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[Cancer Research 66, 8448-8454, September 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Gadd45a Suppresses Ras-Driven Mammary Tumorigenesis by Activation of c-Jun NH2-Terminal Kinase and p38 Stress Signaling Resulting in Apoptosis and Senescence

Jennifer S. Tront1, Barbara Hoffman1,2 and Dan A. Liebermann1,2

1 Fels Institute for Cancer Research and Molecular Biology and 2 Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania

Requests for reprints: Dan A. Liebermann, Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, PA 19140. Phone: 215-707-6903; Fax: 215-707-2805; E-mail: lieberma{at}temple.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Gadd45 family of proteins is known to play a central role as cellular stress sensors that modulate the response of mammalian cells to stress inflicted by physiologic and environmental stressors. Gadd45a was shown to be a direct target to the p53 and BRCA1 tumor suppressor genes, whose loss of function is known to play a vital role in breast carcinogenesis; however, the role of Gadd45a in the suppression of breast cancer remains unclear. To address this issue, Gadd45a-deficient mice were crossed with breast cancer prone mouse mammary tumor virus–Ras mice to generate mice that express activated Ras and differ in their Gadd45a status. Using this mouse model, we show that the loss of Gadd45a accelerates Ras-driven mammary tumor formation, exhibiting increased growth rates and a more aggressive histologic phenotype. Moreover, it is shown that accelerated Ras-driven tumor formation in the absence of Gadd45a results in both a decrease in apoptosis, which is linked to a decrease in c-Jun NH2-terminal kinase (JNK) activation, and a decrease in Ras-induced senescence, which is correlated with a decrease in p38 kinase activation. Altogether, these results provide a novel model for the tumor-suppressive function of Gadd45a in the context of Ras-driven breast carcinogenesis, showing that Gadd45a elicits its function through activation of the stress-induced JNK and p38 kinases, which contribute to increase in apoptosis and Ras-induced senescence. (Cancer Res 2006; 66(17): 8448-54)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of breast cancer is a multistage process. Mutations and overexpression of proto-oncogenes, such as Ras, are known to cooperate with mutations or deletions of growth suppressor genes, such as p53 and BRCA1, in the development of breast cancer (1–4). The Ras oncogenes harbor point mutations, leading to an amino acid substitution at positions 12, 13, 59, and 61, which confer transforming activity in various human cancers. Activating Ras mutations are found in human malignancies with an overall frequency of 15% to 20% and are found in 10% to 12% of breast carcinomas (5, 6).

The Gadd45 family of genes (growth arrest and DNA damage) plays an important role in cell cycle control, survival, and apoptosis. There is evidence that the proteins encoded by these genes play a pivotal role as stress sensors that modulate the cellular response to a variety of stressors (7, 8). Gadd45a is one member of the three-member family that is regulated by important tumor suppressor proteins, such as p53 (9) and BRCA1 (10). Gadd45a is also known to interact with key cell regulators, such as p21 (11), cdc2/cyclin B1 (12), proliferating cell nuclear antigen (9), p38 (13), and mitogen-activated protein (MAP) three kinase 1 (MTK1)/MAP kinase (MAPK) kinase kinase (MEKK) 4 (14, 15). The cellular function of Gadd45a is dependent on its interacting partner. For example, physical interaction between Gadd45a and MTK1/MEKK4 results in the activation of c-Jun NH2-terminal kinase (JNK), which can lead to cell growth inhibition or apoptosis (14, 15). Additionally, the physical interaction between Gadd45a and the MAPK p38 may play a pivotal role in preventing oncogene-induced growth in part by regulating p53 tumor suppression (13). Gadd45a-null mice were found to display increased susceptibility to radiation-induced carcinogenesis (16). Therefore, it was of interest to investigate the role that Gadd45a may play in breast tumorigenesis and to examine possible cooperation between oncogenic Ras and loss of Gadd45a in breast tumor formation and progression.

