistance to TRAIL-induced apoptosis.

1. The authors demonstrate that HS at the tumor surface promotes TRAIL binding, and that HS promotes TRAIL-induced breast cancer and myeloma cell apoptosis. These findings are based on pre-treatment with heparinase to remove cell-surface HS prior to TRAIL-treatment, or presence of soluble heparin to compete with cell-surface HS for TRAIL binding. A more direct way to establish such new HS function could have been genetic manipulation of cancer cells to overexpress HS (e.g. by Syndecan 1 core-protein transgene expression in resistant cell types, e.g. IM-9 cells) or to express less or undersulfated HS (by interfering with the biosynthetic pathway in apoptosis-prone cells, for example by targeted inactivation/RNAi of the HS co-polymerase or sulfotransferases in RPMI-8226 cells). Changed susceptibility of these cells to TRAIL-induced apoptosis would greatly support the authors claim and suggest one promising new strategy to decrease tumor resistance against TRAIL-induced apoptosis.

2. The mechanistics of TRAIL-induced, HS-modulated tumor cell apoptosis could be more clearly defined. For example, the authors demonstrate convincingly that cell surface HS is essential for TRAIL-induced apoptosis in MDA-MB-453 breast cancer cells, and later show that a tumor cell line (IM-9 cells) that expresses HS and the core protein to which HS is attached to only limited degrees is the most resistant to TRAIL-induced apoptosis. Yet, these findings suggest, but do not prove a dose-dependent role of HS in TRAIL-induced apoptosis. Indeed, the authors later report that cell surface HS promotes TRAIL-induced myeloma cell apoptosis regardless of the sensitivity levels, and that other factors - the degree of TRAIL multimerization or DR4/DR5 receptor internalization - are also important. A cartoon could be added to the final figure to sort through these possible mechanisms and their relative importance.

3. The authors show that RPMI-8226 cell-surface HS promotes DR5 internalization despite the absence of direct DR5/heparin interactions. This is important because, as the authors are certainly aware of, HS manipulation at the cell surface (for example by heparinase treatment) changes a plethora of signaling pathways that may also indirectly affect apoptosis. HS-dependent internalization of TRAIL-receptor complexes, however, provides an important direct link from HS expression to TRAIL-induced apoptosis. To further support this possible link, it would therefore be worthwhile to include the binding characteristics and HS-dependent internalization of DR4 into this study.

Reviewer #2 (Public Review):

Summary:
In the manuscript by Luo et al, the authors investigated the nature and function of TRAIL-HS binding for the regulation of TRAIL-mediated apoptosis in cancer cells. The authors discovered that TRAIL binds to 12mer HS and identified the amino acid residues critical for the binding. The authors further nicely showed that 12mer HS binds to TRAIL homotrimer and larger HS can further promote the formation of larger TRAIL oligomers. Structural analyses were conducted to characterize the binding of TRAIL/HS complexes. At functional level, the authors demonstrated that HS promotes the cell surface binding of TRAIL to enhance TRAIL-mediated apoptosis in a variety of cancer cells. Moreover, the ability of TRAIL to induce apoptosis is correlated with cell surface HS level. Lastly, the authors showed that HS forms complex with TRAIL and its receptor DR5 and promotes DR5 internalization.

Strengths:
Overall, this is a nicely executed study providing both mechanistic and functional insight for TRAIL-mediated apoptosis. It conducted detailed characterization on the direct binding between HS and TRAIL and provided solid evidence supporting the role of such interaction for the regulation of TRAIL-induced apoptosis. The experiments were well-designed with proper controls included. The data interpretation is accurate. The manuscript was clearly written and easy to follow by general readers.

Weaknesses:
There is no major weakness identified from this study. However, the role of HS for the formation of TRAIL homotrimer needs to be further clarified. In addition, the current relationship between cell surface HS level and sensitivity to TRAIL-mediated apoptosis is still correlative, as the authors indicated. Additional evidence to support the regulatory function of HS would further strengthen the significance of the study.