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The role of the transcription factor FOXP1 in the immune response to breast cancer

par De Silva, Jasenthu L. P.
Président du jury Braun, Michel Y
Promoteur Piccart-Gebhart, Martine
Co-Promoteur Willard-Gallo, Karen
Publication Non publié, 2018-01-23

Thèse de doctorat

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Résumé :

Breast cancer (BC) was not initially considered an immunogenic tumor; however, recent data show that immune-related factors are associated with patient prognosis and the response to treatment. Several large adjuvant clinical trials have shown that tumor infiltrating lymphocytes (TIL) are significantly associated with a better prognosis and can also predict responsiveness to pre-operative chemotherapy, particularly in the triple negative (TN) & HER2+ BC subtypes (Carsten Denkert et al., 2010; Loi et al., 2013a). Recently, the presence of ectopic lymph node-like structures characterized by distinct T and B cell zones, called tertiary lymphoid structures (TLS), were identified adjacent to the tumor (Gu-Trantien et al., 2013) in 60% of BC (Buisseret et al., 2017b) and linked with a good prognosis (Gu-Trantien et al., 2013). The mechanisms involved in TLS formation and activities and their impact on tumor immunity is relatively unknown. TIL infiltration and TLS formation are likely regulated, in part, by transcription factors (TF) that control cytokine/chemokine production within the tumor microenvironment (TME) (Pimenta and Barnes, 2014). One such TF, the forkhead box protein 1 (FOXP1) is abnormally expressed in various human tumors and has a known role in regulating immune cell functions. Contradictory data on FOXP1 expression together with a lack of information on its immune regulation led us to explore its role in this tumor type. The first part of this thesis research focused on FOXP1-mediated regulation in BC. Gene/protein analysis was examined in the four BC molecular subtypes, revealing its enriched expression in estrogen receptor positive (ER+) tumors (Luminal A/B). Luminal BC is generally less infiltrated compared with frequently high TIL infiltration in ER negative (ER-) tumors (i.e. HER2+ and TN) [reviewed in (Solinas et al., 2017a) and (Loi et al., 2014)]. We found that high FOXP1 expression in a cohort of untreated primary BC was significantly associated with a lower TIL and fewer TLS compared to FOXP1 low (FOXP1lo) tumors. This observation led us to investigate the effect of FOXP1 on cytokines and chemokines potentially involved in TIL recruitment and/or TLS formation. BC cancer cell lines were used to silence [MCF7; FOXP1hi] or overexpress [MDA-MB-231; FOXP1lo] FOXP1 expression. FOXP1 repression upregulated a number of cytokines and chemokines involved in T and B cell migration and function, while FOXP1 overexpression repressed a majority of the same factors. Expression analysis of the major T and B cell cytokine and chemokine genes was performed for FOXP1lo and FOXP1hi primary BC. These data reveal that FOXP1hi BCs have significant decreases in CXCL9, CXCL10, CXCL11, CXCL13, CX3CL1, CCL20, IL2, IL21, granzyme B and IFNγ and high levels of the immunosuppressive cytokines, IL10 and TGFβ. We next performed a lymphocyte migration assay using primary tumor supernatants prepared from FOXP1lo and FOXP1hi BC finding significantly decreased migration of total CD45+ lymphocytes, B cells, helper (CD4+) and cytotoxic (CD8+) T cells using FOXP1hi compared to FOXP1lo SN. Overall, our data suggest that FOXP1 plays an important role in repressing anti-tumor immune responses by negatively regulating TIL migration directed by specific cytokines and chemokines.The second part of this thesis research focused on the role FOXP1 plays in BC TLS. FOXP1 expression in T and B cell TIL and TLS was evaluated using RT-qPCR, multicolor flow cytometry, immunofluorescence (IF) and immunohistochemistry (IHC) and fresh, fixed and frozen breast tissues. Based on the FOXP1 expression two types of TLS were identified in BC: 1) TLS containing a germinal center (GC-TLS) and 2) TLS lacking a GC (non-GC-TLS). Examination of proteins specifically associated with active humoral immune responses allowed us to identify GC-TLS but not non-GC-TLS as functional. Gene expression analysis of micro-dissected tissues revealed distinct immune profiles that characterize B cell follicles in tonsils and spleen as well as aggregates, non-GC-TLS and GC-TLS in BC. This analysis further demonstrates that ongoing cell-mediated immune responses are associated with GC-TLS. The findings from this thesis research add important information to our understanding of how immune responses are initiated and maintained in BC and provide further insight into the identification and organization of functional immune responses at the tumor site.