To achieve this goal, we have taken advantage of the established breast cancer prone mouse mammary tumor virus (MMTV)-v-Ras transgenic mice, where the v-Ha-Ras oncogene, which contains an activation mutation in codon 12 (Gly to Arg) and 59 (Ala to Thr), is under the control of the MMTV promoter (17). Gadd45a-deficient mice and MMTV-v-Ras transgenic mice were interbred to generate mice that express the oncogenic Ras transgene and differ in their Gadd45a status. Using these mice, we show that Gadd45a deficiency significantly accelerates the onset of breast tumorigenesis. These tumors exhibit increased growth rates and a more aggressive histologic phenotype compared with their Gadd45a wild-type (WT) counterparts. The increased growth rate of Ras-driven breast tumors lacking Gadd45a can be accounted for by an increase in the fraction of cells progressing through the cell cycle, which may be due to the observed decreases in both apoptosis and oncogene-induced senescence (OIS). Mechanistically, it is shown that the decrease in apoptosis associated with loss of Gadd45a is linked to a decrease in JNK activation and abrogation of Ras-induced senescence is linked to a decresase in p38 activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. MMTV-Ras transgenic mice in an inbred FVB genetic background were originally obtained from Charles River Laboratories (Wilmington, MA). Gadd45a–/– mice (in a C57BL/6 x 129Sv background) were graciously provided by Albert Fornace (Harvard University, Boston, MA). Offspring from interbreeding Gadd45a–/– and MMTV-Ras mice were generated as littermates from common matings so that all animals were maintained in a mixed genetic background. Offspring from crosses between MMTV-Ras and Gadd45a–/– mice were screened by PCR for their Ras and Gadd45a status. At the time of weaning, a small piece of tail was cut from each animal that was then used to isolate genomic DNA by standard procedures for PCR analysis. Primers for the detection of MMTV-Ras were 5'-GAGGCAGGGACCAGCAAGACATC-3' (5' sense) and 5'-ACAGACCCTGAACCACGCATCAAC-3' (3' antisense). To determine the Gadd45a status, PCRs using three primers allowed for simultaneous detection of the WT and mutant Gadd45a allele. These primers consisted of a 5' upstream primer (5'-CACCTCTGCTTACCTCTGCACAAC-3'), a common 3' downstream primer (5'-CCAGAAGACCTAGACAGCACGGTT-3'), and a neo-specific primer (5'-AAGCGCATGCTCCAGACTGCCTT-3'). Reactions were run for 37 cycles of 94°C for 1 minute, 63°C for 14 seconds, and 72° for 12 seconds.

Tumor formation and onset. At 4 weeks of age, female mice from all genotypes were observed twice weekly for the formation of visible tumor masses. On detection of a mass, the tumor growth properties were monitored every other day for ~14 days or until the general health of the animal was compromised, at which time the mouse was sacrificed according to standard protocols. Tumor measurements were taken with hand calipers to evaluate tumor volume [calculated tumor volume (mm3) = W2 x L, where W is width and L is length]. Tumor growth curves were generated by plotting the average daily tumor growth against time. After 14 days, the animal was sacrificed and the tumor was collected in accordance with Temple University (Philadelphia, PA) and NIH guidelines.

Tumor onset was plotted using a Kaplan-Meier survival curve. Differences between Kaplan-Meier curves were determined using a Mantel-Cox log-rank statistical test. Differences in tumor incidence were determined by the {chi}2 test.

Histologic evaluation. Tumor samples were fixed in 10% buffered formalin and then embedded in paraffin for sectioning. Several sections were then stained with H&E to examine histologic differences (University of Pennsylvania Core Histology Facility, Philadelphia, PA).

Apoptosis. Tumor tissue sections, fixed in 10% buffered formalin and then embedded in paraffin, were in situ labeled for apoptotic cells using the ApoAlert DNA fragmentation Assay kit (BD Biosciences, Franklin Lakes, NJ). Cells were analyzed by light microscopy. Necrotic regions of the tumor were avoided. Using a 10 x 450 field range, the number of terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)–positive stained cells and the total number of propidium iodide (PI)–stained cells was determined with Image J photo program. A minimum of five samples per genotype were analyzed. Differences in percentage apoptosis between different genotypes were evaluated using the Student's t test.

