Deregulated CDC25A Expression Promotes Mammary Tumorigenesis with Genomic Instability (original) (raw)

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Molecular Biology, Pathobiology, and Genetics| February 05 2007

Dipankar Ray;

1Department of Molecular Pharmacology and Biological Chemistry and

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Yasuhisa Terao;

3Biochemistry and Molecular Genetics and

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Peter G. Fuhrken;

6Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois;

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Zhi-Qing Ma;

7Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; and

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Francesco J. DeMayo;

7Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; and

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Nyla A. Heerema;

8Department of Pathology, Ohio State University, Columbus, Ohio

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Roberta Franks;

5Research Resources Center, University of Illinois College of Medicine, Chicago, Illinois;

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Sophia Y. Tsai;

7Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; and

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Eleftherios T. Papoutsakis;

Eleftherios T. Papoutsakis

6Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois;

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Hiroaki Kiyokawa

1Department of Molecular Pharmacology and Biological Chemistry and

2Robert H. Lurie Comprehensive Cancer Center, Northwestern University; Departments of

3Biochemistry and Molecular Genetics and

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Requests for reprints: Hiroaki Kiyokawa, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, 303 East Superior Street, Lurie 3-113, Chicago, IL 60611. Phone: 312-503-0699; Fax: 312-503-0700; E-mail: kiyokawa@northwestern.edu.

Received: October 24 2006

Revision Received: November 21 2006

Accepted: November 29 2006

Online ISSN: 1538-7445

Print ISSN: 0008-5472

2007

Cancer Res (2007) 67 (3): 984–991.

Article history

Received:

October 24 2006

Revision Received:

November 21 2006

Accepted:

November 29 2006

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Citation

Dipankar Ray, Yasuhisa Terao, Peter G. Fuhrken, Zhi-Qing Ma, Francesco J. DeMayo, Konstantin Christov, Nyla A. Heerema, Roberta Franks, Sophia Y. Tsai, Eleftherios T. Papoutsakis, Hiroaki Kiyokawa; Deregulated CDC25A Expression Promotes Mammary Tumorigenesis with Genomic Instability. _Cancer Res 1 February 2007; 67 (3): 984–991. https://doi.org/10.1158/0008-5472.CAN-06-3927

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Abstract

Checkpoint pathways help cells maintain genomic integrity, delaying cell cycle progression in response to various risks of fidelity, such as genotoxic stresses, compromised DNA replication, and impaired spindle control. Cancer cells frequently exhibit genomic instability, and recent studies showed that checkpoint pathways are likely to serve as a tumor-suppressive barrier in vivo. The cell cycle–promoting phosphatase CDC25A is an activator of cyclin-dependent kinases and one of the downstream targets for the CHK1-mediated checkpoint pathway. Whereas CDC25A overexpression is observed in various human cancer tissues, it has not been determined whether deregulated CDC25A expression triggers or promotes tumorigenesis in vivo. Here, we show that transgenic expression of CDC25A cooperates markedly with oncogenic ras or neu in murine mammary tumorigenesis. MMTV-CDC25A transgenic mice exhibit alveolar hyperplasia in the mammary tissue but do not develop spontaneous mammary tumors. The MMTV-CDC25A transgene markedly shortens latency of tumorigenesis in MMTV-ras mice. The MMTV-CDC25A transgene also accelerates tumor growth in MMTV-neu mice with apparent cell cycle miscoordination. CDC25A-overexpressing tumors, which invade more aggressively, exhibit various chromosomal aberrations on fragile regions, including the mouse counterpart of human 1p31-36, according to array-based comparative genomic hybridization and karyotyping. The chromosomal aberrations account for substantial changes in gene expression profile rendered by transgenic expression of CDC25A, including down-regulation of Trp73. These data indicate that deregulated control of cellular CDC25A levels leads to in vivo genomic instability, which cooperates with the neu-ras oncogenic pathway in mammary tumorigenesis. [Cancer Res 2007;67(3):984–91]

Introduction

Development of cancer involves accumulation of genetic changes that lead to malignant transformation of normal cells. DNA damage, induced by various genotoxic stresses, compromised DNA replication, or impaired spindle control, activates checkpoint pathways and delays cell cycle progression to facilitate repair functions. Most cancer cells exhibit genomic instability, and recent studies have provided evidence that checkpoint pathways serve as a tumor-suppressive barrier in vivo (1). Although normal cells possess multiple signaling pathways for checkpoint, essentially all signals converge at negative regulation of cyclin-dependent kinases (CDK), which form central parts of the cell cycle machinery (2). CDK2, activated by association with cyclin E and cyclin A during the G1 and S phase, respectively, is critical for initiation and completion of DNA replication. CDK1, formally known as CDC2, associates with cyclin A or cyclin B during the G2 phase and plays an essential role in initiation and completion of mitosis. Activation of the checkpoint pathways leads to inhibition of these CDKs, resulting in arrest at different phases of the cell cycle.

