My research project focused on Triple Negative Breast Cancer (TNBC), which is the most aggressive breast cancer subtype, characterized by the absence of ER, PR and HER2 receptors. Up to now, no targeted therapeutic opportunities for patients are available. One of the key players in the promotion of TNBC aggressiveness is the oncofetal protein HMGA1, an architectural chromatin factor involved in the regulation of gene transcription. In this work we aimed to identify the molecular pathways through which HMGA1 could promote invasiveness in TNBC. By bioinformatic analysis in a cohort of TCGA breast cancer samples, we found an inverse correlation between HMGA1 expression and p27 protein, a well-known CDK inhibitor that has CDK-independent cytoplasmic functions such as the modulation of cell migration. Moreover, in patients with high levels of HMGA1, we observed an enrichment in the expression of a molecular partner of p27, the microtubule-destabilizing protein stathmin. Then, we confirmed the functional relationship observed by TCGA analysis demonstrating that p27 was upregulated at the protein level following the silencing of HMGA1 in two TNBC cell lines (MDA-MB-231 and MDA-MB-157), while stathmin was down-modulated. Moreover, via qRT-PCR, we observed that p27 mRNA level was not modulated by HMGA1, implying a post-translational level of regulation. In fact, by cycloheximide assay, we showed that p27 protein was stabilized after HMGA1 silencing. Moreover, we found that p27 localized in the cytoplasm and, after HMGA1 depletion, was phosphorylated in the cytoplasmic-retaining sites S10 and T198. Looking at stathmin involvement in TNBC, we determined that it is implicated in the promotion of MDA-MB-231 microtubule dynamicity and migration. By silencing the expression of HMGA1 in the same cell line, we demonstrated that stathmin diminishes its interaction with tubulin and it is responsible of motility promotion downstream HMGA1. Thus, we co-silenced HMGA1, p27 and stathmin in MDA-MB-231 cells and we analysed the trans-well migratory abilities of cells showing that HMGA1 promotes the migration through the regulation of p27 and stathmin. One of the chemotherapeutic drugs in the first line treatment for TNBC is paclitaxel, an antineoplastic drug that acts interfering with microtubule function. In literature, both HMGA1 and stathmin have been shown to be involved in paclitaxel chemoresistance. Therefore, we tried to sensitize TNBC cells by targeting the HMGA1-p27-stathmin axis. Upon HMGA1-silencing of MDA-MB-231 cells, we were able to reduce the motility of cells treated with paclitaxel, sensitizing them to the treatment. Finally, we determined that the HMGA1 depletion in MDA-MB-231 cells injected in the mammary fat pad of nude mice, was able to sensitize the primary tumor volume to paclitaxel treatments.
Overall, we identify an HMGA1/p27/stathmin axis involved in the promotion of migration that could be considered as a possible target in combination with standard paclitaxel chemotherapy in TNBC patients.