Flow cytometry. At the time of sacrifice, a 25- to 50-mg section of tumor tissue was minced and treated with dispase (0.6 units/mL; Invitrogen, Carlsbad, CA) and collagenase I (100 units/mL; Invitrogen) for 1 hour at 37° with slight agitation. Following incubation, the sample was passed through a cell strainer and washed with PBS. The sample was then treated with RBC lysis buffer (Cambrex, East Rutherford, NJ) to eliminate RBCs from the culture. The tumor cells were fixed with ethanol and then stained with PI (12). A minimum of five samples from each genotype were analyzed. Statistical significance was determined using a Student's t test.

Immunohistochemistry and immunofluorescence. Paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to antigen unmasking by sodium citrate (10 mmol/L; pH 6.0) for 30 minutes at a sub-boiling temperature. (For immunohistochemistry only: endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 minutes). Sections were then blocked with 5% serum for 1 hour at room temperature followed by incubation with primary antibody overnight at 4°C [phosphorylated JNK (Cell Signaling Technology, Danvers, MA), phosphorylated p38 immunohistochemistry preferred (Cell Signaling Technology), and ß-galactosidase (Abcam, Cambridge, MA)]. For immunohistochemistry, sections were then incubated with a peroxidase-conjugated secondary antibody for 30 minutes at room temperature followed by treatment with avidin-biotin complex method reagent (Vector Laboratories, Burlingame, CA) for 30 minutes. Sections were stained with 3,3'-diaminobenzidine substrate and counterstained with hematoxylin (Vector Laboratories, Burlingame, CA). For immunofluorescence, sections were incubated with either fluorescein- or Texas red–conjugated secondary antibody (TI-1000; Vector Laboratories) for 2 hours at room temperature in the dark and mounted for viewing. For single-staining experiments, samples were stained using fluorescein secondary antibody followed by incubation with PI stain for 20 minutes at room temperature. Using a 10 x 450 field range, the number of positive-stained cells and the total number of cells were determined with Image J photo program for both immunohistochemistry and immunofluorescence samples. A minimum of five samples from each genotype were analyzed for each analysis. Differences between genotypes were evaluated using the Student's t test. For the Annexin V and phosphorylated JNK double staining, the samples were stained with phosphorylated JNK primary antibody and Texas red–conjugated secondary antibody as described above immediately followed by the Annexin V protocol as described above. For the phosphorylated p38 and ß-galactosidase double staining, both antibodies were mixed at equal concentrations and incubated overnight at 4°C. Fluorescein-conjugated anti-rabbit secondary antibody was used to detect phosphorylated 38. Texas red–conjugated anti-mouse secondary antibody was used to detect ß-galactosidase. Images were acquired and merged through Spot Imaging software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gadd45a is up-regulated during Ras-driven breast carcinogenesis. Our working hypothesis was that Gadd45a is a stress sensor protein, which is up-regulated by oncogenic stress during breast carcinogenesis and functions to modulate tumor development. To assess the validity of our hypothesis, Gadd45a expression was examined in normal and tumor mammary tissue obtained from the three genotypes (Ras/Gadd45a+/+, Ras/Gadd45a+/–, and Ras/Gadd45–/–). For comparison, Gadd45a expression was also assessed in nonmammary tissue (i.e., spleen tissue).

Gadd45a expression was undetectable in normal mammary and spleen tissue obtained from all three genotypes. In comparison, detectable levels of Gadd45a were observed in breast tumor tissue obtained from Ras/Gadd45a+/+ and Ras/Gadd45+/– mice but not Ras/Gadd45a–/–. The highest level of Gadd45a expression was observed in Ras/Gadd45a+/+ tumors with an intermediate level of expression in the heterozygote tissue. Taken together, these data support the hypothesis that Gadd45a expression is up-regulated during breast carcinogenesis.

Gadd45a deficiency results in accelerated mammary tumorigenesis in MMTV-Ras mice. To assess the effect of Gadd45a deficiency on breast carcinogenesis, Gadd45a-deficient mice were crossed with mammary tumor prone MMTV-Ras transgenic mice to generate animals that carried oncogenic Ras and differed in their Gadd45a status (Ras/Gadd45a–/–, Ras/Gadd45a+/–, and Ras/Gadd45a+/+). Female animals from each genotype were monitored twice weekly for the formation of tumors. It was observed that tumorigenesis is accelerated in Ras/Gadd45a–/– mice when compared with Ras/Gadd45a+/+ mice (P < 0.005; Fig. 1A ). The median tumor onset, measured as the time, in which 50% of animals develop tumors, was 5 months for Ras/Gadd45a–/– mice, whereas the tumor onset for Ras/Gadd45a+/+ mice was 8 months. Ras/Gadd45a+/– mice had an intermediate median tumor onset of 7 months. The loss of Gadd45a increased overall tumor incidence, from 74% of Ras/Gadd45a+/+ mice developing tumors within 12 months to 94% of Ras/Gadd45a–/– mice developing tumors within the same time frame. Again, Ras/Gadd45a+/– mice had an intermediate incidence of 92%.


