Six1 promotes epithelial-mesenchymal transition and malignant conversion in human papillomavirus type 16-immortalized human keratinocytes

Abstract

Six1, a member of the Six family of homeodomain transcription factors, is overexpressed in various human cancers, and SIX1 overexpression is associated with tumor progression and metastasis. Six1 messenger RNA levels increase during in vitro progression of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we show that HKc/DRoverexpressing Six1 exhibited a more mesenchymal phenotype, 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/DR. We found that overexpression of Six1 downregulated epithelial-related genes and upregulated mesenchymalrelated genes, which suggests that Six1 overexpression induces epithelial-mesenchymal transition (EMT). Pathway analysis of the microarray data showed alterations in the transforming growth factor-beta (TGF-β) pathway, including enhanced expression of the TGF-β receptor type II (TßRII), and activation of the mitogen-activated protein kinase (MAPK) pathway in HKc/DRoverexpressing Six1, suggesting that Smad-independent pathways of TGF-β signaling may be involved in Six1-mediated EMT. p38 MAPK activation was required for sustained Six1-induced EMT and TβRII overexpression. Finally, we determined that Six1 overexpression in HKc/DR resulted in malignant conversion and increased the cancer stem cell (CSC)-like population. Thus, Six1 overexpression promotes EMT, CSCs properties and malignant conversion in HKc/DR through MAPK activation, which supports the possible use of p38-TßRII inhibitors for the treatment of cancers overexpressing Six1.