Hyaluronic acid binding to TLR4 promotes proliferation and blocks apoptosis in colon cancer.

Hyaluronic acid (HA), a constituent of the extracellular matrix, promotes colorectal cancer (CRC) growth. CD44 is a relevant HA receptor in this context. However, HA is also a ligand for TLR4, a receptor of significance in CRC. In this study we examine the relative contribution of HA interactions with CD44 and TLR4 in colon tumorigenesis. CRC models included ApcMin/+ mice, azoxymethane/dextran sodium sulfate (AOM-DSS) and CT26 tumor isografts. We used knockout mice and CT26 CRC cells with CRISPR knockdown of CD44 and TLR4. HA activity was modulated by PEP1 (a 12-mer peptide that blocks HA from binding its receptors), hyaluronidase (which promotes HA degradation), or 4-MU (HA synthesis inhibitor). Blockade of HA binding via PEP1 decreased growth in all CRC models and in cell culture. The effects were significant in WT and with CD44 deletion, but not with TLR4 deletion. In the AOM-DSS model, mice deficient in CD44 or TLR4 had fewer tumors. CD44 and TLR4 deficient CT26 isografts grew more slowly, exhibiting decreased tumor cell proliferation and increased apoptosis. In vitro, endogenous HA blocked LPS binding to TLR4 suggesting that HA is a relevant TLR4 ligand in colon cancer. Finally, PEP1 enhanced tumor radiation sensitivity in the isograft model. Together these results indicate that HA binding to TLR4, as well as CD44, plays a key role in colon tumorigenesis. These findings also raise the possibility that an agent that blocks HA binding, like PEP1, may be useful as an adjuvant therapy in colon cancer.