6495

SIX1

PURPOSE: Lung adenocarcinoma often manifests as tumors with mainly lepidic growth. The size of invasive foci determines a diagnosis of in situ, minimally invasive adenocarcinoma, or invasive types and suggests that some adenocarcinomas undergo malignant progression in that order. This study investigates how transcriptional aberrations in adenocarcinoma cells at the early stage define the clinical phenotypes of adenocarcinoma tumors at the advanced stage. EXPERIMENTAL DESIGN: We comprehensively searched for differentially expressed genes between preinvasive and invasive cancer cells in one minimally invasive adenocarcinoma using laser capture microdissection and DNA microarrays. We screened expression of candidate genes in 11 minimally invasive adenocarcinomas by reverse transcriptase PCR and examined their involvement in preinvasive-to-invasive progression by transfection studies. We then immunohistochemically investigated the presence of candidate molecules in 64 samples of advanced adenocarcinoma and statistically analyzed the findings, together with clinicopathologic variables. RESULTS: The transcription factors Notch2 and Six1 were upregulated in invasive cancer cells in all 11 minimally invasive adenocarcinomas. Exogenous Notch2 transactivated Six1 followed by Smad3, Smad4, and vimentin, and enlarged the nuclei of NCI-H441 lung epithelial cells. Immunochemical staining for the transcription factors was double positive in the invasive, but not in the lepidic growth component of a third of advanced Ads, and the disease-free survival rates were lower in such tumors. CONCLUSIONS: Paired upregulation of Notch2 and Six1 is a transcriptional aberration that contributes to preinvasive-to-invasive adenocarcinoma progression by inducing epithelial-mesenchymal transition and nuclear atypia. This aberration persisted in a considerable subset of advanced adenocarcinoma and conferred a more malignant phenotype on the subset.CI - (c)2011 AACR.

epithelial-mesenchymal transition (EMT) has a major role in cancer progression, as well as normal organ development and human pathology such as organ fibrosis and wound healing. Here, we performed a gene expression array specialized in EMT of colorectal cancer (CRC). From a comprehensive gene expression analysis using epithelial- and mesenchymal-like CRC cell lines, and following the ontology (GO) analysis, SIX1 gene was identified to be an EMT-related gene in CRC. Using SW480 cells stably transfected with a SIX1 expression construct and their control counterparts, we demonstrated that SIX1 overexpression represses CDH1 expression and promotes EMT in CRC. SIX1-induced CDH1 repression and EMT in CRC cells were correlated at least in part with posttranscriptional ZEB1 activation and miR-200-family transcriptional repression. In primary tumors of CRC, in accord with the functional findings, aberrant expression of SIX1 in cancer cells was observed at the disruption of the basement membrane and at the tumor invasive front, where tumor cells underwent EMT in vivo. Taken together, SIX1 overexpression is suggested to occur in carcinogenesis, and contribute to repression of CDH1 expression and promotion of EMT partly through repression of miR-200-family expression and activation of ZEB1 in CRC.

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/DR-overexpressing 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 mesenchymal-related 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-beta) pathway, including enhanced expression of the TGF-beta receptor type II (TbetaRII), and activation of the mitogen-activated protein kinase (MAPK) pathway in HKc/DR-overexpressing Six1, suggesting that Smad-independent pathways of TGF-beta signaling may be involved in Six1-mediated EMT. p38 MAPK activation was required for sustained Six1-induced EMT and TbetaRII 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-TbetaRII inhibitors for the treatment of cancers overexpressing Six1.CI - (c) The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial-mesenchymal transition (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. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.