Soluble TNFα induced mucin 4 is a mediator of trastuzumab resistance and of an immunosuppressive tumor microenvironment in HER2+ breast cancer

DE MARTINO, MARA; ELIZALDE, PATRICIA V.; BRUNI, SOFÍA; SANTA MARIA DE LA PARRA, LUCIA; MAURO, FLORENCIA L.; SCHILLACI, ROXANA

Congreso; Society of Immunotherapy and Cancer Annual Meeting; 2019

About 13-20% of breast cancer (BC) patients are HER2 positive (HER2+) and receive trastuzumab (T), an anti-HER2 monoclonal antibody, but 40-60% of them relapse. We have demonstrated that tumor necrosis factor alpha (TNFα) induces the expression of the transmembrane glycoprotein mucin 4 (MUC4), that shields T epitope in HER2, impairing its antitumor effects. Also, we have shown that Etanercept (E), an inhibitor of soluble and transmembrane TNFα (sTNFα, tmTNFα), downregulated MUC4 expression and sensitized de novo T-resistant BC xenografts to T (1). The aim of this work was to study the participation of MUC4 on T resistance in vivo, on T-mediated antitumor innate immune response, and to evaluate the role of sTNFα on MUC4 expression.We used the T-resistant JIMT-1 cell line transduced with a doxycycline (Dox)-inducible MUC4 shRNA construct (JIMT-shMUC4). To block TNFα, we used E or the dominant negative-TNFα protein INB03 (DN). Nude mice bearing JIMT-1-shMUC4 tumors (~50 mm3), were randomly assigned to the experimental (+Dox 2mg/ml in drinking water) or control group (−Dox). Both groups were treated with IgG, T, E (all 5 mg/kg), DN (10 mg/kg), T+E or T+DN i.p. twice a week and tumor volume was monitored routinely.  MUC4 expression was determined by Western Blot in tumor extracts. Tumor-infiltrating immune cells were evaluated by immunofluorescence and analyzed by flow cytometry.   In control groups, only T+E and T+DN administrations were able to inhibit tumor growth (72% and 75%, respectively, P<0.0001 vs. IgG) in line with our previous results (1). Knockdown of MUC4 expression revealed that T treatment was effective in inhibiting JIMT-shMUC4 tumor growth (62%, P<0.0001) at comparable levels to T+E and T+DN administration (78% and 76%, respectively, P<0.0001 vs. IgG). DN treatment decreased MUC4 expression in control tumors similar to our previous report with E treatment (1). Analysis of tumor-infiltrating immune cells showed that tumor growth inhibition was accompanied by an increase in NK cells activation and degranulation, and an increase in M1/M2 macrophages ratio. Interestingly, upon MUC4 downregulation, myeloid-derived suppressor cells infiltration was reduced and macrophage recruitment was enhanced in the tumor bead of IgG+Dox vs IgG-Dox groups. This is the first report to show that sTNFα is responsible for MUC4 regulation and that MUC4 is the major player in TNFα-induced T resistance in vivo. In addition, MUC4 favors an immunosuppressive tumor microenvironment. We propose that patients with HER2+ and MUC4+ tumors should be treated with T and TNFα-blocking agents to avoid resistance.