Date of Award

8-9-2014

Document Type

Open Access Dissertation

First Advisor

Kim E. Creek

Abstract

Inappropriate expression of embryonic genes, particularly homeodomain transcription factors, contributes to tumorigenesis and tumor progression. The overexpression of Six1, a member of the Six family of homeodomain transcription factors, has been found in various human cancers, and is associated with tumor progression and metastasis. We have previously determined that the expression of SIX1 mRNA increased during in vitro progression of human papillomavirus type 16 (HPV16)- immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. However, the mechanism(s) of how Six1 promotes HPV16- mediated transformation remain unknown. In this study, we explored the role of Six1 at early stages and late stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16 and HKc/DR. We found that HKc/HPV16 and HKc/DR overexpressing Six1 exhibited a more mesenchymal phenotype compared to vector controls, as characterized by a fibroblastic appearance and increased invasion. We utilized Whole Human Genome Microarrays to explore the gene expression changes associated with Six1 overexpression in HKc/HPV16 and HKc/DR. Using a combination of KEGG pathway analysis, realtime PCR, and Western blotting, we determined that overexpression of Six1 downregulated epithelial-related genes and upregulated mesenchymal-related genes, which suggests that Six1 overexpression induces epithelialmesenchymal transition (EMT). In addition, we observed alterations in the transforming growth factor-beta (TGF-β) pathway and activation of the Mitogen-activated protein kinase (MAPK) pathway in response to Six1 overexpression, indicating that Smadindependent pathways of TGF-β signaling may be involved in Six1-mediated EMT. Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype; while Six1 overexpression in HKc/DR resulted in malignant conversion and increased the cancer stem cell (CSC)-like population. The activation of MAPK is linked to Six1- mediated resistance to calcium-induced differentiation in HKc/HPV16 and Six1- associated features of CSCs in HKc/DR. Finally, we used a series of MAPK inhibitors to further demonstrate the important role MAPK activation plays in Six1-induced EMT and CSCs properties. Taken together, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation, and Six1 overexpression promotes EMT, CSCs properties, and malignant conversion at late stages of HPV16-mediated transformation of HKc.

We also used an orthotopic mouse model and a splenic injection metastasis model to investigate the role of Six1 overexpression in colorectal cancer (CRC) progression and metastasis. We found that overexpression of Six1 dramatically promoted CRC tumor growth and metastasis in vivo, increased features of cancer stem cells (CSCs), and stimulated angiogenesis by up-regulating the expression of vascular endothelial growth factor (VEGF). Moreover, we determined that Six1 overexpression results in the recruitment of tumor-associated macrophages (TAM) by increasing the expression of macrophage-specific colony-stimulating factor-1 (CSF-1), chemokine (C-C motif) ligand 2/5 (CCL2/5) and VEGF, which further facilitates CRC tumor growth and metastasis. Furthermore, we determined that Six1 activated ERK and p38 MAPK signaling in MC38 CRC cells. In summary, our studies strongly suggest that Six1 overexpression promotes malignant progression in models of cervical and colon cancer, and MAPK activation may play a pivotal role in Six1-associated tumor progression.

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