Gadd45a Suppresses Ras-Driven Mammary Tumorigenesis By

Research Article

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. Tront,1 Barbara Hoffman,1,2 and Dan A. Liebermann1,2

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

Abstract genes play a pivotal role as stress sensors that modulate the cellular Gadd45a The Gadd45 family of proteins is known to play a central role response to a variety of stressors (7, 8). is one member of as cellular stress sensors that modulate the response of the three-member family that is regulated by important tumor mammalian cells to stress inflicted by physiologic and suppressor proteins, such as p53 (9) and BRCA1 (10). Gadd45a is environmental stressors. Gadd45a was shown to be a direct also known to interact with key cell regulators, such as p21 (11), target to the p53 and BRCA1 tumor suppressor genes, whose cdc2/cyclin B1 (12), proliferating cell nuclear antigen (9), p38 (13), loss of function is known to play a vital role in breast and mitogen-activated protein (MAP) three kinase 1 (MTK1)/MAP carcinogenesis; however, the role of Gadd45a in the suppres- kinase (MAPK) kinase kinase (MEKK) 4 (14, 15). The cellular function of Gadd45a is dependent on its interacting partner. For sion of breast cancer remains unclear. To address this issue, Gadd45a-deficient mice were crossed with breast cancer example, physical interaction between Gadd45a and MTK1/MEKK4 prone mouse mammary tumor virus–Ras mice to generate results in the activation of c-Jun NH2-terminal kinase (JNK), which mice that express activated Ras and differ in their Gadd45a can lead to cell growth inhibition or apoptosis (14, 15). Addi- status. Using this mouse model, we show that the loss of tionally, the physical interaction between Gadd45a and the MAPK Gadd45a p38 may play a pivotal role in preventing oncogene-induced growth accelerates Ras-driven mammary tumor formation, Gadd45a exhibiting increased growth rates and a more aggressive in part by regulating p53 tumor suppression (13). -null histologic phenotype. Moreover, it is shown that accelerated mice were found to display increased susceptibility to radiation- Gadd45a induced carcinogenesis (16). Therefore, it was of interest to Ras-driven tumor formation in the absence of results Gadd45a in both a decrease in apoptosis, which is linked to a decrease investigate the role that may play in breast tumorigenesis and to examine possible cooperation between oncogenic Ras and in c-Jun NH2-terminal kinase (JNK) activation, and a Gadd45a decrease in Ras-induced senescence, which is correlated with loss of in breast tumor formation and progression. a decrease in p38 kinase activation. Altogether, these results To achieve this goal, we have taken advantage of the established provide a novel model for the tumor-suppressive function of breast cancer prone mouse mammary tumor virus (MMTV)-v-Ras Gadd45a in the context of Ras-driven breast carcinogenesis, transgenic mice, where the v-Ha-Ras oncogene, which contains an Gadd45a activation mutation in codon 12 (Gly to Arg) and 59 (Ala to Thr), is showing that elicits its function through activation Gadd45a of the stress-induced JNK and p38 kinases, which contribute to under the control of the MMTV promoter (17). -deficient increase in apoptosis and Ras-induced senescence. (Cancer Res mice and MMTV-v-Ras transgenic mice were interbred to generate 2006; 66(17): 8448-54) 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 tumorigen- Introduction esis. These tumors exhibit increased growth rates and a more The development of breast cancer is a multistage process. aggressive histologic phenotype compared with their Gadd45a Mutations and overexpression of proto-oncogenes, such as Ras, are wild-type (WT) counterparts. The increased growth rate of Ras- known to cooperate with mutations or deletions of growth driven breast tumors lacking Gadd45a can be accounted for by an suppressor genes, such as p53 and BRCA1, in the development of increase in the fraction of cells progressing through the cell cycle, breast cancer (1–4). The Ras oncogenes harbor point mutations, which may be due to the observed decreases in both apoptosis and leading to an amino acid substitution at positions 12, 13, 59, and 61, oncogene-induced senescence (OIS). Mechanistically, it is shown which confer transforming activity in various human cancers. that the decrease in apoptosis associated with loss of Gadd45a is Activating Ras mutations are found in human malignancies with an linked to a decrease in JNK activation and abrogation of Ras- overall frequency of 15% to 20% and are found in 10% to 12% of induced senescence is linked to a decresase in p38 activation. breast carcinomas (5, 6). The Gadd45 family of genes (growth arrest and DNA damage) plays an important role in cell cycle control, survival, and Materials and Methods apoptosis. There is evidence that the proteins encoded by these Mice. MMTV-Ras transgenic mice in an inbred FVB genetic background were originally obtained from Charles River Laboratories (Wilmington, À À MA). Gadd45a / mice (in a C57BL/6 Â 129Sv background) were graciously Requests for reprints: Dan A. Liebermann, Fels Institute for Cancer Research and provided by Albert Fornace (Harvard University, Boston, MA). Offspring À À Molecular Biology and Department of Biochemistry, Temple University School of from interbreeding Gadd45a / and MMTV-Ras mice were generated as Medicine, 3307 North Broad Street, Philadelphia, PA 19140. Phone: 215-707-6903; littermates from common matings so that all animals were maintained in a Fax: 215-707-2805; E-mail: [email protected]. I2006 American Association for Cancer Research. mixed genetic background. Offspring from crosses between MMTV-Ras and À À doi:10.1158/0008-5472.CAN-06-2013 Gadd45a / mice were screened by PCR for their Ras and Gadd45a status.