DNA damage activates the proximal checkpoint kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), which transduce the signal to several downstream effectors, such as the CHK1 and CHK2 kinases and the tumor suppressor p53 (3–5). In response to the damage-induced signals, p53 transactivates the Kip family CDK inhibitor p21Cip1/Waf1 and a cyclin B/CDK1 inhibitor, 14-3-3σ. This p53-mediated transcriptional control induces G1 or G2 arrest, representing sustained or sometimes permanent checkpoint responses. In contrast, acute control of cell cycle checkpoint is mediated by inactivation of CDC25 family phosphatases. CDC25 proteins activate CDKs by dephosphorylation at the ATP-binding domains (6). Of three members of the family, CDC25A plays critical roles in promoting cell cycle progression at multiple phases by regulating CDK2-cyclin E, CDK2-cyclin A, and CDK1-cyclin B (7). Knockdown of CDC25A by RNA interference delays both G1-S and G2-M transitions (8), whereas overexpression of CDC25A can induce aberrant mitotic events (9). In response to DNA damage, CHK1 phosphorylates CDC25A protein on multiple sites, resulting in ubiquitination by the Skp1-Cullin-β-TrCP E3 ligase and rapid proteasome-dependent degradation (10, 11). It has been shown that CHK1-mediated phosphorylation of CDC25A helps cells maintain appropriate levels of this protein even during unperturbed cell cycle progression. On the other hand, CHK2-mediated phosphorylation of CDC25C inactivates this phosphatase by cytoplasmic sequestration, which forms another G2 checkpoint mechanism. Thus, multiple modes of CDC25 inactivation play key roles in cell cycle checkpoint (6).

Deregulation of checkpoint pathways, which could lead to genomic instability, has been implicated for initiation and progression of tumorigenesis. Loss of ATM strongly predisposes humans and mice to tumorigenesis (12, 13). Whereas homozygous disruption of Atr causes lethality in mice (14), Atr haploinsufficiency promotes tumorigenesis in mice with defective DNA mismatch repair (15). Chk1 homozygous mutant mice are also lethal, and Chk1 haploinsufficiency modestly enhances tumorigenesis induced by transgenic expression of Wnt-1 (16). Consistently with the importance of CDC25A as a checkpoint target, CDC25A levels are increased in tissues and tumors in Chk1 heterozygous mice (17). Overexpression of CDC25A has been reported in a variety of human cancers, including breast, head and neck, liver, thyroid, ovary, and lung cancers as well as non–Hodgkin's lymphomas (18–23), although the mechanisms of cancer-associated overexpression have been poorly understood. A recent report described that 47% of breast cancer tissues at early T1 stages overexpressed CDC25A, which correlated with poor prognosis of the patients (24). These data imply that perturbed control of cellular CDC25A levels is a critical step of in vivo tumorigenesis; however, this hypothesis has not been evaluated directly. In the present study, we examined the effect of deregulated CDC25A expression on tumor susceptibility using a new transgenic mouse model.

Materials and Methods

Generation of transgenic mice. MMTV-CDC25A transgenic mice were developed in the transgenic core at Baylor College of Medicine (Houston, TX) by conventional DNA injections into single-cell stage FVB/N mouse embryos as described previously (25). MMTV-H-ras mice and MMTV-neu mice in the FVB/N background have been described previously (26, 27). MMTV-CDC25A mice were crossed with MMTV-H-ras or MMTV-neu mice, and double transgenic offspring were compared with single transgenic littermates about spontaneous tumorigenesis. Mice were maintained according to protocols approved by the Institutional Animal Care Committees at Northwestern University (Evanston, IL), University of Illinois (Chicago, IL), and Baylor College of Medicine according to the American Association for Laboratory Animal Science regulation. To detect tumorigenesis, each mouse was physically examined twice weekly and the date of detecting palpable tumors was recorded. Mice were sacrificed when diameters of primary tumors reached 2 cm. To figure out tumor volumes, the following formula was used: V = a × _b_2 / 2 (a = longer diameter; b = shorter diameter). The first identified tumor was measured in each animal.

Karyotyping and pathologic examinations. To establish cell cultures from tumors developed in transgenic mice, tumor tissues were minced with scissors and placed on culture dishes containing DMEM with 15% fetal bovine serum. When cells proliferated on the dish up to 90% confluency, they were trypsinized and subcultured in 1:3 dilution. For karyotyping, cells were treated with 770 ng/mL colcemid for 2 h and then treated with hypotonic KCl solution followed by fixation with methanol/acetic acid (3:1). Fixed cells were dropped onto a slide glass, treated with trypsin, stained with Wright stain, and subjected to karyotyping. More than 10 metaphase cells per sample were analyzed for karyotype data. These procedures were done in the Cytogenetics Core at Ohio State University (Columbus, OH). For histochemical analyses of in vivo tumors, mice carrying palpable tumors were i.p. injected with 50 μg/mL bromodeoxyuridine (BrdUrd), and 2 h later, animals were euthanized by CO2 gas. Tumor tissues were then dissected and fixed in 10% buffered formalin. Fixed tissues were paraffin embedded and sectioned using standard procedures.

