Abstract 3694: FOXP1 suppresses immune cell migration in breast tumors
Authors: Pushpamali De Silva, Soizic Garaud, Roland de Wind, Gert Van den Eynden, Anaïs Boisson, Cinzia Solinas, Edoardo Migliori, Hugues Duvillier, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo
Journal: Cancer Research (2017)

Abstract Tumor infiltrating lymphocytes (TIL) play an essential role in mediating response to chemotherapy and improving clinical outcomes in breast cancer (BC). Extensive TIL infiltration is characterized by their organized into tertiary lymphoid structures (TLS). TIL infiltration and TLS formation may be regulated, in part, by transcription factors (TF) controlling cytokine/chemokine production within the tumor microenvironment. The forkhead box protein 1 (FOXP1) is a TF shown to be abnormally expressed in a variety of human tumors and play an important role in T cell cytokine production. Therefore we aimed to study FOXP1-mediated regulation of TIL in BC. Investigation of FOXP1 expression in public microarray data from untreated BC patients, BC cell lines [Luminal A (MCF7), HER2+ (BT474) and triple negative (TN; MDA-MB231)] and prospectively collected formalin-fixed paraffin-embedded (FFPE) primary breast tissues showed that FOXP1 is repressed at transcript and protein level in HER2+ or TN breast tumors compared to estrogen receptor positive tumors (Luminal A and B). Moreover HER2+ and TN subtypes, which showed decreased FOXP1 levels, are 2 well known highly infiltrated BC. Based on our hypothesis that FOXP1 could play a role on immune cell infiltration in breast tumors, data analysis of the prospective BC cohort showed that high FOXP1 (FOXP1hi) expression is significantly associated with a lower percentage of TIL and number of TLS compared to FOXP1 low (FOXP1lo) tumors. To investigate the impact on specific cytokines/chemokines involved in TIL recruitment and/or TLS formation, FOXP1 was silenced in MCF7 (FOXP1hi tumor cell line) or upregulated in MDA-MB-231 (FOXP1lo tumor cell line) followed by gene expression analysis using a RT-qPCR based human cytokine/chemokine array. FOXP1 repression upregulated major T and B cell chemoattractant chemokines and overexpression repressed most of these molecules in the cell line experiments. Next we analyzed major chemoattractant molecule expression in FOXP1lo and FOXP1hi prospective breast tumors and found that FOXP1hi tumors having a significant decrease in CXCL9, CXCL10, CXCL11, CXCL13, CX3CL, CCL20, IL2, and IL21. A migration assay (Transwell chambers) done using healthy donor (HD) PBMC showed a significant increase in total lymphocytes migrated towards FOXP1 repressed tumor conditioned media (TCM) of MCF7 compared to the TCM of control or medium alone. Finally analysis of lymphocyte migration to FOXP1lo and FOXP1hi tumor supernatants (SN) from primary tumors that we consistently prepare without enzymatic digestion, showed that there was a significant decrease in number of lymphocytes migrated towards FOXP1hi tumor SN including the migration rates of individual T and B lymphocytes populations compared to FOXP1lo tumor SN. These data suggest that FOXP1 could play a critical role in establishing effective anti-tumor immune responses by negative regulation of TIL via suppression of cytokine/chemokine expression in breast tumors. Citation Format: Pushpamali De Silva, Soizic Garaud, Roland de Wind, Gert Van den Eynden, Anaïs Boisson, Cinzia Solinas, Edoardo Migliori, Hugues Duvillier, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo. FOXP1 suppresses immune cell migration in breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2017-3694