Figure 1
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  		Figure 1. Gadd45a deficiency results in accelerated mammary tumorigenesis in MMTV-Ras mice. A, Kaplan-Meier curve showing the proportion of tumor-free mice as a function of time for Ras/Gadd45a–/– (n = 26), Ras/Gadd45a+/– (n = 25), and Ras/Gadd45a+/+ (n = 25; P < 0.005). B, average tumor growth rate for tumors arising in Ras/Gadd45a–/– (n = 8), Ras/Gadd45a+/– (n = 7), and Ras/Gadd45a+/+ (n = 6) mice. Tumor growth was monitored every other day for 13 days by caliper measurement.

 
To determine if the loss of Gadd45a contributed to an increase in the rate of tumor growth, tumor volume was determined every 2 days by caliper measurements, starting on first tumor visualization and continuing for ~2 weeks or until the general well being of the animal was compromised. Mammary tumors arising from Ras expressing Gadd45a-deficient mice displayed significantly increased rates of tumor growth compared with those mice that express Gadd45a (P < 0.05; Fig. 1B). For example, at 14 days, the average Ras+/Gadd45a–/– tumor had a volume of 4,809 ± 1,373 mm3 (n = 8), whereas the Gadd45a-expressing tumors had an average tumor volume of 2,684 ± 791 mm3 (n = 6). Once again, Gadd45a+/– mice displayed an intermediate phenotype.

Gadd45a-deficient tumors display a more aggressive histologic phenotype. Tumor sections from the three genotypes were analyzed to determine if the loss of Gadd45a contributes to histologic changes within the mammary tumors, which correlate with tumor aggressiveness. Higher histologic tumor grades are associated with a loss of cellular shape and size uniformity, increased nuclear size, hyperchromatic nuclei, and presence of multinucleated cells. For grading, nuclear size and shape, nuclear/cytoplasmic ratio, presence of hyperchromatic nuclei, and presence of multinucleated cells were evaluated and incorporated into a final score of 0 to 4, with 4 representing the highest grade. As shown in Fig. 2A and summarized in Fig. 2B, the loss of Gadd45a is associated with a higher histologic grade compared with WT controls. All of the Ras/Gadd45–/– samples had a grade range of 2 to 3, whereas the Ras/Gadd45+/+ samples had a grade range of 1 to 2 (n = 12 and 10, respectively). Ras expressing Gadd45a-deficient tissue samples displayed a loss of cellular uniformity as well as multinucleated cells and the presence of hyperchromatic nuclei, which were not seen in the WT samples. Interestingly, the Ras/Gadd45a+/– tumor samples displayed elevated levels of fibrosis, which results in an overproduction of connective tissue by fibroblast cells due to a stress response. This was not observed in either of the other genotypes. Further investigation into this phenomenon is planned.


Figure 2
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  		Figure 2. Gadd45a-deficient tumors display a more aggressive histologic phenotype. A, histologic characteristics of formalin-fixed, paraffin-embedded tumor tissue arising from Ras expressing Gadd45a+/+, Gadd45a+/–, and Gadd45a–/–, respectively (magnification, x40). Ras+/Gadd45a–/– sample displaying hyperchromatic nuclei and a multinucleated cell at x100 magnification. B, ten formalin-fixed, paraffin-embedded tumors from each genotype were examined for histologic characteristics. +, if all 10 samples displayed the individual characteristic; ++, if all 10 samples displayed multiple occurrences of the characteristic.

