Abstract 2047: MUC4 enables immune tumor evasion in HER2+ breast cancer
Authors: Bruni Sofia , Mauro Florencia L. , Mercogliano Maria F. , Proietti Cecilia J. , Adami Carla , Dupont Agustina , Inurrigarro Gloria , Russo Rosalia Cordo , Elizalde Patricia V. , Schillaci Roxana 
Published: June 2022

Abstract
HER2+ is a breast cancer (BC) subtype characterized by the overexpression/amplification of HER2. Patients receive trastuzumab (Tz) but many (27-42%) do not achieve an objective response. We demonstrated that the overexpression of TNFɑ induces Tz resistance in tumors by upregulating the membrane glycoprotein MUC4, which masks Tz epitope on HER2, impairing its binding and reducing its therapeutic effects. We have also proved that blocking the soluble TNFɑ isoform with INB03 (DN) reduces MUC4 expression, overcomes Tz resistance and unleashes an antitumor innate immune response characterized by an increase in NK cell-activation and degranulation and a macrophage (Mφ) polarization to the M1 subtype.
This study aims to determine the impact of MUC4 expression on Mφ and NK cells antitumor activity in the presence of Tz or Tz+DN, and on human T-lymphocyte recruitment and differentiation. Tz-resistant HER2+ BC human cell lines, JIMT-1 and KPL-4, were genetically modified to stably express a doxycycline-inducible (Dox) MUC4 shRNA (shMUC4) or a scramble one (shControl), and injected s.c. into female nude mice. After tumor establishment, the animals were randomly divided into a control group (-Dox) or an induced group (+Dox), and treated twice a week i.p. with IgG (5 mg/kg) or Tz+DN (5 and 10 mg/kg each, respectively) (-Dox) and with IgG or Tz (+Dox). Chlodronate or anti-asialo GM1 was used to deplete Mφ or NK cells, respectively. Tumor volume was measured routinely and, at the end point, tumors were processed and infiltrating immune cells were analyzed by flow cytometry.
In -Dox tumors, both Mφ and NK cells are needed to achieve Tz+DN antitumor effect (p&lt;0.01 and p&lt;0.05, respectively). However, upon MUC4 silencing, only Mφ are required to mediate Tz antitumor effect (p&lt;0.01). To address MUC4 role on T-lymphocyte differentiation, we isolated human T-cells from peripheral blood, activated them with CD3/CD28 beads and exposed them to conditioned mediums obtained from JIMT-1 and KPL-4 cells with or without MUC4 expression. The absence of MUC4 generated a secretome that promoted the differentiation of naïve T-cells to an effector subtype. Finally, we studied the tumor infiltrating lymphocytes (TILs) in a cohort of 90 patients with HER2+ BC and found a negative correlation between the presence of TILs and MUC4 expression (p=0.01).
We conclude that Mφ are key players in the Tz-mediated antitumor innate immune response. Moreover, MUC4 expression impairs T-cell effector function and promotes immunologically cold HER2+ tumors, an additional resistance mechanism of this glycoprotein. We propose that women with HER2+MUC4+ BC could benefit from the combined treatment of Tz+DN to enhance innate and adaptive antitumor immune responses and prevent or overcome Tz resistance. Because MUC4 expression can be easily determined from biopsy tissue, early determination of MUC4 status in HER2+ BC could guide therapeutic choices to improve treatment outcomes.
Citation Format: Sofia Bruni, Florencia L. Mauro, Maria F. Mercogliano, Cecilia J. Proietti, Carla Adami, Agustina Dupont, Gloria Inurrigarro, Rosalia Cordo Russo, Patricia V. Elizalde, Roxana Schillaci. MUC4 enables immune tumor evasion in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2047.