HMGA1 AND FOXM1 SYNERGISTICALLY REGULATE A COMMON GENE NETWORK MODULATING THE ANGIOGENESIS IN BREAST CANCER

My research project was focused on the study of triple negative breast cancer (TNBC), which is highly aggressive and unresponsive to common treatments. One of the factors responsible for the aggressive features of TNBC is the High mobility group A1 (HMGA1) protein, a master regulator of the gene transcription, whose high expression level has been correlated with a higher grade in breast cancer. To deepen the knowledge about HMGA1 in breast cancer, we sequenced the RNA collected from a model of TNBC, the MDA-MB-231 cell line, upon HMGA1 silencing; thanks to a bioinformatic analysis, we unraveled molecular partners HMGA1 could cooperate with in regulating common downstream gene networks in breast cancer and among them we selected Forkhead box M1 (FOXM1) transcription factor, chosen for its role in breast carcinogenesis. Firstly, we validated several HMGA1/FOXM1 targets by qRT-PCR after HMGA1 and FOXM1 silencing in TNBC cell lines. Moreover, the depletion of FOXM1 in TNBC cells leads to the acquisition of a more epithelial-like phenotype, with a disruption of the vimentin network, a typical marker of mesenchymal phenotype, results similar to those we obtained upon HMGA1 silencing, and further accentuated by the co-silencing of the two factors. In addition, upon HMGA1 depletion in TNBC cell lines and HEK-293T, FOXM1 translocates from the nucleus to the cytoplasm, suggesting that HMGA1 could modulate FOXM1 subcellular localization and activity. To validate this hypothesis, we performed a luciferase reporter assay, by transfecting the HEK293T cells with a construct containing a portion of the promoter of a known HMGA1/FOXM1 target, the Vascular Endothelial growth factor A (VEGFA). By co-transfecting the two factors, we found that HMGA1 increases the transcriptional activity of FOXM1 on VEGFA promoter and with several deletion constructs we restricted the region responsible for this activity. Considering that the VEGFA is one of the main inducer of angiogenesis, a hallmark in breast cancer, we investigated how HMGA1 and FOXM1 are involved. Firstly, we demonstrated that the amount of VEGFA secreted by MDA-MB-231 cells after HMGA1 silencing is decreased. Afterwards, we treated the HUVEC endothelial cells with supernatants of MDA-MB-231 depleted of HMGA1 and/or FOXM1 and we observed that the proliferation, the migration, and the ability to form a vessel-like network of HUVEC cells were affected. We finally validated in vivo that HMGA1 and FOXM1 synergistically regulate the neo-angiogenesis in Zebrafish larvae. Hence, we demonstrated that HMGA1 and FOXM1 are involved in the regulation of the same gene network in TNBC and that HMGA1 influences the FOXM1 cellular localization and transcriptional activity. We showed also that the tumor cells, on the driving force of HMGA1 and FOXM1, modulate the angiogenic process carried out by endothelial cells, both in vitro and in vivo.