CUX1 transcription factor is required for optimal ATM/ATR-mediated responses to DNA damage

The p110 Cut homeobox 1 (CUX1) transcription factor regulates genes involved in DNA replication and chromosome segregation. Using a genome-wide-approach, we now demonstrate that CUX1 also modulates the constitutive expression of DNA damage response genes, including ones encoding ATM and ATR, as well as proteins involved in DNA damage-induced activation of, and signaling through, these kinases. Consistently, RNAi knockdown or genetic inactivation of CUX1 reduced ATM/ATR expression and negatively impacted hallmark protective responses mediated by ATM and ATR following exposure to ionizing radiation (IR) and UV, respectively. Specifically, abrogation of CUX1 strongly reduced ATM autophosphorylation after IR, in turn causing substantial decreases in (i) levels of phospho-Chk2 and p53, (ii) γ-H2AX and Rad51 DNA damage foci and (iii) the efficiency of DNA strand break repair. Similarly remarkable reductions in ATR-dependent responses, including phosphorylation of Chk1 and H2AX, were observed post-UV. Finally, multiple cell cycle checkpoints and clonogenic survival were compromised in CUX1 knockdown cells. Our results indicate that CUX1 regulates a transcriptional program that is necessary to mount an efficient response to mutagenic insult. Thus, CUX1 ensures not only the proper duplication and segregation of the genetic material, but also the preservation of its integrity

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

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

Genetic inactivation of Cux1 reduces the DNA repair efficiency of MEFs.

<p>(A) MEFs from Cux1^+/+, Cux1^+/−, and Cux1^−/− mice were exposed to 10 µm H₂O₂ for 20 min on ice, allowed to recover at 37°C for the indicated time. DNA damage before and after treatment was measured by comet assay at pH>13 as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001807#pbio-1001807-g003" target="_blank">Figure 3F</a>, except that the time course was extended since recovery takes longer in MEFs. Each bar represents the average of at least 30 comets. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001. (B) Expression of the wild-type Cux1 gene was analyzed by RT-qPCR. Below is a schematic representation of the wild-type CUX1 protein and the CUX1/lac Z fusion protein present in the knockout cells <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001807#pbio.1001807-Ellis1" target="_blank">[51]</a>. Shown at the top are the functional domains: Inh, auto-inhibitory domain; CC, coiled-coil; CR1, CR2, and CR3, Cut repeat 1, 2, and 3; HD, cut homeodomain; R1 and R2, repression domains 1 and 2. Arrows indicate the forward and reverse primers used. (C) Expression of CUX1 (using CUX1–1300 antibody), OGG1, and APE1 was verified by immunoblotting.</p

RAS Transformation Requires CUX1-Dependent Repair of Oxidative DNA Damage

<div><p>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 <i>Cux1^+/−</i> MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. <i>In vitro</i> 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 <i>Kras</i> 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.</p></div

p200 CUX1 transgenic mice develop mammary gland tumors of various histopathologies.

<p>(A) Kaplan-Meier survival curves evaluating risk of developing mammary gland tumors in wild-type and p200 CUX1 cohorts. The indicated <i>p</i> values were calculated by the log rank test. Using the Cox proportional hazards test, the probability of developing a mammary gland tumor was determined to be 24.7 higher in p200 CUX1 mice (<i>p</i> value: 3.33×10⁻⁶) than in wild-type nontransgenic mice. (B) H&E staining of mammary tumors from p200 CUX1 transgenic mice. Histopathological types were classified as adenosquamous carcinoma (i), solid carcinoma (ii), carcinoma papillary (iii), and carcinoma cribiform (iv). (C) Expression of p200 CUX1 transgenes in mammary tumors (T) and adjacent mammary glands (A) of transgenic mice and normal mammary glands tissues (N) of transgenic mice was analyzed by RT-qPCR. Mouse-specific β2-microglobulin was used as controls for the mammary gland tissues and GAPDH were used for Hs578T and NIH3T3 cell lines.</p

Genetic inactivation of Cux1 reduces the DNA repair efficiency of MEFs.

<p>(A) MEFs from Cux1^+/+, Cux1^+/−, and Cux1^−/− mice were exposed to 10 µm H₂O₂ for 20 min on ice, allowed to recover at 37°C for the indicated time. DNA damage before and after treatment was measured by comet assay at pH>13 as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001807#pbio-1001807-g003" target="_blank">Figure 3F</a>, except that the time course was extended since recovery takes longer in MEFs. Each bar represents the average of at least 30 comets. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001. (B) Expression of the wild-type Cux1 gene was analyzed by RT-qPCR. Below is a schematic representation of the wild-type CUX1 protein and the CUX1/lac Z fusion protein present in the knockout cells <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001807#pbio.1001807-Ellis1" target="_blank">[51]</a>. Shown at the top are the functional domains: Inh, auto-inhibitory domain; CC, coiled-coil; CR1, CR2, and CR3, Cut repeat 1, 2, and 3; HD, cut homeodomain; R1 and R2, repression domains 1 and 2. Arrows indicate the forward and reverse primers used. (C) Expression of CUX1 (using CUX1–1300 antibody), OGG1, and APE1 was verified by immunoblotting.</p

KRAS mutations in mammary tumors from MMTV-p200 CUX1 transgenic mice.

a<p>These normal tissues were from the adjacent mammary gland of tumor-bearing mice.</p

Total number of tumors from wild-type and p200 CUX1 transgenic mice.

<p>Results from wild-type mice are shown from a previous study as well as from the present study. All wild-type mice were taken into account to calculate the <i>p</i> values. Other tumors include hematopoietic tumors, liver tumors, uterine tumors, pancreatic tumors, and intestine tumors.</p><p>* <i>p</i> value≤0.05;</p><p>*** <i>p</i> value≤0.0001.</p

CUX1 knockdown is synthetic lethal for RAS-transformed human cancer cell lines.

<p>Lentivirus expressing a doxycycline-inducible shRNA against CUX1 was introduced in a paired cell line DLD-1 [KRAS^G13D] or DKO-4 [KRAS^WT] and Hs578T [HRAS^G12D]. (A) DLD-1 and DKO-4 cells: doxycycline was added to the medium and after 4 d CUX1 protein expression was analyzed by Western blotting. Cells expressing shRNA CUX1 or not were seeded in triplicate and counted daily for 7 d. Each point represents the average ± SD. The graph is a representative example of two independent experiments. (B and C) On day 6, DNA strand breaks were quantified in either alkaline pH (pH 14), pH 10 (detection of single and double strand breaks), or in the presence of the FPG) (detection of single, double, and oxidized purines and formamidopyrimidine). ** <i>p</i> value<0.001; *** <i>p</i><0.0001 on a student's <i>t</i> test. (D) Hs578T cells: CUX1 protein expression and cell proliferation were analyzed as in Figure 4A. (E) Hs578T cells were cultured in the absence (−) or presence of doxycycline for 4 days (+), followed by a 4-d withdrawal period (w). CUX1 protein expression was analyzed by immunoblotting, and DNA strand breaks for each condition were quantified as described in Figure 4B. * <i>p</i> value<0.05; *** <i>p</i><0.0001 on a student's <i>t</i> test. (F) 8-OHdG levels were measured in DLD-1 and Hs578T cells expressing shRNA CUX1 or not.</p