 
Loss of Gadd45a results in an increase in the percentage of breast tumor cells in the S phase of the cell cycle and decrease in cells in G0-G1. Loss of Gadd45a was observed to accelerate tumor development and growth (Fig. 1). Therefore, it was asked if the increased growth of Ras-driven breast tumors lacking Gadd45a is associated with acceleration in cell cycle progression. All mammary tumor samples, regardless of Gadd45a status, displayed multiple aneuploidy peaks, indicative of compromised genomic integrity (Fig. 3A ). To examine cell cycle properties, we chose, however, to focus our investigation on the diploid population of cells, which comprised >90% of the cell population for all samples. Data obtained show that 13% of Ras tumor cells expressing Gadd45a are in the S phase of the cell cycle, where the loss of Gadd45a resulted in a 2.5-fold increase in the percentage of cells in the S-phase fraction (i.e., 36%; Fig. 3B). To correlate the increased percentage of cells in the S phase to the increased tumor growth rates in the Ras/Gadd45a–/– tumors, we also did Ki67 immunohistochemistry staining to confirm increased proliferation rates. We found an increase in Ki67 staining in the Ras/Gadd45a–/– samples compared with the Ras/Gadd45a+/+ control (data not shown).


Figure 3
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  		Figure 3. Loss of Gadd45a results in an increase in the percentage of breast tumor cells in the S phase of the cell cycle and decrease in cells in G0-G1. A, histogram from a representative PI-stained tumor, showing a diploid peak as well as multiple aneuploidy peaks from a Ras/Gadd45a+/+ sample. B, flow cytometric analysis of PI-stained tumor cells, showing averages for tumors with different genotypes. G0-G1, S, and G2-M fractions were 81 ± 5.1%, 13 ± 1.3%, and 6 ± 0.9% for Ras+/Gadd45a+/+ (n = 8); 73 ± 3.5%, 24 ± 2.1%, and 3 ± 0.4% for Ras+/Gadd45a+/– (n = 12); 61 ± 2.9%, 36 ± 2.0%, and 3 ± 0.5% for Ras+/Gadd45a–/– (n = 14).

 
Concomitantly, it can be seen that loss of Gadd45a also resulted in a significant decrease in cells in the G0-G1 phase of the cell cycle. Taken together, these data show that the increased growth rate of Ras/Gadd45a–/– tumors compared with Ras/Gadd45a+/+ tumors is due in part to an increase in the fraction of cells that progress through the cell cycle.

Gadd45a-deficient breast tumors display a decreased level of apoptosis, which is associated with impaired JNK activation. Acceleration of tumor development as a consequence of Gadd45a deficiency may also reflect a decrease in apoptosis of breast cancer cells. Gadd45a has been implicated in programmed cell death via activation of the stress-induced JNK kinase (15, 18). Thus, it was of interest to assess how the loss of Gadd45a affects apoptosis of Ras-driven breast tumor cells and if this involved failure to activate JNK.

To determine the number of tumor cells undergoing apoptosis, TUNEL analysis was done on formalin-fixed, paraffin-embedded tumor tissue samples from each of the genotypes (Fig. 4B ). The loss of Gadd45a was observed to significantly decrease total tumor cell apoptosis >4.5-fold. To understand mechanistically how Gadd45a deficiency results in decreased apoptosis, we explored the activation status of JNK in Ras-driven breast tumor cells, which are either WT or null for Gadd45a. As shown by both Western blotting (Fig. 4A) and immunohistochemistry (Fig. 4C), the level of phosphorylated JNK, indicative of activated JNK, and the percentage of cells expressing phosphorylated JNK was significantly lower (P ≤ 0.05) in Ras/Gadd45a–/– tumor cells compared with Ras/Gadd45+/+ cells. Ras/Gadd45a+/– tumor cells displayed an intermediate expression level (data not shown). There was no detectable phosphorylated JNK in normal mammary and spleen tissue (data not shown).