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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¶-GAGGCAGG- GACCAGCAAGACATC-3¶ (5¶ sense) and 5¶-ACAGACCCTGAACCACGCAT- CAAC-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 94jC for 1 minute, 63jC for 14 seconds, and 72j 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 f14 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 Â 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. Differ- ences between Kaplan-Meier curves were determined using a Mantel-Cox log-rank statistical test. Differences in tumor incidence were determined by the m2 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 Figure 1. Gadd45a deficiency results in accelerated mammary tumorigenesis in Lakes, NJ). Cells were analyzed by light microscopy. Necrotic regions of the MMTV-Ras mice. A, Kaplan-Meier curve showing the proportion of tumor-free tumor were avoided. Using a 10 Â 450 field range, the number of terminal mice as a function of time for Ras/Gadd45aÀ/À (n = 26), Ras/Gadd45a+/À +/+ deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)– (n = 25), and Ras/Gadd45a (n = 25; P < 0.005). B, average tumor growth rate À/À n +/À n positive stained cells and the total number of propidium iodide (PI)–stained for tumors arising in Ras/Gadd45a ( = 8), Ras/Gadd45a ( = 7), and Ras/Gadd45a+/+ (n = 6) mice. Tumor growth was monitored every other day for cells was determined with Image J photo program. A minimum of five 13 days by caliper measurement. samples per genotype were analyzed. Differences in percentage apoptosis between different genotypes were evaluated using the Student’s t test. the dark and mounted for viewing. For single-staining experiments, samples Flow cytometry. At the time of sacrifice, a 25- to 50-mg section of tumor were stained using fluorescein secondary antibody followed by incubation tissue was minced and treated with dispase (0.6 units/mL; Invitrogen, with PI stain for 20 minutes at room temperature. Using a 10 Â 450 field Carlsbad, CA) and collagenase I (100 units/mL; Invitrogen) for 1 hour at 37j range, the number of positive-stained cells and the total number of cells with slight agitation. Following incubation, the sample was passed through were determined with Image J photo program for both immunohistochem- a cell strainer and washed with PBS. The sample was then treated with RBC istry and immunofluorescence samples. A minimum of five samples from lysis buffer (Cambrex, East Rutherford, NJ) to eliminate RBCs from the each genotype were analyzed for each analysis. Differences between culture. The tumor cells were fixed with ethanol and then stained with PI genotypes were evaluated using the Student’s t test. For the Annexin V and (12). A minimum of five samples from each genotype were analyzed. phosphorylated JNK double staining, the samples were stained with Statistical significance was determined using a Student’s t test. phosphorylated JNK primary antibody and Texas red–conjugated secondary Immunohistochemistry and immunofluorescence. Paraffin-embed- antibody as described above immediately followed by the Annexin V ded tissue sections were deparaffinized, rehydrated, and subjected to protocol as described above. For the phosphorylated p38 and h-galacto- antigen unmasking by sodium citrate (10 mmol/L; pH 6.0) for 30 minutes at sidase double staining, both antibodies were mixed at equal concentrations a sub-boiling temperature. (For immunohistochemistry only: endogenous and incubated overnight at 4jC. Fluorescein-conjugated anti-rabbit secon- peroxidase activity was blocked by incubation in 3% hydrogen peroxide for dary antibody was used to detect phosphorylated 38. Texas red–conjugated 10 minutes). Sections were then blocked with 5% serum for 1 hour at room anti-mouse secondary antibody was used to detect h-galactosidase. Images temperature followed by incubation with primary antibody overnight at were acquired and merged through Spot Imaging software. 4jC [phosphorylated JNK (Cell Signaling Technology, Danvers, MA), phosphorylated p38 immunohistochemistry preferred (Cell Signaling Technology), and h-galactosidase (Abcam, Cambridge, MA)]. For immuno- Results histochemistry, sections were then incubated with a peroxidase-conjugated Gadd45a is up-regulated during Ras-driven breast carcino- secondary antibody for 30 minutes at room temperature followed by genesis. Our working hypothesis was that Gadd45a is a stress treatment with avidin-biotin complex method reagent (Vector Laboratories, Burlingame, CA) for 30 minutes. Sections were stained with 3,3¶- sensor protein, which is up-regulated by oncogenic stress during diaminobenzidine substrate and counterstained with hematoxylin (Vector breast carcinogenesis and functions to modulate tumor develop- Laboratories, Burlingame, CA). For immunofluorescence, sections were ment. To assess the validity of our hypothesis, Gadd45a expression incubated with either fluorescein- or Texas red–conjugated secondary was examined in normal and tumor mammary tissue obtained À antibody (TI-1000; Vector Laboratories) for 2 hours at room temperature in from the three genotypes (Ras/Gadd45a+/+, Ras/Gadd45a+/ , and www.aacrjournals.org 8449 Cancer Res 2006; 66: (17). September 1, 2006

