Transcriptional activation of the Lats1 tumor suppressor gene in tumors of CUX1 transgenic mice

<p>Abstract</p> <p>Background</p> <p><it>Lats1 </it>(large tumor suppressor 1) codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog <it>wts </it>(warts) in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of <it>Lats1 </it>inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation.</p> <p>Results</p> <p>Genome-wide location arrays in cell lines of various cell types revealed that <it>Lats1 </it>was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of <it>Lats1</it>. Expression of <it>Lats1 </it>was reduced in cux1^-/-MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of <it>Lats1 </it>was sufficient to confer transcriptional activation by CUX1. <it>Lats1 </it>was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1.</p> <p>Conclusion</p> <p>While inactivation of <it>Lats1</it>/<it>wts </it>in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that <it>Lats1 </it>is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of <it>LATS1 </it>in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be associated with either loss-of-function or overexpression of <it>Lats1</it>.</p

Deciphering the oncogenic role of CUX1 through the study of mouse models

There is increasing evidence that CUX1 can act as an oncogene in various cell types. First of all, it was found to be overexpressed in different human tumors, such as in uterine leiomyomas, breast tumors and pancreatic tumors. Both p75 and p110 were also found to be overexpressed in tumor cell lines of different origins. My research project has focused on characterizing the oncogenic potential of short CUX1 isoforms through the study of transgenic mice. The first model we used was to overexpress p75 CUX1 under the control of the cytomegalovirus enhancer-chicken β-actin promoter. Interestingly, these mice developed polycystic kidneys at variable penetrance and severity, correlating with transgene expression levels. Cystic kidneys were found to display enhanced proliferation and also showed an upregulation of c-myc and downregulation of p27KIP1. The second model we used was to overexpress p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat in the hypoxanthine phosphorybosyltransferase locus. The first phenotype we observed was that p75 CUX1 transgenic virgin female mice of the first backcross generations developed a myeloproliferative-disease-like myeloid leukemia. These results indicate that overexpression of p75 CUX1 can alter the proliferation and differentiation of some hematopoietic cells. The second phenotype we observed in these mice was that both p75 CUX1 and p110 CUX1 mice of pure FVB background developed mammary tumors of various histopathologies. Metastasis to the lung was observed in the p75 CUX1 transgenic mice only. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested CUX1 DNA binding was increased. Interestingly, higher expression of erbB2 mRNA was seen in most solid carcinomas, while adenosquamous carcinomas displayed higher expression of various WntDe plus en plus d'études suggèrent que le facteur de transcription CUX1 soit un oncogène. Premièrement, CUX1 est surexprimé dans certaines tumeurs humaines, dont les léiomyomes utérins, les tumeurs du sein et les tumeurs du pancréas. Les isoformes courtes CUX1 p75 et p110 sont de plus surexprimées dans plusieurs lignées cellulaires provenant de différents types de tumeurs humaines. L'objectif de mon projet de recherche était de vérifier le potentiel oncogénique des isoformes courtes de CUX1 dans des souris transgéniques. Dans le premier modèle de souris, CUX1 p75 était exprimé sous le contrôle du promoteur du Cytomégalovirus. Ces souris ont développé des reins cystiques avec un degré de pénétrance et de sévérité directement proportionnel aux niveaux d'expression du transgène. Les cellules épithéliales des reins cystiques proliféraient davantage que les cellules normales et montraient une expression élevée de c-myc mais réduite de p27KIP1. Dans le second modèle de souris, CUX1 p75 ou p110 était exprimé sous le contrôle du promoteur du virus MMTV (Mouse Mammary Tumor Virus), spécifiquement intégré dans le locus hprt (hypoxanthine phosphoribosyltransferase). Des rétrocroisements successifs dans la souche FVB ont permis d'uniformiser le bagage génétique des souris transgéniques. Les souris CUX1 p75 des premières générations, donc de bagage génétique mixte, ont développé des leucémies caractérisées par une hyperprolifération de cellules myéloïdes neutrophiles. Ces résultats suggèrent que CUX1 p75 peut affecter la prolifération et la différenciation de certaines cellules hématopoïétiques. Dans la souche FVB pure, les souris CUX1 p75 et p110 ont développé des tumeurs mammaires de diverses histopathologies. Des métastases aux poumons ont été observées dans trois cas de souris CUX1 p75 avec des carcinomes solides mammaires. Les transgènes

Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

Autocrine activation of the Wnt/β-catenin pathway occurs in several cancers, notably in breast tumors, and is associated with higher expression of various Wnt ligands. Using various inhibitors of the FZD/LRP receptor complex, we demonstrate that some adenosquamous carcinomas that develop in MMTV-CUX1 transgenic mice represent a model for autocrine activation of the Wnt/β-catenin pathway. By comparing expression profiles of laser-capture microdissected mammary tumors, we identify Glis1 as a transcription factor that is highly expressed in the subset of tumors with elevated Wnt gene expression. Analysis of human cancer datasets confirms that elevated WNT gene expression is associated with high levels of CUX1 and GLIS1 and correlates with genes of the epithelial-to-mesenchymal transition (EMT) signature: VIM, SNAI1 and TWIST1 are elevated whereas CDH1 and OCLN are decreased. Co-expression experiments demonstrate that CUX1 and GLIS1 cooperate to stimulate TCF/β-catenin transcriptional activity and to enhance cell migration and invasion. Altogether, these results provide additional evidence for the role of GLIS1 in reprogramming gene expression and suggest a hierarchical model for transcriptional regulation of the Wnt/β-catenin pathway and the epithelial-to-mesenchymal transition

Cut homeobox 1 causes chromosomal instability by promoting bipolar division after cytokinesis failure

Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2+ subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome

RAS Transformation Requires CUX1-Dependent Repair of Oxidative DNA Damage

The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1⁺/⁻ MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras(G12V) and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway