Abstract 1306: New therapeutic approach for triple negative breast cancer: soluble TNFα blockade and MUC4 expression as a prognostic biomarker
Authors: Mauro Florencia , Bruni Sofia , Dupont Agustina , Inurrigarro Gloria , Figurelli Silvina , Barchuk Sabrina , Vecchia Daniel Lopez Della , Russo Rosalia Cordo , Deza Ernesto Gil , Mercogliano María Florencia , Schillaci Roxana 
Published: June 2023

Abstract
Among all breast cancers, triple negative (TNBC) subtype has the worst survival rate. We have demonstrated that the proinflammatory cytokine TNFα induces trastuzumab resistance through Mucin4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein expressed in several tumors and it is involved in metastasis dissemination. Here, we evaluated the role of TNFα and MUC4 in metastasis in TNBC cell lines and we assessed the clinical impact of MUC4 expression in TNBC patients. TNFα blockade was achieved using etanercept (E), which blocks the soluble (sTNFα) and transmembrane isoforms of TNFα, or dominant negative protein INB03 (DN) which only neutralizes sTNFα. BT-549 and MDA-MB-231 TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. TNFα blockade decreases the expression of the mesenchymal markers vimentin and snail in both cell lines. To assess the impact of TNFα blockade on tumor cells and its effect on the tumor microenvironment (TME), we collected conditioned media (CM) of 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 was impaired with CM-Eta (p&lt;0.01) and CM-DN (p&lt;0.01), and MDA-MB-231 invasive capacity was reduced only with CM-DN (p&lt;0.01). We evaluated MUC4 participation on invasion in MDA-MB-231 cell line transfected with a siRNA targeting MUC4 with or without DN treatment, and an invasion assay was performed. MUC4 knockdown impaired cell invasion (p=0.0001 vs siRNA control transfected cells). The treatment with DN of the MUC4-silenced cells did not further enhanced the effect, demonstrating that the impairment of the invasion is due to the decrease in MUC4. 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 55 TNBC patients. MUC4 expression inversely correlated with TILs (p=0.00013). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM on T cell migration. CM-DN increased T cell migration compared to control CM (p&lt;0.01). MUC4 expression inversely correlated with Ki67 (p=0.016), 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 metastasis risk (p=0.005).In conclusion, TNFα blocking agents decrease MUC4, mesenchymal markers and the invasive capacity of TNBC cells, and in turn increase human T cell migration. MUC4 is an independent biomarker of poor overall survival, it is associated with an increased risk of metastasis and immune desert tumors. We propose TNFα as a new target for the treatment of TNBC, and MUC4 as a predictive marker to guide a combined treatment of TNFα blockers with chemotherapy or immunotherapy.
Citation Format: Florencia Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, María Florencia Mercogliano, Roxana Schillaci. New therapeutic approach for triple negative breast cancer: soluble TNFα blockade and MUC4 expression as a prognostic biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1306.