À À Ras/Gadd45 / ). For comparison, Gadd45a expression was also incidence, from 74% of Ras/Gadd45a+/+ mice developing tumors À À assessed in nonmammary tissue (i.e., spleen tissue). within 12 months to 94% of Ras/Gadd45a / mice developing À Gadd45a expression was undetectable in normal mammary and tumors within the same time frame. Again, Ras/Gadd45a+/ mice spleen tissue obtained from all three genotypes. In comparison, had an intermediate incidence of 92%. detectable levels of Gadd45a were observed in breast tumor tissue To determine if the loss of Gadd45a contributed to an increase À obtained from Ras/Gadd45a+/+ and Ras/Gadd45+/ mice but not in the rate of tumor growth, tumor volume was determined every À À Ras/Gadd45a / . The highest level of Gadd45a expression was 2 days by caliper measurements, starting on first tumor observed in Ras/Gadd45a+/+ tumors with an intermediate level of visualization and continuing for f2 weeks or until the general expression in the heterozygote tissue. Taken together, these data well being of the animal was compromised. Mammary tumors support the hypothesis that Gadd45a expression is up-regulated arising from Ras expressing Gadd45a-deficient mice displayed during breast carcinogenesis. significantly increased rates of tumor growth compared with those Gadd45a deficiency results in accelerated mammary tumor- mice that express Gadd45a (P < 0.05; Fig. 1B). For example, at À À igenesis in MMTV-Ras mice. To assess the effect of Gadd45a 14 days, the average Ras+/Gadd45a / tumor had a volume of deficiency on breast carcinogenesis, Gadd45a-deficient mice were 4,809 F 1,373 mm3 (n = 8), whereas the Gadd45a-expressing tumors crossed with mammary tumor prone MMTV-Ras transgenic mice to had an average tumor volume of 2,684 F 791 mm3 (n = 6). Once À generate animals that carried oncogenic Ras and differed in their again, Gadd45a+/ mice displayed an intermediate phenotype. À À À Gadd45a status (Ras/Gadd45a / , Ras/Gadd45a+/ ,andRas/ Gadd45a-deficient tumors display a more aggressive histo- Gadd45a+/+). Female animals from each genotype were monitored logic phenotype. Tumor sections from the three genotypes were twice weekly for the formation of tumors. It was observed that analyzed to determine if the loss of Gadd45a contributes to À À tumorigenesis is accelerated in Ras/Gadd45a / mice when histologic changes within the mammary tumors, which correlate compared with Ras/Gadd45a+/+ mice (P < 0.005; Fig. 1A). The with tumor aggressiveness. Higher histologic tumor grades are median tumor onset, measured as the time, in which 50% of animals associated with a loss of cellular shape and size uniformity, À À develop tumors, was 5 months for Ras/Gadd45a / mice, whereas increased nuclear size, hyperchromatic nuclei, and presence of the tumor onset for Ras/Gadd45a+/+ mice was 8 months. Ras/ multinucleated cells. For grading, nuclear size and shape, nuclear/ À Gadd45a+/ mice had an intermediate median tumor onset cytoplasmic ratio, presence of hyperchromatic nuclei, and presence of 7 months. The loss of Gadd45a increased overall tumor of multinucleated cells were evaluated and incorporated into a

