Neutralizing soluble tumor necrosis factor alpha overcomes trastuzumab-resistant breast cancer immune evasion by downregulating mucin 4, improving NK cell function and decreasing myeloid-derived suppressor cells in tumor microenvironment

SCHILLACI R,; MERCOGLIANO MF; PROIETTI CJ; DE MARTINO M; FRAHM I; BRUNI S; INURRIGARRO G; ELIZALDE PV.

San Antonio

Congreso; San Antonio Breast Cancer Symposium; 2018

Background: Novel strategies aimed to overcome trastuzumab (Tz) resistance of HER2+ breast cancer (BC ) areneeded. Recently, we demonstrated a novel immune evasion strategy used by BC where tumor necrosis factor alpha(TNF) induces upregulation of the transmembrane glycoprotein mucin 4 (MUC 4) via NF-kB activation to impair Tz bindingthat prevents antibody mediated killing of BC cells. Etanercept, a non-selective inhibitor of soluble and transmembraneTNF (sTNF, tmTNF), downregulated MUC 4 expression and sensitized de novo Tz-resistant BC xenografts to Tz. Moreover,we showed that MUC 4 expression is an independent predictor of poor disease-free survival in patients treated with Tz inthe adjuvant setting (C lin C ancer Res 2017, 23:636). Etanercept is immunosuppressive due to off-target effects on tmTNFwhile selective inhibition of sTNF improves the immune response to the tumor (C ancer Immunol Res 2016, 4:441).Because of the immunosuppressive properties of etanercept, we wanted study if the dominant negative-TNF proteinXPro1595 (DN-TNF; also known as INB03) that neutralizes sTNF without affecting tmTNF is able to downregulate MUC 4 toinhibit Tz-resistant tumor growth and improve innate antitumor immune response.Methods: To assess the effect of DN-TNF on Tz-resistant HER2+ tumor growth, JIM T-1 cells were s.c. injected in nudemice. When tumors were established, animals were treated with IgG, DN-TNF, Tz or DN-TNF+Tz, i.p. twice a week for onemonth. Innate immune response was determined by flow cytometry analysis of NK cells activation and degranulation andmyeloid-derived suppressor cells (MDSC ) subtypes in tumor microenvironment (TME) and in spleen. Tz-dependent NKcells degranulation was assessed in splenocytes using HER2+, Tz-sensitive cell line BT-474 as the target. MUC 4 andphospho NF-kB expression was determined by Western blot.Results: Treatment with Tz or DN-TNF had no impact on JIM T-1 tumor growth. However, co-treatment with DN-TNF andTz resulted in significantly less growth. At day 21s t, tumor volume was 75mm3 in DN-TNF+Tz vs 300mm3 control groups.DN-TNF+Tz treatment showed a decrease in myeloid cell infiltration and MDSC phenotype was enriched in thegranulocytic-MDSC vs monocytic-MDSC suggesting a less immunosuppressive TME. DN-TNF+Tz administrationsignificantly increased activation and degranulation of tumor infiltrating NK cells. In addition, spleen NK cells from theseanimals exhibited enhanced Tz-dependent degranulation vs control groups. MUC 4 expression was downregulated intumors treated with DN-TNF and NF-kB phosphorylation was inhibited (all comparisons p