Glycosylation of HER2+ human breast cancer cells control sensitivity to Trastuzumab

MARIÑO KV; PERROTA RM; RABINOVICH GA; DALOTTO-MORENO T; SALATINO M; CAGNONI A

Congreso; Annual Meeting of the AACR; 2018

Galectins decode glycan-containing information in a number of cell receptors adjusting signaling thresholds and modulating cellular functions. Upon specific binding to cell surface glycans these lectins control receptor clustering, endocytosis and signaling, influencing many physiological and pathological conditions. In particular, galectin-1 (Gal1), a 14kD prototype galectin, binds to terminal N-acetyllactosamine residues on glycosylated proteins in the absence of α2-6 sialic acid capping (Gal1 permissive glycophenotype). In early studies, our group demonstrated that tumors usurp the Gal1 pathway to evade immune surveillance and to promote aberrant angiogenesis, influencing sensitivity to anti-VEGF therapy. Here we aim to explore the glycosylation signature of HER2+ breast cancer cells and to investigate the effects of tumor-derived Gal1 on RTK activation and resistance to trastuzumab (TZ). We first selected three HER2+ human breast cancer cell lines with different response to TZ: JIMT-1 (resistant-TZR), BT-474 and SK-BR-3 (sensitive-TZS). To identify specific glycan structures, we used a panel of biotinylated plant lectins with different carbohydrate specificities. We found that TZS cell lines exhibited a Gal-1 restrictive glycophenotype characterized by high α2,6 sialic acid capping. Profiling of N-type glycans (WAX-HPLC) confirmed that TZSs cell lines exhibit higher frequency of α2,6 sialic acid residues compared to TZR JIMT-1. This effect was further substantiated by RT-PCR analysis of specific glycosyltransferases responsible of Gal1 ligands biosynthesis. These results were supported by analysis of raw data from public databases arrays (GSE62327) showing that patients who presented complete response to TZ exhibited higher levels of ST6GAL1, a glycosyltransferase responsible for α2-6 sialic acid capping. In accordance with the glycophenotype, TZR cell line bound and expressed higher levels of Gal1 when compared to TZS cell lines by Western blot, RT-PCR and ELISA of conditioned medium, suggesting a positive autocrine loop that could modulate cell behavior. Moreover, in silico analysis of raw data from the Long HER Study (GSE44272) revealed that patients with poor response to TZ expressed higher levels of Gal1 mRNA than long-term responders, reinforcing our hypothesis from a clinical standpoint. Finally, in order to elucidate Gal1 implications in TZ resistance, we knocked down Gal1 in JIMT-1 cell line using shRNA strategies. Remarkably, absence of Gal1 expression sensitized JIMT-1 cells to TZ-inhibition in vitro, and the resistant phenotype was further reestablished by addition of human recombinant Gal1. In summary, our study suggests that individual HER2+ human breast cancer cells display particular ?glycosylation signatures? which, in association with Gal1 expression pattern, may control resistance to anti-HER2 targeted therapy and may predict breast cancer clinical outcome.