Immunologic analyses. For immunoblotting, cells were scraped on culture dishes filled with lysis buffer followed by sonication as described previously (28). Anti-CDC25A monoclonal antibody (clone Ab3) was obtained from NeoMarkers (Fremont, CA). Anti-Ki67 antibody was purchased from Novocastra Laboratories (New Castle, United Kingdom), and anti-BrdUrd monoclonal antibody was purchased from PharMingen (San Diego, CA). Phosphorylated-specific anti-histone H3 rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoblotting, immunohistochemistry, and immunocytochemistry were done as described previously (29). For histologic analyses, similar sizes of tumors (∼1 cm) were sampled from MMTV-H-ras mice at 18 weeks and from MMTV-H-ras; MMTV-CDC25A mice at 10 weeks of age. For MMTV-neu mice and MMTV-neu; MMTV-CDC25A mice, tumors were sampled at ∼25 weeks of age. To determine percentages of cells in S and M phases, BrdUrd and phosphorylated histone H3 immunoreactivities were scored in >500 tumor cells per animal, and at least three animals per group were analyzed.

Comparative genomic hybridization microarray. Genomic DNA was isolated using DNeasy Tissue kit (Qiagen, Valencia, CA) and labeled using the BioPrime Array CGH Genomic Labeling Module (Invitrogen, Carlsbad, CA). Samples were hybridized to Mouse CGH 60-mer oligonucleotide microarrays (Agilent G4414A, Agilent Technologies, Wilmington, DE) in a dye-swap replicate configuration (MMTV-CDC25A; MMTV-neu tumor-derived cell culture versus normal mouse mammary tissue). Microarrays were washed with Agilent CGH wash buffers and scanned on an Agilent Microarray Scanner (G2565BA) as described previously. Images were analyzed using Agilent Feature Extraction (version 8.5) and data were analyzed using Agilent CGH Analytics (version 3.2) software with data averaging across a 1 Mbp window and default variables as described previously (30). Raw and normalized data were deposited in the Gene Expression Omnibus (accession GSE4767).9

Gene expression microarray. Gene expression microarray analysis was done using the materials and protocols from the Agilent Technologies two-color gene expression platform. Briefly, total RNA was isolated from snap-frozen tumor samples, linearly amplified, labeled, and hybridized to Mouse Whole Genome 60-mer oligonucleotide microarrays (G4122A) using a dye-swap replicate configuration (MMTV-CDC25A; MMTV-neu tumors versus MMTV-neu tumors) to eliminate dye bias and maximize statistical power (31). After scanning and feature extraction, duplicate spots were averaged and data were normalized (32). Raw and normalized data, along with completed protocol information, were deposited in the Gene Expression Omnibus (accession GSE4114).9 The Student's t test was done using the MultiExperiment Viewer 3.0 (Institute for Genomic Research, Rockville, MD; ref. 33).10

For clustering by chromosomal location, a “sliding window” method was used to assess regional differential expression. Briefly, for each probe, the median expression ratio was calculated for all probes within ±5 × 105 bp of the probe location. Thresholds for differential expression were chosen to exceed 98% of medians calculated based on analogous analysis with gene locations scrambled. Agilent Array CGH Analytics version 3.2 software package was used to identify chromosomal regions with aberrant gene expression. The hypergeometric Z-score was used to flag statistically differentially expressed regions using the default variables of the program and a 1 Mbp window size.

Statistical analyses. Kaplan-Meier tumor-free survival curves were compared between strains by the log-rank test analysis, and P values were determined. The significance did not change when we used the Wilcoxon test instead. Other statistical analyses were done using ANOVA and Student's t test.

Results

Transgenic expression of CDC25A results in alveolar hyperplasia in mammary glands. To examine whether ectopic expression of CDC25A results in tumorigenesis, we generated a transgenic mouse line expressing human CDC25A under the mouse mammary tumor virus (MMTV) promoter (Fig. 1A). To maximize transgenic expression, a rabbit β-globin gene fragment containing splice donor and acceptor sites (25) was inserted between the promoter and the full-length human CDC25A cDNA. Immunoblotting with extracts from mammary tissues showed that levels of CDC25A protein, as combination of transgenic and endogenous expression, were significantly higher in transgenic mice compared with those in nontransgenic control (Fig. 1B). To determine the effect of CDC25A overexpression in mammary gland development, we did whole-mount and histologic examinations on mammary glands of 8-month-old virgin transgenic mice (Fig. 1C). Transgenic mammary glands displayed hyperplastic changes, with increased numbers of alveolar buds, compared with age-matched nontransgenic tissues. H&E-stained sections confirmed hyperplastic changes in mammary epithelia, and BrdUrd incorporation assays showed that percentages of cells in the S phase were increased in transgenic mammary glands (Fig. 1D). These data suggest that increased expression of CDC25A accelerates developmental proliferation of mammary epithelial cells.

