| The vast majority of cancer deaths are caused by the metastatic spread of primary tumors. Understanding metastasis, the process by which cells migrate from a localized tumor to distant organs, is crucial for the development of novel anticancer agents. Despite significant advances in recent years, the molecular mechanisms involved in cell migration and invasion still remain unclear. To explore the basis of metastasis, we used the Boyden chamber invasion assay to select for highly invasive cells from the low to moderately invasive breast cancer cell lines, MCF7 and MDA-MB-453. The invasive lines, named MCF7-I4 and MDA-MB-453-I4 respectively, displayed epithelial-mesenchymal transition (EMT) and dramatically enhanced invasive ability. EMT changes were correlated with a decrease of E-cadherin and an increase of fibronectin and beta1-integrin. Examining molecular changes between I4 and parental cell lines revealed increased expression of Twist and AKT2, as well as increased STAT3 activation, in the invasive cells compared to the parental lines. Our hypothesis is that Twist, AKT2, and STAT3 may be regulating each other, to mediate the invasive properties of breast cancer cells. This work focuses on elucidating the mechanism of action, and signaling network, of Twist in our I4 system and breast cancer progression.; The first part of our work focused on the relationship between Twist and AKT2. We demonstrated that ectopic expression and siRNA knockdown of Twist resulted in a significant increase and reduction, respectively, of AKT2 protein and mRNA expression. Twist bound to E-box elements on the AKT2 promoter and enhanced the promoter activity. Moreover, blocking AKT2 decreased Twist-promoted migration, invasion and paclitaxel-resistance. Reintroducing AKT2 largely rescued the phenotype resulted from knockdown of Twist in I4 cells, suggesting that AKT2 is a downstream target and functional mediator of Twist. In addition, we observed a 68.8% correlation of elevated Twist and AKT2 expression in late stage as opposed to 13% in early stage breast cancers. Hence, we identified Twist as a positive transcriptional regulator of AKT2 expression which leads to an increase in invasive and survival abilities of breast cancer cells.; The second part of our study focused on the mechanism of upregulation of Twist during tumor progression. In our I4 cells, we observed significantly increased STAT3 phosphorylation levels, while total STAT3 protein levels remained unchanged between parental and I4 cell lines. Further, we demonstrated that activation of STAT3 induces Twist expression at protein and mRNA levels. IL-6 stimulation or expression of constitutively active Src also upregulated Twist. Blocking of STAT3 either by a small molecule inhibitor, shRNAs, or DN-STAT3 resulted in significant reduction of Twist protein and mRNA expression. We have also cloned the human Twist promoter and shown that STAT3 directly binds to and transactivates the Twist promoter. Furthermore, inhibition of STAT3 reduced cell migration, invasion and colony formation of the I4 cells, and ectopic expression of Twist rescued these phenotypes resulting from blocking STAT3. Finally, we also observed a strong correlation between phospho-STAT3 (Y705) and Twist levels in primary breast carcinomas.; In summary, our work established a direct link between STAT3, Twist and AKT2 pathways and demonstrated that the STAT3/Twist/AKT2 axis contributed to the control of breast cancer cell migration, invasion and survival. Therefore, pharmacological disruption of this axis could potentially interrupt breast cancer progression. |
