FOXP3 EXPRESSION IN BREAST CARCINOMA CELLS AND METASTATIC SPREAD

Forkhead box P3 (FOXP3), a gene member of the forkhead/winged-helix family of transcription regulators, is implicated in regulating immune system development and function. This gene has been found to be of crucial importance for the generation of CD4+CD25+ regulatory T cells (Tregs). Tregs express both the full-length FOXP3 and Δ2FOXP3 natural splice variant. In addition to its expression in the lymphocyte lineage, studies have recently described FOXP3 expression in non-hematopoietic-derived cells, including cancerous or normal epithelial cells of multiple lineages and origins. The role of FOXP3 in cancer cells is still unclear. Our immunohistochemical (IHC) and statistical analyses of archival material from two old series of breast cancer patients indicated that the expression of FOXP3 in tumor cells is an independent strong prognostic factor for distant metastases. The impact of FOXP3 on patient survival has been confirmed by our IHC analysis on chemotherapy-treated breast cancer patients. In fact, FOXP3 positive patients had poorer disease-free survival compared to FOXP3-negative patients. To investigate FOXP3 role in breast cancer, its expression was assessed in a panel of breast carcinoma cell lines and in human primary breast carcinoma samples by Western blot analyses. Full lenght FOXP3 was detected in all human breast cancer samples and breast cancer cell lines analyzed, whereas the Δ2FOXP3 isoform was visible solely in human breast tumors. The involvement of ∆2FOXP3 in breast cancer and the possibility that this isoform could have a different role from that of full length FOXP3 in breast cancer progression has been investigated. WTFOXP3 or ∆2FOXP3 overexpression was induced in MDA-MB-231 breast cancer cell line. Both WTFOXP3 and Δ2FOXP3 overexpression significantly increased in vitro migration and invasion capability of breast cancer cells, whilst inhibiting breast cancer cell proliferation. Taking advantage of these in vitro results and to further investigate in vivo role of FOXP3 in breast cancer metastasis, the metastatic capability of WTFOXP3- or Δ2FOXP3-overexpressing MDA-MB-231 breast cancer cells was investigated. The mean number of spontaneous lung metastases was superimposable in WTFOXP3- and Δ2FOXP3-overexpressing tumor bearing mice (mean±SD: 12.5±27.4 and 10.3±18.1, for WTFOXP3- and Δ2FOXP3-MDA-MB-231-injected mice, respectively). These findings do not support a role of Δ2FOXP3 isoform in promoting breast cancer metastasis. In our IHC analyses of breast carcinoma specimens subcellular staining of FOXP3 was observed to be cytoplasmic or cytoplasmic/nuclear. Since the role of FOXP3 is transcription regulation, which mainly occurs in the nucleus, a cytoplasmic FOXP3 localization could affect its biological role. Thus the hypothesis that FOXP3 in tumor cells may have distinct biological activities and prognostic values according to its subcellular localization was investigated. Metastatic capability of two breast cancer cell clones with inducible FOXP3 expression and with different FOXP3 subcellular localization was evaluated. FOXP3 overexpression in breast cancer cells with a predominantly nuclear FOXP3 localization led to a significant reduction in the number of both spontaneous and experimental lung metastases compared to controls (mice in which FOXP3 overexpression was not induced). Contrarily, in mice injected with breast cancer cells which showed a predominantly cytoplasmic FOXP3 localization, FOXP3 overexpression in tumor cells led to a significant increase in the number of lung metastatic lesions compared to control group. These results suggested that nuclear FOXP3 localization enable its transcriptional activity, resulting in an onco-suppressive effect, while its cytoplasmic localization unable this transcription factor to perform its biological functions, resulting in an opposite in vivo effect. Our findings indicate that FOXP3 subcellular localization in breast tumor cells is an important determinant of prognosis, supporting the involvement of this transcription factor in breast cancer metastasis. Additional studies are in progress to confirm these data and to better understand the molecular mechanisms involved in FOXP3 role in driving breast cancer metastasis.