Figure 4
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  		Figure 4. Gadd45a-deficient breast tumors display a decreased level of apoptosis, which is associated with impaired JNK activation. A, Western blot analysis for phosphorylated JNK using representative samples from each genotype. Total JNK levels were analyzed as a control. B, quantitation of percentage apoptotic cells of each genotype determined by TUNEL analysis on formalin-fixed, paraffin-embedded tissue sections. Positive cells were visualized by light microscopy and quantitated as described in Materials and Methods. (Ras+/Gadd45a+/+, n = 8; Ras+/Gadd45a–/–, n = 14). C, average percentage of phosphorylated JNK-positive cells from Ras+/Gadd45a+/+ (n = 5) and Ras+/Gadd45a–/– (n = 6). D, formalin-fixed, paraffin-embedded tumor tissue sections were concomitantly analyzed for levels of apoptosis (by TUNEL analysis) and phosphorylated JNK (by immunofluorescence), Ras+/Gadd45a+/+; Ras+/Gadd45a–/–. Representative tumor sections are for TUNEL staining, phosphorylated JNK staining, and the merged images.

 
To examine if apoptosis is directly correlated with JNK activation, we did double-immunofluorescence staining (Fig. 4D). By merging the immunofluorescent images, it is shown that tumor cells undergoing apoptosis do indeed express activated JNK. The fact that some cells positive for phosphorylated JNK were not positive for TUNEL staining raises the possibility that JNK activation may be necessary but not sufficient for the onset of apoptosis; alternatively, these cells may represent cells at early stages of apoptosis where DNA degradation may not yet have taken place. Similar analysis has shown that apoptotic cells do not express activated p38 (data not shown). Taken together, these data imply that Gadd45a-mediated activation of JNK, which in turn results in apoptosis of tumor cells, contributes to the tumor-suppressive function of Gadd45a in Ras-driven breast carcinogenesis.

Gadd45a deficiency results in a decrease in Ras-induced senescence, which correlates with impaired p38 activation in breast cancer cells. Recently, it has become evident that cellular senescence, first discovered in cell culture, is in fact a vital mechanism that constrains tumor development in vivo (19–21). Furthermore, Gadd45a-mediated activation of p38 MAPK has been implicated in H-Ras-induced cell cycle arrest in mouse embryo fibroblasts (13). Clearly, it was of interest to explore whether the tumor-suppressive function of Gadd45a on Ras-driven breast carcinogenesis is in part due to senescence resulting from Gadd45a-mediated p38 activation.

To test this hypothesis, tumors from Ras/Gadd45a+/+ and Ras/Gadd45a–/– mice were compared for the relative number of senescent cells using immunohistochemistry to assess the expression of the senescence marker ß-galactosidase. As shown in Fig. 5B , there was a significant decrease (P ≤ 0.05) in the percentage of breast tumor cells that expressed ß-galactosidase in the Ras/Gadd45a–/– mice compared with tumors obtained from the Ras/Gadd45a+/+ mice.


Figure 5
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  		Figure 5. Gadd45a deficiency results in a decrease in Ras-induced senescence, which correlates with impaired p38 activation in breast cancer cells. A, Western blot analysis for phosphorylated p38 using representative samples from each genotype. Total p38 levels were analyzed as a control. B and C, average percentage of ß-galactosidase-positive (B) and phosphorylated p38-positive (C) cells from Ras+/Gadd45a+/+ (n = 5) and Ras+/Gadd45a–/– (n = 5). D, formalin-fixed, paraffin-embedded tumor tissue sections were analyzed by immunofluorescence for levels of ß-galactosidase and phosphorylated p38 concomitantly (Ras+/Gadd45a+/+ and Ras+/Gadd45a–/–). Representative tumor tissue sections are for ß-galactosidase, phosphorylated p38, and the merged images.

 
Next, the activation status of p38 was examined to determine if loss of Gadd45a reduced p38 activation. Phosphorylated p38, indicative of activated p38, was assessed by both Western blotting (Fig. 5A) and immunohistochemistry (Fig. 5C). The level of expression of phosphorylated p38 was lower, and percentage of phosphorylated p38-expressing cells was significantly reduced (P ≤ 0.05) in Ras/Gadd45a–/– tumor cells compared with Ras/Gadd45a+/+ cells. Ras/Gadd45a+/– tumor cells had an intermediate level of expression (data not shown). There was no detectable level of phosphorylated p38 in normal mammary and spleen tissue (data not shown).

