When stem-like mammosphere cells were used to initiate xenografts, tumor growth and initiation was much faster than whole cell population. miR-140 overexpression was again able to almost
completely eliminate growth of order Alvocidib DCIS tumors[28]. Role of miR-140 in IDC stem cells In order to interrogate the role miR-140 plays in breast cancer, we investigated miR-140 expression in estrogen receptor positive invasive breast tumor cells. We found that miR-140 expression is inversely related with SOX2 expression. Tissue staining of ERα+ IDC revealed a significant increase in SOX2 expression, and qRT-PCR revealed a dramatic downregulation in miR-140 expression. A luciferase reporter assay for the 3’-UTR of SOX2 showed that miR-140 directly targets and inhibits SOX2 expression, and mammosphere assays demonstrated that miR-140 targeting regulates stem cell signaling in tumors. While examining the molecular mechanisms regulating miR-140 expression we identified predicted estrogen response
elements (ERE) in the miR-140 promoter region. Due to the previous reports linking ERα and self-renewal signaling, we investigated a potential ERα miR-140 relationship. In non-tumorigenic cells engineered to express ERα, E2 treatment significantly inhibited miR-140 expression, while also stimulating SOX2 expression. We examined the miR-140 promoter using a luciferase reporter and found that E2-mediated miR-140 downregulation was decreased when the ERE at -79/50 in the miR-140 promoter was mutated. Binding of ERα to the miR-140 promoter was validated using ChIP. In the absence of estrogen, miR-140 expression had very little effect on cancer stem cell frequency. There was a significant decrease in the CD44+/CD24- population when miR-140 was overexpressed following estrogen stimulation, indicating miR-140 plays an important role in the regulation of estrogen stimulated tumor-initiation cells,
potentially through inhibition of SOX2[27]. EXOSOMES Exosomes are spherical membrane vesicles between 50-100 nm, secreted by the majority of cells. Multivescular bodies fuse with the cellular membrane, releasing exosomes into the extracellular matrix[31]. They contain a variety of protein, RNA, products of signaling pathways and miRNAs, some common to all exosomes and some cell specific[32]. The common set of proteins Dacomitinib consists of the tetraspanin family (CD9, CD63, CD82), members of the endosomal sorting complexes required for transport (ESCRT) complex (TSG101, ALix) and heat shock proteins (Hsp60, Hsp70, Hsp90)[33]. Several of these proteins are used for exosome detection in Western blotting or FACS, including CD63 and CD9[34,35]. Exosome function in tumorigenesis There are three known functions of exosomes in tumorigenesis; restructuring of microenvironment, modulation of tumor immune response and direct modification of tumor cells via delivery of protein or genetic material[31,36].