Abstract A30: FOXP3 regulates metastatic spread of breast cancer via control of expression of CXCR4 chemokine receptor

Abstract

Abstract Background: The transcription factor FOXP3 can regulate T cell migration by inhibiting expression of CXCR4, the receptor for the chemokine CXCL12. The increased expression of CXCR4 by breast cancer cells can drive metastatic migration towards sites which express CXCL12. Intracellular trafficking of FOXP3 to the nucleus is required in order for this protein to function. There are three distinct Foxp3 domains that contribute to its nuclear transport. Importantly, human T-cells express at least two splice variants of FOXP3 protein what may affect the structure of nuclear localization regions. We hypothesized that FOXP3 is inactivated in breast cancer due to dysfunctional FOXP3 isoforms causing failure of nuclear localization with subsequent increase in CXCR4 and development of metastasis. Methods: The expression patterns of FOXP3 and CXCR4 were measured at mRNA (qPCR) and protein (immunohistochemistry, immunofluorescence, FACS) levels. 100 sections of benign tissue, cancers, lymph node metastases and 7 breast cell lines were examined with traditional and digital microscopy techniques. The results were correlated with pathological and clinical prognostic indicators of breast cancer. Following comparison of FOXP3 sequences of cancer and normal cells, cancer cells were transfected with FOXP3 vector and changes in their chemotaxis and invasion were investigated. Results: Normal breast epithelial cells (both patient-derived tissues and laboratory cultured cell lines) expressed FOXP3 in their nuclei and at the same time fail to express CXCR4. Breast cancer cells significantly (p<0.05) overexpressed CXCR4, whereas FOXP3 expression was significantly decreased (p<0.05) and confined to the cytoplasm with negligible nuclear expression. Metastases expressed significantly less FOXP3 and more CXCR4 than primaries (p<0.05). We identified a novel FOXP3I12 isoform on cancer cells with insertion of 120bp within first domain of nuclear localization. Transfection of cancer cells with normal FOXP3 plasmid restored physiological nuclear FOXP3 expression. Furthermore, experiments performed with CXCR4-expressing MDA-MB-231 breast cancer cells showed that FOXP3-transfection of this cells effectively inhibited the chemotactic response within a CXCL12 diffusion gradient. Discussion: We demonstrated failure of FOXP3 nuclear localization in breast cancer cells and an inverse correlation between this failure and CXCR4 expression. Isoform FOXP3I12 accumulates outside of the nucleus where it cannot maintain its physiological properties what may lead to increased CXCR4 expression and acquisition of the capacity for the cancer cells to metastasize. Normal FOXP3 reverses its malignant expression pattern and inhibits migration of cancer cells towards their metastatic sites expressing CXCR4. Conclusion: Our findings suggest disruption of FOXP3 nuclear localization due to the dysfunctional isoform FOXP3I12 what may be a novel mechanism of breast cancer invasion and metastases development. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A30.