Figure 1.

Generation and characterization of MMTV-CDC25A transgenic mice. A, the transgenic construct contains the 1.54 kb MMTV long terminal repeat (MMTV-LTR) and the full-length human CDC25A cDNA. rBGpA, rat β-globin polyadenylation sequence. B, immunoblotting for CDC25A and β-actin in mammary tissues with indicated genotypes. WT, wild-type nontransgenic. C and D, hyperplastic changes in mammary glands of MMTV-CDC25A transgenic mice. Mammary tissues from mice with the indicated genotypes were examined by whole-mount analyses, H&E staining on paraffin sections, and anti-BrdUrd immunohistochemistry. Mice were injected with BrdUrd at 2 h before sacrifice. Low, (×10) magnification; High, (×40) magnification.

Figure 1.

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Transgenic expression of CDC25A cooperates with oncogenic ras or neu in mammary tumorigenesis. To determine whether CDC25A overexpression results in mammary tumorigenesis, we monitored virgin MMTV-CDC25A transgenic females over 2 years. None of 22 transgenic mice monitored developed mammary tumors, suggesting that ectopic expression of CDC25A is not sufficient for spontaneous mammary tumorigenesis. However, when crossed with MMTV-H-ras transgenic mice, which have been widely used as a mammary tumor model (26), we found that MMTV-H-ras; MMTV-CDC25A double transgenic mice developed tumors with much shorter latency than MMTV-H-ras single transgenic mice (12 versus 20 weeks; Fig. 2A). Histologic examinations showed that mammary tumors with MMTV-CDC25A transgene were more aggressive in invasion into adjacent stromal tissues (Fig. 2B). To further characterize tumor tissues, we did immunohistochemistry for the S-phase marker BrdUrd and for the mitotic marker phosphorylated histone H3 (Fig. 2C). Tumors in MMTV-H-ras; MMTV-CDC25A mice showed 1.9-fold higher percentages of cells in S phase (26.9 ± 2.4% versus 14.1 ± 1.8%; mean ± SE) and 2.7-fold higher percentages of cells in mitosis (29.6 ± 5.8% versus 10.8 ± 3.4%) compared with tumors in MMTV-H-ras single transgenic mice. These data indicate that overexpression of CDC25A facilitates tumor initiation with enhanced proliferative capability.

Figure 2.

MMTV-CDC25A transgene cooperates with MMTV-H-ras in murine tumorigenesis. A, Kaplan-Meier tumor-free survival curves of MMTV-H-ras single transgenic mice and MMTV-H-ras; MMTV-CDC25A double transgenic mice. The median time of tumor-free survival was 147 and 85 d, respectively. Log-rank test analysis showed significant difference between the two groups (P = 0.0003). B, invasive tumors with enhanced proliferation in MMTV-H-ras; MMTV-CDC25A mice. Top, H&E-stained sections with low (×10) and high (×40) magnification. Increased cells at S and M phases in tumors are shown by immunohistochemistry using anti-BrdUrd and anti–phosphorylated histone H3 (Phospho-H3) antibodies. Mice were injected with BrdUrd at 2 h before sacrifice.

Figure 2.

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We then examined the effect of the MMTV-CDC25A transgene on tumorigenesis induced by the ErbB2/neu oncogene. This well-established oncogene is amplified in human breast cancer tissues of 25% to 30% patients, and its overexpression correlates with poor prognosis (34). We crossbred MMTV-CDC25A mice with MMTV-neu transgenic mice (27) and monitored tumorigenesis. Tumor latency in MMTV-neu; MMTV-CDC25A double transgenic mice was slightly shorter than that in MMTV-neu single transgenic mice, although the difference was not statistically significant (Fig. 3A). Nonetheless, the rates of tumor growth after initial identification were remarkably higher in MMTV-neu; MMTV-CDC25A mice (Fig. 3B). Of 22 MMTV-neu; MMTV-CDC25A mice examined, 80% exhibited tumors in multiple mammary glands, whereas only 25% of MMTV-neu mice (n = 26) displayed multifocal tumors. Mammary tumors in double transgenic mice frequently invaded into adjacent stromal tissues and sometimes showed micropapillary changes (Fig. 3C), which are analogous to micropapillary ductal adenocarcinomas, an aggressive form of human breast cancer. In contrast, most tumors in MMTV-neu single transgenic mice were well-confined adenocarcinomas with less obvious peripheral involvement. Consistently with the histologic observations, cells cultured from mammary tumors in MMTV-neu; MMTV-CDC25A mice showed markedly increased ability to invade into Matrigel compared with cells from tumors in MMTV-neu mice (data not shown). Immunohistochemistry for incorporated BrdUrd and phosphorylated histone H3 showed that tumors expressing the MMTV-CDC25A transgene were highly proliferative. The percentages of cells in S phase observed in MMTV-neu; MMTV-CDC25A tumors were 2.6-fold higher than those in MMTV-neu tumors (34.3 ± 6.5% versus 13.0 ± 1.3%; mean ± SE), whereas the percentages of cells in M phases were increased by 4.0-fold in double transgenic tumors (25.5 ± 2.6% versus 6.1 ± 0.9%; Fig. 3C). Interestingly, several cells in MMTV-neu; MMTV-CDC25A tumors costained with anti-BrdUrd and anti–phosphorylated histone H3 antibodies (Fig. 3D), suggesting miscoordination of the cell cycle, possibly premature mitotic changes, as previously shown in tumors in _Chk1_-deficient mice (17). These observations suggest that CDC25A overexpression promotes development of more malignant phenotypes in _neu_-induced tumors.

