To Investigate the Mode of Galectin-1 and Neuropilin-1 Interactions in Carcinoma-Associated Fibroblasts and Its Implications in Oral Cancer Therapy

The coevolution of cell-surface glycans and glycan-binding proteins regulate the progression of physiologic and pathologic processes. Carcinoma-associated fibroblasts (CAFs) are activated myofibroblasts that promote cancer progression and metastasis. However, it is unclear whether the changes of glycan structure and glycan-binding protein regulate CAF activation. We previously reported that galectin-1 (Gall), a P-galactoside binding lectin, is highly expressed in CAFs and regulates carcinoma-associated fibroblast (CAF)-promoted cancer progression. Our preliminary results showed that CAFs have higher amounts of Gall binding glycan [P1,6 N-acetylglucosamine (GlcNAc)-branched N-glycans and poly-N-acetylactosamine (LacNAc)] but lower amounts of a2,6 sialylation (which masks the Gall binding glycan) compared to the normal counterpart-fibroblasts (NFs, a kind of quiescent fibroblasts). Accordingly, CAFs have a significant increase of the Gall binding compared to NFs and Gall induced higher migration ability in CAFs than in NFs. ST6Gall (a2-6 sialyltransferase) which masks Gall binding through addition of the a2,6 sialic acid to poly LacNAc is strongly suppressed in CAFs. The results indicate the changes of glycosyltransferases and glycan structures in CAFs favor Gall binding and its functions. In addition, because neuropilin-l (NRPl) is a high affinity receptor of Gall (Kd=109±31 nM) and a2,6 sialylation is decreased in CAFs, it is hypothesized that the changes of NRPl sialylation regulate the Gall functions in CAFs. In this study, the Gall binding domain and glycan structure of NRPl in NFs and CAFs will be dissected. We will develop an antibody against the “glycan epitope of NRPl” and evaluate whether specific disruption of the Gall-NRPl interactions is an effective strategy for oral cancer therapy. Four specific aims are proposed to verify our hypothesis. Aim l. To identify which domain of NRPl interacts with Gall in CAFs and to pinpoint the glycosylation sites in this domain. Aim 2. To dissect the NRP1 glycan structures in NFs and CAFs using mass spectrometry and to investigate whether the changes of glycan structure regulates the interaction between Gall and NRP1. Aim 3. To examine whether disruption of the Gal1-NRP1 interactions affect Gal1 induced fibroblast activation and CAF-promoted oral cancer progression. Aim 4. To develop an antibody specifically disrupt the Gal1-NRP1 interactions and evaluate the therapeutic efficacy. The current Gall inhibitors are developed to target the CRD (carbohydrate-recognition domain) region of Gall. However, these kinds of inhibitors are not specific to Gall because of the similarity of the CRD domain in galectin family members. The side effect of these inhibitors is hard to evaluate because the functional redundancy and specificity within the galectin family is unclear. We found that the Gall-NRPl interaction is important because: (1) it is a high affinity binding (Kd=109±31 nM) compared to Gall and lactose interactions (Kd〜200pM). (2) In hepatic stellate cells, NRPl is like a scaffold for Gall to interact with other glycosylated receptors such as TGF- P, PDGF and VEGF receptors based on our current studies (unpublished data). Therefore, the dissection of the Gall-NRPl interactions will be a strong foundation for the design of Gall inhibitors.