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

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

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

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

Acceleration of DNA repair does not require the transcriptional functions of CUX1.

<p>(A) DLD-1 cells were stably transfected with a plasmid expressing CUX1 CR1CR2 fused to a nuclear localization signal (NLS), or with empty vector (vector). Expression of recombinant CUX1 protein expression and OGG1 levels were analyzed by immunoblotting. (B) DLD-1 cells were exposed to 10 µm H₂O₂ for 20 min on ice, allowed to recover at 37°C for the indicated time, and then submitted to Single Cell Gel Electrophoresis (comet assay) to quantify DNA damage as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001807#pbio-1001807-g003" target="_blank">Figure 3F,G,H</a>. 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. (C) RT-PCR analysis was performed to measure mRNA levels of transcriptional targets of p110 CUX1 involved in DDRs and genes involved in base excision repair. Primers are listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001807#pbio.1001807.s009" target="_blank">Table S2</a>. All mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The values are the mean of three measurements, and error bars represent standard deviation.</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

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

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 prevents RAS-induced cell senescence.

<p>(A) IMR90 cells stably expressing p200 CUX1 or carrying an empty vector were infected with a retrovirus expressing HRAS^G12V or an empty vector. Following selection, cells were seeded in triplicate and counted for 7 d. The graph is a representative example of three independent experiments. CUX1-HA and HRAS expression were verified by immunoblotting analyses. (B) The percentage of cells exhibiting SA-βgal activity on day 7 was measured. At least 120 cells were analyzed in each case. (C) On day 6 postselection, IMR90 cells were collected and DNA strand breaks quantified by alkaline (pH>13) single-cell gel electrophoresis. The graph is a representative example of three independent experiments. * Indicates <i>p</i> value<0.05, **<i>p</i><0.001, ***<i>p</i><0.0001 on a student's <i>t</i> test. (D) Cells were fixed and stained for γ-H2AX by immunofluorescence. The histograms show the number of cells with 0, 1 to 5, 6 to 10, or more than 10 γ-H2AX foci. At least 80 cells were counted and the percentage of cells with more than 10 foci were used to calculate the <i>p</i> value; ** <i>p</i><0.001. (E) Cells were stained with CM-DCF-DA to measure their relative ROS levels via the geometric mean of the fluorescence intensity. (F, G, and H) Cells were treated with 200 µM H₂O₂ for 30 min on ice and allowed to recover for 0, 15, 30, and 60 min at 37°C and DNA damage was quantified at pH (pH>13) (F), pH 10 (G), or at pH 10 in the presence of the FPG (H). The graph is a representative example of three independent experiments. (I) IMR90 cells stably expressing p200 CUX1, human OGG1, or carrying an empty vector were infected with a retrovirus expressing HRAS^G12V or an empty vector. Proliferation was measured as in (A) and SA-βgal activity was assessed on day 5 as in (B).</p