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+/À, and Gadd45aÀ/À, respectively (magnification, Â40). Ras+/ Gadd45aÀ/À sample displaying hyperchromatic nuclei and a multinucleated cell at Â100 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.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Gadd45a in MMTV-Ras Mammary Tumorigenesis 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 À À 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 1to2(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 À 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. 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 Sphase 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 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. percentage of cells in the Sphase to the increased tumor growth A, histogram from a representative PI-stained tumor, showing a diploid peak as À/À rates in the Ras/Gadd45a tumors, we also did Ki67 immunohis- well as multiple aneuploidy peaks from a Ras/Gadd45a+/+ sample. B, flow cytometric analysis of PI-stained tumor cells, showing averages for tumors with tochemistry staining to confirm increased proliferation rates. We F F À/À different genotypes. G0-G1, S, and G2-M fractions were 81 5.1%, 13 1.3%, found an increase in Ki67 staining in the Ras/Gadd45a samples and 6 F 0.9% for Ras+/Gadd45a+/+ (n = 8); 73 F 3.5%, 24 F 2.1%, and compared with the Ras/Gadd45a+/+ control (data not shown). 3 F 0.4% for Ras+/Gadd45a+/À (n = 12); 61 F 2.9%, 36 F 2.0%, and 3 F 0.5% + À/À n Concomitantly, it can be seen that loss of Gadd45a also resulted for Ras /Gadd45a ( = 14). in a significant decrease in cells in the G0-G1 phase of the cell cycle. À Taken together, these data show that the increased growth rate of Ras/Gadd45+/+ cells. Ras/Gadd45a+/ tumor cells displayed an À À Ras/Gadd45a / tumors compared with Ras/Gadd45a+/+ tumors is intermediate expression level (data not shown). There was no due in part to an increase in the fraction of cells that progress detectable phosphorylated JNK in normal mammary and spleen through the cell cycle. tissue (data not shown). Gadd45a-deficient breast tumors display a decreased level To examine if apoptosis is directly correlated with JNK activation, of apoptosis, which is associated with impaired JNK activa- we did double-immunofluorescence staining (Fig. 4D). By merging tion. Acceleration of tumor development as a consequence of the immunofluorescent images, it is shown that tumor cells Gadd45a deficiency may also reflect a decrease in apoptosis of undergoing apoptosis do indeed express activated JNK. The fact breast cancer cells. Gadd45a has been implicated in programmed that some cells positive for phosphorylated JNK were not positive cell death via activation of the stress-induced JNK kinase (15, 18). for TUNEL staining raises the possibility that JNK activation may be Thus, it was of interest to assess how the loss of Gadd45a affects necessary but not sufficient for the onset of apoptosis; alternatively, apoptosis of Ras-driven breast tumor cells and if this involved these cells may represent cells at early stages of apoptosis where failure to activate JNK. DNA degradation may not yet have taken place. Similar analysis has To determine the number of tumor cells undergoing apoptosis, shown that apoptotic cells do not express activated p38 (data not TUNEL analysis was done on formalin-fixed, paraffin-embedded shown). Taken together, these data imply that Gadd45a-mediated tumor tissue samples from each of the genotypes (Fig. 4B). The loss activation of JNK, which in turn results in apoptosis of tumor cells, of Gadd45a was observed to significantly decrease total tumor cell contributes to the tumor-suppressive function of Gadd45a in Ras- apoptosis >4.5-fold. To understand mechanistically how Gadd45a driven breast carcinogenesis. deficiency results in decreased apoptosis, we explored the Gadd45a deficiency results in a decrease in Ras-induced activation status of JNK in Ras-driven breast tumor cells, which senescence, which correlates with impaired p38 activation in are either WT or null for Gadd45a. As shown by both Western breast cancer cells. Recently, it has become evident that cellular blotting (Fig. 4A) and immunohistochemistry (Fig. 4C), the level of senescence, first discovered in cell culture, is in fact a vital phosphorylated JNK, indicative of activated JNK, and the mechanism that constrains tumor development in vivo (19–21). percentage of cells expressing phosphorylated JNK was significantly Furthermore, Gadd45a-mediated activation of p38 MAPK has been À À lower (P V 0.05) in Ras/Gadd45a / tumor cells compared with implicated in H-Ras-induced cell cycle arrest in mouse embryo www.aacrjournals.org 8451 Cancer Res 2006; 66: (17). September 1, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research fibroblasts (13). Clearly, it was of interest to explore whether the expression of phosphorylated p38 was lower, and percentage of tumor-suppressive function of Gadd45a on Ras-driven breast phosphorylated p38-expressing cells was significantly reduced À À carcinogenesis is in part due to senescence resulting from (P V 0.05) in Ras/Gadd45a / tumor cells compared with Ras/ À Gadd45a-mediated p38 activation. Gadd45a+/+ cells. Ras/Gadd45a+/ tumor cells had an intermediate To test this hypothesis, tumors from Ras/Gadd45a+/+ and Ras/ level of expression (data not shown). There was no detectable À À Gadd45a / mice were compared for the relative number of level of phosphorylated p38 in normal mammary and spleen tissue senescent cells using immunohistochemistry to assess the expression (data not shown). of the senescence marker h-galactosidase. As shown in Fig. 5B, To determine if Ras-induced senescence of breast tumor cells is there was a significant decrease (P V 0.05) in the percentage of breast directly correlated with activation of p38, we did double-immuno- À À tumor cells that expressed h-galactosidase in the Ras/Gadd45a / fluorescence staining experiments (Fig. 5D). By merging the mice compared with tumors obtained from the Ras/Gadd45a+/+ mice. immunofluorescent images, it is shown that tumor cells expressing Next, the activation status of p38 was examined to determine if h-galactosidase also express activated p38. That some cells positive loss of Gadd45a reduced p38 activation. Phosphorylated p38, for phosphorylated p38 were not positive for h-galactosidase indicative of activated p38, was assessed by both Western blotting staining raises the possibility that p38 activation is necessary, but (Fig. 5A) and immunohistochemistry (Fig. 5C). The level of not sufficient, for the onset of apoptosis; alternatively, these cells

