Transcriptional regulation and functional characterization of the tumor suppressor genes Hugl-1 and Hugl-2

Abstract

Cancer is a multi-step process in which both the activation of oncogenes and the inactivation of tumor suppressor genes alter the normal cellular programs to a state of proliferation and growth. The regulation of a number of tumor suppressor genes and the mechanism underlying the tumor suppression have been intensively studied. Hugl-1 and Hugl-2, the human homologues of Drosophila lgl are shown to be down-regulated in a variety of cancers including breast, colon, lung and melanoma, but the mechanism responsible for loss of expression is not yet known. The regulation of gene expression is influenced by factors inducing or repressing transcription. The present study was focused on the identification and characterization of the active promoters of Hugl-1 and Hugl-2. Further, the regulation of the promoter and functional consequences of this regulation by specific transcription factors was analyzed. Experiments to delineate the function of the mouse homologue of Hugl-2, mgl2 using transgenic mice model were performed. This study shows that the active promoter for both Hugl-1 and Hugl-2 is located 1000bp upstream of transcription start sites. The study also provides first insight into the regulation of Hugl-2 by an important EMT transcriptional regulator, Snail. Direct binding of Snail to four E-boxes present in Hugl-2 promoter region results in repression of Hugl-2 expression. Hugl-1 and Hugl-2 plays pivotal role in establishment and maintenance of cell polarity in a diversity of cell types and organisms. Loss of epithelial cell polarity is a prerequisite for cancer progression and metastasis and is an important step in inducing EMT in cells. Regulation of Hugl-2 by Snail suggests one of the initial events towards loss of epithelial cell polarity during Snail-mediated EMT. Another important finding of this study is the induction of Hugl-2 expression can reverse the Snail-driven EMT. Inducing Hugl-2 in Snail expressing cells results in the re-expression of epithelial markers E-cadherin and Cytokeratin-18. Further, Hugl-2 also reduces the rate of tumor growth, cell migration and induces the epithelial phenotype in 3D culture model in cells expressing Snail. Studies to gain insight into the signaling pathways involved in reversing Snail-mediated EMT revealed that induction of Hugl-2 expression interferes with the activation of extracellular receptor kinase, Erk. Functional aspects of mammalian lgl in vivo was investigated by establishing mgl2 conditional knockout mice. Though disruption of mgl2 gene in hepatic tissues did not alter the growth and development, ubiquitous disruption of mgl2 gene causes embryonic lethality which is evident by the fact that no mgl2-/- mice were born.