Ricerc@Sapienza

Several studies have been performed with the aim of identifying drugs able in inhibiting Epithelial-Mesenchymal Transition (EMT), chiefly by blocking PI3K/Akt pathway. We have already demonstrated that treatment with myo-Inositol at the pharmacological dose can block EMT in breast cancer cells by downregulating PI3K/Akt and inducing changes in cytoskeletal architecture. Herewith, we investigated the mechanism of action of myo-inositol in both highly (MDA-MB-231) and low (MCF-7) invasive human breast cancer cells. After 30’ and 24h from treatment, gene expression analysis revealed a significant downregulation of Pi3k and Psen1 after 30’ in both cell lines. Psen1 downregulation was maintained in MDA-MB-231 at 24h. Likewise, we explored the modulation of Ip6k1, Dnmt3b, Isyna1 and p53. In MDA-MB-231, a strong downregulation of Ip6k1 expression was recorded at 30’ and 24h, whilst Dnmt3b was reduced only at 30’. On the contrary, in MCF-7, Ip6k1 downregulation was unexpectedly associated to the upregulation of Dnmt3b at 30’. IP6K1 is a key enzyme of inositol metabolism, inhibits ISYNA1, probably inducing de novo DNA methylation (i.e., DNMT3B). Furthermore, IP6K1 inhibition correlates with a decrease of cancer cells motility. The upregulation of Isyna1 was observed in both cell lines at 30’, together with p53. ISYNA1 activates myo-Inositol intracellular biosynthesis starting from glucose-6-phosphate. In this activation, p53 plays a key role in binding Isyna1 promoter and eventually enabling its expression. Western-blot of MDA-MB-231 confirmed that changes in gene expression were also mirrored by concurrently modifications in IP6K1 and p53 protein levels, altogether with a decrease of both MDM2 and YAP/TAZ. It is worth noting that in MCF-7, no changes were observed in protein levels. In-silico analysis was performed using TCGA miRNA-Seq data to identify differentially expressed miRNAs between normal and tumoral tissue in breast cancer patients. To further gain mechanistic insights on myo-Inositol effects, we compared these data with main differentially expressed cancer-related miRNAs in MDA-MB-231 cells after 30’ from treatment. This analysis allowed to identify two mRNAs, downregulated in tumor tissues, that were significantly increased with myo-Inositol: miR-92a-3p and miR-125a-5p. Using DIANA tools, miR-92a-3p was predicted to interact with Notch-1 and PI3K, linking it to cytoskeletal rearrangement. Moreover, a strong interaction was predicted between miR-125a-5p and IP6K1 in 3’-UTR site. Indeed, the upregulation of miR-125a-5p is usually correlated with metastasis inhibition in breast cancer. In MDA-MB-231, miR-125a-5p upregulation was maintained at 24h, while in MCF-7 was slightly upregulated at 30’ and downregulated at 24h. Our results suggest that myo-Inositol causes early changes in gene expression, probably led by miRNAs and methylation remodeling. Elucidation of the role of miR-125a-5p/IP6K1 axis will reveal strategies for molecular targeted therapies in breast cancer.