Figure 3.

MMTV-CDC25A transgene promotes mammary tumor development in MMTV-neu transgenic mice. A, Kaplan-Meier curves of MMTV-neu single transgenic mice and MMTV-neu; MMTV-CDC25A double transgenic mice. The median time of tumor-free survival was 176 and 168 d, respectively. Log-rank test analysis did not show significant difference between the two groups (P = 0.58). B, accelerated growth of mammary tumors in MMTV-neu; MMTV-CDC25A mice. Tumor volumes were measured every 2 d after detection by palpation. Points, mean of three mice per group; bars, SE. C, increased proliferation in tumors from MMTV-neu; MMTV-CDC25A mice. Tumor sections were analyzed by H&E staining and immunohistochemistry for BrdUrd and phosphorylated histone H3. Low, (×10) magnification; High, (×40) magnification. D, cell cycle miscoordination in tumors of MMTV-neu; MMTV-CDC25A mice. Tumor sections were analyzed by immunofluorescent staining for BrdUrd and phosphorylated histone H3.

Figure 3.

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CDC25A overexpression destabilizes fragile chromosomal regions. To examine how ectopic CDC25A expression affects chromosomal stability, cells cultured from tumors in MMTV-neu; MMTV-CDC25A mice were analyzed by array-based comparative genomic hybridization (aCGH; Fig. 4A). Tumors at early stages, with diameters of <1 cm, were isolated from mice at 26 weeks of age. One of consistent changes in double transgenic tumor cells was homozygous deletion at the telomeric region of chromosome 4 around D2.3 (Fig. 4B). This region of the mouse chromosome 4 is homologous to human chromosome 1p31-36, in which loss of heterozygosity (LOH) has been described in breast cancer tissues (35, 36). aCGH also detected a deletion in the centromeric region of chromosome 2. Karyotyping showed that MMTV-neu; MMTV-CDC25A tumor cells were mostly tetraploid with complex abnormalities, including deletions on chromosome 4, a deletion of chromosome 13 at region D1, additional genetic material on chromosome 17 at region D, and a t(X;18)(F5;A2) translocation (Fig. 4C). In addition, aCGH and karyotyping consistently showed several changes in chromosomal numbers, such as increased copies of chromosomes 10 and 15 and decreased copies of chromosome X. In contrast, tumor cells from MMTV-neu single transgenic mice exhibited mostly normal karyotypes, except for tetraploidy, and no major changes were identified by aCGH (data not shown). A minority of those cells exhibited t(X;18) translocation according to karyotyping. To further assess biological effects of the chromosomal aberrations in CDC25A-overexpressing tumors, we did a genome-wide gene expression microarray analysis to compare RNA samples of freshly isolated tumors from MMTV-neu; MMTV-CDC25A mice and those from MMTV-neu mice. RNA samples from two mice of each group were analyzed using a dye-swap replicate design (31) for a total of four hybridizations. This identified 597 differentially expressed genes with a t test cutoff of P < 0.001 and fold change cutoff of ±1.4-fold (Supplementary Data). To correlate the changes in gene expression with chromosomal alterations, cluster analyses based on chromosomal location of each gene were done (Fig. 5A). Genes located over an ∼21 Mbp section in the telomeric region of chromosome 4 were down-regulated in tumors with the CDC25A transgene (Fig. 5B). The down-regulated region of chromosome 4 includes several genes that control cell proliferation and death, such as Jun, Casp9 (death receptor associated caspase-9), and Trp73 (the p53-related p73 tumor suppressor). In particular, marked down-regulation of Trp73 was confirmed in tumors from five other double transgenic mice by quantitative reverse transcription-PCR (RT-PCR; Fig. 5C). These data suggest that CDC25A overexpression destabilizes fragile chromosomal regions, such as the telomeric end of chromosome 4, which could play an important role in altered gene expression associated with tumor initiation and progression.

Figure 4.

