The function of transforming growth factor β (TGF-β) in cancer is notoriously complex. Initially TGF-β limits tumorigenesis, but at later stages in tumor progression TGF-β promotes the malignant spread of tumor cells. Past studies to understand the pro-metastasis utility of TGF-β centered upon its ability to regulate protein-coding genes. Recently, a small class of non-coding RNAs known as microRNAs (miRNAs) emerged as novel posttranscriptional regulators of gene expression. The significance of miRNA function in cellular processes from embryonic development to the maintenance of homeostasis in adult tissues is becoming increasingly clear. Also apparent is the strong association between aberrant miRNA expression and human diseases, such as cancer. The contribution of miRNAs to TGF-β-mediated cellular responses remains an open question. Thus, I became interested if miRNAs offered an additional layer of regulation in TGF-β signaling through which this cytokine exerts its pro-metastasis function.
To address this inquiry, in the first part of this dissertation I investigated whether miRNAs influenced the ability of TGF-β to induce cellular responses directly involved with carcinoma metastasis, such as epithelial-mesenchymal transition (EMT). Here, I identified two miRNAs, miR-21 and miR-31, that are upregulated during EMT in LIM 1863 organoids, a colon carcinoma model of EMT driven by TGF-β. We performed in vitro studies to characterize the function of miR-21 and miR-31 and found that these two miRNAs positively impact the induction of EMT, migration and invasion by TGF-β. Furthermore, we uncovered TIAM1 (T lymphoma and metastasis gene 1) as a novel target of both miR-21 and miR-31 and show that downregulation of TIAM1 is critical for the pro-migration and pro-invasion activities of miR-21 and miR-31. Together these findings reveal miR-21 and miR-31 as downstream effectors of TGF-β signaling by facilitating EMT, migration and invasion of colon carcinoma cells.
How TGF-β regulates miR-21 and miR-31 became important questions and thus the focus of the second part of this thesis. Interestingly, I found that TGF-β and TNF-α synergize to increase miR-21 and miR-31 levels in LIM 1863 organoids and that the synthesis of new factors induced by TGF-β/TNF-α are required for this upregulation. Moreover, I report that regulation of miR-21 by TGF-β/TNF-α occurs at multiple levels of biogenesis. More specifically data provided here show that Smad4 binds to the promoter of miR-21 to upregulate its expression thereby specifying miR-21 as a typical TGF-β target gene. This mechanism is different from one recently observed in smooth muscle cells in which TGF-β did not stimulate miR-21 transcription, but interestingly, Smad4 enhanced the Drosha-mediated processing of the miR-21 precursor. These two mechanisms suggest that TGF-β regulation of miR-21 is contextual and highlight the complexity of TGF-β signaling.
As a whole, my findings establish important roles for miR-21 and miR-31 in TGF-β-mediated cellular responses that facilitate the pro-metastasis utility of TGF-β in colon cancer. Also, I describe a novel mechanism by which TGF-β/TNF-α signaling elevates the level of miR-21 and miR-31. Future studies that identify additional targets of miR-21 and miR-31 may offer further insight into the molecular mechanisms underlying cellular regulation by TGF-β. This information will be vital for the design of therapeutic interventions for colon cancer patients
