Abstract 2753: Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy
Authors: Mauro Florencia Luciana , Bruni Sofia , Dupont Agustina , Inurrigarro Gloria , Figurelli Silvina , Barchuk Sabrina , Vecchia Daniel Lopez Della , Russo Rosalia Cordo , Deza Ernesto Gil , Mercogliano Maria Florencia , Schillaci Roxana 
Published: March 2024

Abstract
Triple negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and has the worst survival rate. Immunotherapy is a new treatment option but resistance is frequent. We have demonstrated that TNF induces trastuzumab resistance through mucin 4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein involved in metastasis (MTS) dissemination. Here, we evaluated the impact of the axis TNF/MUC4 on the invasive capacity of TNBC cell lines and in a preclinical model of spontaneous MTS. Finally, we assessed the clinical impact of MUC4 expression in TNBC patients. TNF blockade was achieved with etanercept (E), which blocks the soluble (sTNF) and transmembrane isoform of TNF, or with the dominant negative protein INB03 (DN) which neutralizes only sTNF. BT-549 and MDA-MB-231 human TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. To assess the impact of TNF blockade on tumor cells and its effect on the tumor microenvironment, we collected conditioned media (CM) from MDA-MB-231 and BT-549 cells treated with E or DN which were used to evaluate the invasive capacity of the TNBC cell lines. The invasion of BT-549 and MDA-MB-231 was impaired with CM-Eta (p≤0.01) and CM-DN (p≤0.01 and p≤0.05, respectively). Female BALB/c mice bearing the TNBC LMM3 tumors were treated with IgG, DN, anti-PD-1 antibodies or DN+anti-PD-1 for two weeks. After the treatments we performed surgery to remove the primary tumor and the animals were sacrificed 2 weeks later. No effect on tumor growth was observed. However, the DN+anti-PD-1 group was the only one with less of 3 MTS per lung (p≤0.05). We obtained tumor extracts and found that the tumors of the animals treated with DN or DN+anti-PD-1 had decreased MUC4 expression measured by Western blot. We determined the presence of TILs by H & E and the expression of MUC4, Ki67, PD-L1 (SP142), androgen receptor (AR) and cytokeratin 5 by immunohistochemistry in a cohort of 56 early TNBC patients. MUC4 expression was inversely correlated with TILs presence (p=0.0003). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM and MDA-MB-231-CM on T cell migration. CM-DN from both cell lines increased T cell migration compared to control CM (p≤0.01). MUC4 expression was inversely correlated with Ki67 (≥30%, p=0.036), PD-L1 (p=0.001) and AR (p=0.047) in our cohort. Moreover, MUC4 proved to be an independent predictor of poor overall survival (p=0.02), and is associated with a higher MTS risk (p=0.005). MUC4 is associated with poorly-infiltrated TNBC, and sTNF blockade downregulates its expression decreasing MTS when combined with anti-PD-1. We propose the TNF as a new target for the treatment of TNBC, and MUC4 as a predictive biomarker to guide a combined treatment of TNF blockers with immunotherapy.
Citation Format: Florencia Luciana Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, Maria Florencia Mercogliano, Roxana Schillaci. Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2753.