Transgenic expression of CDC25A results in chromosomal instability in MMTV-_neu_–induced tumors. A, aCGH using cell cultures derived from mammary tumors in MMTV-neu; MMTV-CDC25A mice. Representative data from analyses of two independent animals. Arrows, major changes in copy numbers. B, homozygous deletion of chromosome 4 around the D2.3 region and amplification of a short adjacent region detected in MMTV-neu; MMTV-CDC25A mammary tumor cells. C, karyotyping analyses of mammary tumor cells from MMTV-neu mice and those from MMTV-CDC25A; MMTV-neu mice. Cells were prepared and cultured from tumors, and metaphase spreads were analyzed by karyotyping as described in Materials and Methods. Left, apparently normal karyotype observed in cells from a MMTV-neu tumor. A diploid spread is shown, whereas several near-normal tetraploid spreads were also observed. Right, abnormal karyotype observed in all cells from a MMTV-CDC25A; MMTV-neu tumor. The tetraploid karyotype includes deletions on chromosome 4 at region D1; a deletion of chromosome 13 at region D1; additional material on chromosome 17 at region D; a t(X;18)(F5;A2) translocation; decreased copies of chromosomes 2, 3, 8, 9, 12, 16, 18, 19, and X; and increased copies of chromosomes 10 and 15 as well as marker chromosomes.

Figure 4.

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Figure 5.

Mammary tumors in MMTV-neu; MMTV-CDC25A mice exhibit down-regulation of genes in a telomeric region of chromosome 4. A, gene expression microarray data showing down-regulation of genes within the telomeric region of chromosome 4 in tumor tissues from MMTV-neu; MMTV-CDC25A mice compared with those from MMTV-neu mice. Shaded curves, hypergeometric Z-score representing the extent of differential expression within a 1 Mbp sliding window. Negative and positive values denote lower and higher expression in MMTV-neu; MMTV-CDC25A double transgenic mice, respectively. B, hypergeometric Z-score across chromosome 4. C, expression levels of Trp73 in mammary tumors from five individual MMTV-neu; MMTV-CDC25A mice (black columns) relative to those from MMTV-neu mice (white column). Columns, mean (n = 5); bars, SE. The level of each transcript was determined by quantitative RT-PCR.

Figure 5.

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Discussion

The present study is the first in vivo study to establish the causal relationship between CDC25A overexpression and oncogenesis. The cooperation of the MMTV-CDC25A transgene with MMTV-H-ras or MMTV-neu verifies tumor-promoting action of CDC25A in mice. It was reported that ∼50% of T1a,b breast cancer tissues exhibited CDC25A overexpression, which correlated with poor survival (24). Our data on faster tumor growth and more invasive characteristics rendered by the MMTV-CDC25A transgene suggest that CDC25A plays a critical role in malignant phenotypes of human breast cancer. The gene expression microarray showed that _neu_-induced tumors with the MMTV-CDC25A transgene had increased expression of multiple genes that can promote oncogenesis, including Ccnd2 (cyclin D2), Nek1 (NIMA), and Elf4 (an ETS transcription factor also known as MEF; see Supplementary Data). The aggressiveness of CDC25A-overexpressing tumors could result from accumulation of genetic alterations associated with chromosomal instability. The expression microarray and aCGH analyses have revealed that CDC25A overexpression promotes the loss of genes on the telomeric region of chromosome 4. The region of mouse chromosome 4 is orthologous with human 1p31-36, on which LOH and deletions have been described in breast cancers (35–37). Similar genomic imbalances have been previously described in tumors from MMTV-neu mice at late stages (38). Therefore, we hypothesize that rapid proliferation induced by the oncogenic neu-ras signaling pathway makes chromosomal fragile sites (e.g., mouse chromosome 4 or human chromosome 1p) susceptible to DNA damage and impaired checkpoint function with CDC25A overexpression facilitates clonal expansion of cells with allelic imbalance. Interestingly, the chromosomal region contains several genes with possible tumor-suppressive function, such as Trp73 and Casp9, whose expression levels are down-regulated in tumors overexpressing CDC25A. Further investigations are necessary to determine how changes in these genes with CDC25A deregulation affect progression of mammary tumorigenesis.