To determine if Ras-induced senescence of breast tumor cells is directly correlated with activation of p38, we did double-immunofluorescence staining experiments (Fig. 5D). By merging the immunofluorescent images, it is shown that tumor cells expressing ß-galactosidase also express activated p38. That some cells positive for phosphorylated p38 were not positive for ß-galactosidase staining raises the possibility that p38 activation is necessary, but not sufficient, for the onset of apoptosis; alternatively, these cells may represent cells in the process of undergoing senescence where ß-galactosidase expression has not been up-regulated. Similar analysis has shown that ß-galactosidase expression does not correlate with activation of JNK (data not shown). These results show that the tumor-suppressive function of Gadd45a on Ras-induced breast carcinogenesis is partly mediated by p38 activation, which in turn results in Ras-induced senescence of breast tumor cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mouse models of breast cancer have proven invaluable to the investigation of breast tumor initiation and progression. To examine the role of Gadd45a in breast tumor suppression, we generated a mouse model using both the known breast cancer–susceptible MMTV-Ras mouse strain and the Gadd45a-deficient mouse strain. By crossing these two strains, we generated a breast cancer–prone mouse model that lacked Gadd45a expression. Using this novel mouse model, we show that the loss of Gadd45a significantly accelerates the onset of breast tumorigenesis. These tumors exhibit increased growth rates and a more aggressive histologic phenotype compared with their Gadd45a WT counterparts. Furthermore, it is shown that the increased growth rate of Ras-driven breast tumors lacking Gadd45a can be accounted for by an increase in the fraction of cells progressing through the cell cycle, which is likely due to the observed decreases in both apoptosis and OIS. Mechanistically, it is shown that the decrease in apoptosis associated with loss of Gadd45a is linked to a decrease in JNK activation, and abrogation of Ras-induced senescence is linked to a block in p38 activation. Together, these results provide a novel model for Gadd45a breast tumor suppression, showing that Gadd45a functions to suppress Ras-driven tumor growth through a decrease in the fraction of cells progressing through the cell cycle and increases in both tumor cell apoptosis and tumor cell senescence (Fig. 6 ).


Figure 6
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  		Figure 6. Schematic diagram the role Gadd45a plays in Ras-driven mammary tumor suppression Ras. In the presence of Ras, Gadd45a acts as a stress sensor to modulate the induction of activated JNK and p38, which correlates with an increase in apoptosis and senescence, respectively. This suggests that Gadd45a acts as a tumor suppressor in the presences of Ras.

 
Previously, it was shown that deficiency in p53 or p21 accelerates Ras-driven breast carcinogenesis (22–24). Because Gadd45a is a direct target of the p53 and BRCA1 tumor suppressor genes, our data imply that breast tumor suppression afforded by p53 and BRCA1 is partially mediated by Gadd45a.

We have observed that a higher percentage of Ras-driven breast tumor cells are in the S phase and a lower percentage of the cells are in the G0-G1 phase of the cell cycle. This may be due to the role of Gadd45a in modulation of p21 function. Because Gadd45a is known to physically interact with p21, apparently, the effect of loss of Gadd45a on Ras-driven breast tumorigenesis may also involve loss of modulation of p21 function. It will be interesting, in this context, to determine what effect the combined loss of Gadd45a and p21 has on Ras-driven breast carcinogenesis. Regardless, it is likely that the effect on the cell cycle from loss of Gadd45a is due to the observed large reduction in senescence.

Until recently, the concept of OIS has been controversial, whether it is the result of cell culture stresses or an authentic in vivo process to prevent tumorigenesis. Recently, it has been documented that OIS occurs in vivo, playing a role in impeding tumor formation (19–21). Our data provide an important extension of this notion, showing for the first time that Ras-induced senescence in vivo is mediated, at least in part, by Gadd45a through activation of the Gadd45a partner protein, stress-induced p38 kinase.

Additional work is under way focused on determining how the loss of Gadd45a may affect breast carcinogenesis driven by oncogenes other than Ras, as well as assessing what effect loss of each of the other Gadd45 gene family members has on oncogene-driven breast carcinogenesis.


   Acknowledgments
 
Grant support: Department of Defense Breast Cancer Research Program grant DAMD17-02-1-0575 (D.A. Liebermann) and NIH grant RO1 CA081168 (B. Hoffman).

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 A. Fornace for the Gadd45a–/– mice and J. Litvin for careful examination of the histologic samples.

Received 6/ 1/06. Revised 6/20/06. Accepted 6/26/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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