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+/Gadd45aÀ/À, n = 14). C, average percentage of phosphorylated JNK-positive cells from Ras+/Gadd45a+/+ (n = 5) and Ras+/Gadd45aÀ/À (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+/ Gadd45aÀ/À. Representative tumor sections are for TUNEL staining, phosphorylated JNK staining, and the merged images.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Gadd45a in MMTV-Ras Mammary Tumorigenesis may represent cells in the process of undergoing senescence where tumors exhibit increased growth rates and a more aggressive h-galactosidase expression has not been up-regulated. Similar histologic phenotype compared with their Gadd45a WT counter- analysis has shown that h-galactosidase expression does not corre- parts. Furthermore, it is shown that the increased growth rate of late with activation of JNK (data not shown). These results show Ras-driven breast tumors lacking Gadd45a can be accounted for that the tumor-suppressive function of Gadd45a on Ras-induced by an increase in the fraction of cells progressing through the cell breast carcinogenesis is partly mediated by p38 activation, which cycle, which is likely due to the observed decreases in both in turn results in Ras-induced senescence of breast tumor cells. apoptosis and OIS. Mechanistically, it is shown that the decrease in apoptosis associated with loss of Gadd45a is linked to a Discussion decrease in JNK activation, and abrogation of Ras-induced Mouse models of breast cancer have proven invaluable to the senescence is linked to a block in p38 activation. Together, these investigation of breast tumor initiation and progression. To results provide a novel model for Gadd45a breast tumor examine the role of Gadd45a in breast tumor suppression, we suppression, showing that Gadd45a functions to suppress Ras- generated a mouse model using both the known breast cancer– driven tumor growth through a decrease in the fraction of cells susceptible MMTV-Ras mouse strain and the Gadd45a-deficient progressing through the cell cycle and increases in both tumor mouse strain. By crossing these two strains, we generated a breast cell apoptosis and tumor cell senescence (Fig. 6). cancer–prone mouse model that lacked Gadd45a expression. Previously, it was shown that deficiency in p53 or p21 accelerates Using this novel mouse model, we show that the loss of Gadd45a Ras-driven breast carcinogenesis (22–24). Because Gadd45a is a significantly accelerates the onset of breast tumorigenesis. These direct target of the p53 and BRCA1 tumor suppressor genes, our