CDC25A has attracted much attention as a major target of CHK1-mediated response to DNA damage (8, 39, 40). Whereas Chk1 −/− mice are embryonic lethal, Chk1 +/− mice exhibit increased susceptibility to _Wnt_-induced tumorigenesis (16). Chk1 +/− mammary epithelial cells express high levels of CDC25A (17). The increased tumorigenesis in Chk1 +/− mice could depend largely on stabilization of CDC25A protein. Although the mechanisms of CDC25A overexpression in human cancers are not well understood, protein stabilization may play a key role in many cases (41). The present study provides evidence that increased CDC25A expression in vivo leads to genetic alterations in fragile chromosomal loci in _neu_-induced mammary tumors. Forced expression of CDC25A in cultured cell lines can induce premature mitotic changes and DNA damage (1, 42). Thus, maintenance of proper CDC25A levels is important for genomic stability and tumor suppression. One of the critical downstream targets of CDC25A is cyclin E/CDK2, and knock-in mice with highly stable cyclin E (T393A) exhibit genomic instability and greatly increased susceptibility to _ras_-induced lung tumorigenesis (43). Deregulated CDC25A expression can result in ectopic activation of cyclin E/CDK2 (44), which allows cells to override S-phase checkpoint and subsequently leads to genomic instability. Cyclin B/CDK1 may also be activated by CDC25A overexpression (9), leading to perturbed G2-M checkpoint. Better understanding of CHK1-dependent and CHK1-independent pathways that control CDC25A levels is necessary for more effective therapeutic options. MMTV-CDC25A mice are useful tools not only for elucidating the role of cell cycle checkpoint in tumor suppression but also as platforms for testing therapeutic strategies.

This article is dedicated to late Robert H. Costa, who encouraged us continuously even during his own fight against cancer.

Acknowledgments

Grant support: NIH grants R01-CA100204, CA112282, and HD38085 (H. Kiyokawa); CA105352 (F.J. DeMayo and S.Y. Tsai); and HL076448 (S.Y. Tsai); Department of Defense grant DAMD 17-02-1-0413 (H. Kiyokawa); National Science Foundation (P.G. Fuhrken); Searle Leadership Fund (H. Kiyokawa); and Robert H. Lurie Comprehensive Cancer Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Suchitra Prasad, Alba Santana, Lida Aris, Kimberly Hansel, Anne Shilkaitis, Jadwiga Labanowska, the transgenic facilities of the University of Illinois at Chicago and Baylor College of Medicine, the Cytogenetics Core Facility at Ohio State University, and the Center for Women's Health and Reproduction (U54 HD40093) for technical assistance and resources; Dario Sepulveda for technical assistance with the microarray experiments, including RNA extractions; Alfred Rademaker (Biostatistics Core, Robert H. Lurie Comprehensive Cancer Center, Chicago, IL) for statistical analyses; and Hidayatullah Munshi, Nissim Hay, Siwanon Jirawatnotai, Evan Osmundson, and Stephen Sener for helpful suggestions, discussions, and inspiration.

References

Bartkova J, Horejsi Z, Koed K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.

Nature

2005

;

434

864

–70.

Sherr CJ. The Pezcoller lecture: cancer cell cycles revisited.

Cancer Res

2000

;

3689

–95.

Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD. Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage.

Proc Natl Acad Sci U S A

1999

;

13777

–82.

Hirao A, Kong YY, Matsuoka S, et al. DNA damage-induced activation of p53 by the checkpoint kinase Chk2.

Science

2000

;

287

1824

–7.

Shieh SY, Ahn J, Tamai K, Taya Y, Prives C. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.

Genes Dev

2000

;

289

–300.

Donzelli M, Draetta GF. Regulating mammalian checkpoints through Cdc25 inactivation.

EMBO Rep

2003

;

671

–7.

Kallstrom H, Lindqvist A, Pospisil V, Lundgren A, Rosenthal CK. Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import.

Exp Cell Res

2005

;

303

–100.

Zhao H, Watkins JL, Piwnica-Worms H. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints.

Proc Natl Acad Sci U S A

2002

;

14795

–800.

Molinari M, Mercurio C, Dominguez J, Goubin F, Draetta GF. Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis.

EMBO Rep

2000

;

–9.

Busino L, Donzelli M, Chiesa M, et al. Degradation of Cdc25A by β-TrCP during S phase and in response to DNA damage.

Nature

2003

;

426

–91.

Jin J, Shirogane T, Xu L, et al. SCFβ-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase.

Genes Dev

2003

;

3062

–74.

Savitsky K, Sfez S, Tagle DA, et al. The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species.

Hum Mol Genet

1995

;

2025

–32.

Barlow C, Hirotsune S, Paylor R, et al. Atm-deficient mice: a paradigm of ataxia telangiectasia.

Cell

1996

;

159

–71.

Brown EJ, Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality.

Genes Dev

2000

;

397

–402.

Fang Y, Tsao CC, Goodman BK, et al. ATR functions as a gene dosage-dependent tumor suppressor on a mismatch repair-deficient background.

EMBO J

2004

;

3164

–74.

Liu Q, Guntuku S, Cui XS, et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.

Genes Dev

2000

;

1448

–59.

Lam MH, Liu Q, Elledge SJ, Rosen JM. Chk1 is haploinsufficient for multiple functions critical to tumor suppression.

Cancer Cell

2004

;

–59.

Gasparotto D, Maestro R, Piccinin S, et al. Overexpression of CDC25A and CDC25B in head and neck cancers.

Cancer Res

1997

;

2366

–8.

Xu X, Yamamoto H, Sakon M, et al. Overexpression of CDC25A phosphatase is associated with hypergrowth activity and poor prognosis of human hepatocellular carcinomas.

