p110 CUX1 Homeodomain Protein Stimulates Cell Migration and Invasion in Part through a Regulatory Cascade Culminating in the Repression of E-cadherin and Occludin

Abstract

In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.The molecular mechanisms by which transformed cells become migratory and invasive during tumor progression are beginning to be unraveled (reviewed in Ref. 1). Some events are reminiscent of an important developmental process termed epithelial-to-mesenchymal transition (EMT) 3 (reviewed in Refs. 2, 3). During EMT, tumor cells redistribute or down-regulate their epithelium-specific proteins such as adherent and tight-junction proteins, including E-cadherin and occludin, and start to express mesenchymal proteins, such as vimentin and N-cadherin. As a result, cell-cell contacts are disrupted causing a loss of apico-basal polarity, and cells acquire mesenchymal and migratory properties necessary for invasion. Transcriptional repression has emerged as a fundamental mechanism for silencing of E-cadherin and occludin, and several transcriptional repressors have been identified (reviewed in Ref. 4). Snail and Slug, which belong to the Snail superfamily of zinc finger transcriptional repressors, are the most characterized E-cadherin repressors (5-9). The zinc fingers present at the carboxyl terminus of the proteins function as the sequencespecific DNA-binding domains that recognize consensus E2 box-type elements. Their repressor capacity is mediated by the SNAG domain present at the amino-terminal part of the proteins (reviewed in Ref. 10).A requirement for the CUX1 homeodomain ...

The roles of CUX1 homeodomain proteins in the establishment of a transcriptional program required for cell migration and invasion

“…1 In agreement with these results, mouse embryonic fibroblasts (MEFs) from CUX1-knockout mice exhibited a defect in migration and invasion as compared to MEFs from wild type mice. 2 These observations were extended using an in vivo invasion assay. Indeed, pulmonary colonization after tail vein injection was reduced following stable expression of CUX1 shRNA in HT1080 and MDA-MB-231.…”

“…2 The defect was rescued completely by p110 CUX1, but only partially by p200 CUX1. Subsequent experiments showed that cell migration was stimulated by treatments that increase proteolytic processing of p200 CUX1 into p110 CUX1, whereas cell migration was decreased in the presence of a protease inhibitor that prevents the generation of p110 CUX1.…”

“…Transcriptional targets of CUX1 were identified by performing genome-wide location analysis in several cell lines. 2 Targets that play a role in cell migration, invasion and adhesion were then validated in independent chromatin…”

CUX1, a haploinsufficient tumour suppressor gene overexpressed in advanced cancers

“…Using transcription and cellbased assays, a role for p110 CUX1 was shown in many cellular processes, notably in cell cycle progression and cell proliferation 21,22 , strengthening of the spindle assembly checkpoint 19 , establishment of a transcriptional programme that enables efficient DNA damage responses 23 , and cell migration and invasion 13,42 . In addition, from RNA interference (RNAi)-mediated knockdown and genetic inactivation, CUX1 was shown to be required for the resistance to apoptotic signals in pancreatic cancer cells 14 , the repression cytokine genes associated with M1 macrophages 43 , and dendrite branching and spine development in cortical neurons 34 .…”

“…Many transcriptional targets and cellular functions of CUX1 readily suggest mechanisms by which increased p110 or p75 CUX1 expression might promote tumour development and progression, including acceleration of S phase entry 21,22,41,62,63 , stimulation of cell migration and invasion 13,42,[64][65][66] , resistance to apoptosis 14 , and promotion of bipolar mitosis in the presence of supernumerary centrosomes 19 (reviewed in REF. 67).…”