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 h-galactosidase-positive (B) and phosphorylated p38-positive (C) cells from Ras+/Gadd45a+/+ (n =5) and Ras+/Gadd45aÀ/À (n = 5). D, formalin-fixed, paraffin-embedded tumor tissue sections were analyzed by immunofluorescence for levels of h-galactosidase and phosphorylated p38 concomitantly (Ras+/Gadd45a+/+ and Ras+/Gadd45aÀ/À). Representative tumor tissue sections are for h-galactosidase, phosphorylated p38, and the merged images.

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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 Sphase 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 OIShas 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 Received 6/1/2006; revised 6/20/2006; accepted 6/26/2006. Grant support: Department of Defense Breast Cancer Research Program grant Figure 6. Schematic diagram the role Gadd45a plays in Ras-driven DAMD17-02-1-0575 (D.A. Liebermann) and NIH grant RO1 CA081168 (B. Hoffman). mammary tumor suppression Ras. In the presence of Ras, Gadd45a acts The costs of publication of this article were defrayed in part by the payment of page as a stress sensor to modulate the induction of activated JNK and p38, which charges. This article must therefore be hereby marked advertisement in accordance correlates with an increase in apoptosis and senescence, respectively. with 18 U.S.C. Section 1734 solely to indicate this fact. À À This suggests that Gadd45a acts as a tumor suppressor in the presences We thank A. Fornace for the Gadd45a / mice and J. Litvin for careful examination of Ras. of the histologic samples.

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Cancer Res 2006; 66: (17). September 1, 2006 8454 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Gadd45a Suppresses Ras-Driven Mammary Tumorigenesis by Activation of c-Jun NH 2-Terminal Kinase and p38 Stress Signaling Resulting in Apoptosis and Senescence

Jennifer S. Tront, Barbara Hoffman and Dan A. Liebermann

Cancer Res 2006;66:8448-8454.

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