Clin Cancer Res

2003

;

1764

–72.

Ito Y, Yoshida H, Nakano K, et al. Expression of cdc25A and cdc25B proteins in thyroid neoplasms.

Br J Cancer

2002

;

1909

–13.

Broggini M, Buraggi G, Brenna A, et al. Cell cycle-related phosphatases CDC25A and B expression correlates with survival in ovarian cancer patients.

Anticancer Res

2000

;

4835

–40.

Wu W, Fan YH, Kemp BL, Walsh G, Mao L. Overexpression of cdc25A and cdc25B is frequent in primary non-small cell lung cancer but is not associated with overexpression of c-myc.

Cancer Res

1998

;

4082

–5.

Hernandez S, Hernandez L, Bea S, et al. cdc25 cell cycle-activating phosphatases and c-myc expression in human non-Hodgkin's lymphomas.

Cancer Res

1998

;

1762

–7.

Cangi MG, Cukor B, Soung P, et al. Role of the Cdc25A phosphatase in human breast cancer.

J Clin Invest

2000

;

106

753

–61.

Ma ZQ, Chua SS, DeMayo FJ, Tsai SY. Induction of mammary gland hyperplasia in transgenic mice over-expressing human cdc25B.

Oncogene

1999

;

4564

–76.

Sinn E, Muller W, Pattengale P, Tepler I, Wallace R, Leder P. Coexpression of MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo.

Cell

1987

;

465

–75.

Muller WJ, Sinn E, Pattengale PK, Wallace R, Leder P. Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene.

Cell

1988

;

105

–15.

Tsutsui T, Hesabi B, Moons DS, et al. Targeted disruption of CDK4 delays cell cycle entry with enhanced p27Kip1 activity.

Mol Cell Biol

1999

;

7011

–9.

Kiyokawa H, Kineman RD, Manova-Todorova KO, et al. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1).

Cell

1996

;

721

–32.

Brennan C, Zhang Y, Leo C, et al. High-resolution global profiling of genomic alterations with long oligonucleotide microarray.

Cancer Res

2004

;

4744

–8.

Churchill GA. Fundamentals of experimental design for cDNA microarrays.

Nat Genet

2002

;

Suppl:

490

–5.

Yang H, Haddad H, Tomas C, Alsaker K, Papoutsakis ET. A segmental nearest neighbor normalization and gene identification method gives superior results for DNA-array analysis.

Proc Natl Acad Sci U S A

2003

;

100

1122

–7.

Saeed AI, Sharov V, White J, et al. TM4: a free, open-source system for microarray data management and analysis.

Biotechniques

2003

;

374

–8.

Zhou BP, Hung MC. Dysregulation of cellular signaling by HER2/neu in breast cancer.

Semin Oncol

2003

;

–48.

Bieche I, Champeme MH, Matifas F, Cropp CS, Callahan R, Lidereau R. Two distinct regions involved in 1p deletion in human primary breast cancer.

Cancer Res

1993

;

1990

–4.

Millikan RC, Ingles SA, Diep AT, et al. Linkage analysis and loss of heterozygosity for chromosome arm 1p in familial breast cancer.

Genes Chromosomes Cancer

1999

;

354

–61.

Ragnarsson G, Eiriksdottir G, Johannsdottir JT, Jonasson JG, Egilsson V, Ingvarsson S. Loss of heterozygosity at chromosome 1p in different solid human tumours: association with survival.

Br J Cancer

1999

;

1468

–74.

Montagna C, Andrechek ER, Padilla-Nash H, Muller WJ, Ried T. Centrosome abnormalities, recurring deletions of chromosome 4, and genomic amplification of HER2/neu define mouse mammary gland adenocarcinomas induced by mutant HER2/neu.

Oncogene

2002

;

890

–8.

Chen MS, Ryan CE, Piwnica-Worms H. Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding.

Mol Cell Biol

2003

;

7488

–97.

Sorensen CS, Syljuasen RG, Falck J, et al. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A.

Cancer Cell

2003

;

247

–58.

Loffler H, Syljuasen RG, Bartkova J, Worm J, Lukas J, Bartek J. Distinct modes of deregulation of the proto-oncogenic Cdc25A phosphatase in human breast cancer cell lines.

Oncogene

2003

;

8063

–71.

Spruck CH, Won KA, Reed SI. Deregulated cyclin E induces chromosome instability.

Nature

1999

;

401

297

–300.

Loeb KR, Kostner H, Firpo E, et al. A mouse model for cyclin E-dependent genetic instability and tumorigenesis.

Cancer Cell

2005

;

–47.

Blomberg I, Hoffmann I. Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases.

Mol Cell Biol

1999

;

6183

–94.

2007

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Deregulated CDC25A Expression Promotes Mammary Tumorigenesis with Genomic Instability (original) (raw)

Abstract

Introduction

Materials and Methods

Results

Discussion

Acknowledgments

References

Supplementary data

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