Jennifer S. Tront

Conserved DNA methylation in Gadd45a-/- mice

Gadd45a (growth arrest and DNA-damage-inducible protein 45 alpha) plays a pivotal role in cellular stress responses and is implicated in DNA repair, cell cycle arrest and apoptosis. Recently, it was proposed that GADD45A is a key regulator of active DNA demethylation by way of its role in DNA repair. Barreto et al. reported that Gadd45a overexpression activated transcription from methylation-silenced reporter plasmids and promoted global DNA demethylation. siRNA-mediated knockdown of Gadd45a levels resulted in increased levels of DNA methylation at specific endogenous loci. Based on these exciting results, Gadd45a-/- mice might be predicted to have a hypermethylation phenotype. We report here that neither global nor locus-specific methylation is increased in Gadd45a-/- mice.

Gadd45a suppresses Ras-driven mammary tumorigenesis by activation of c-Jun NH2-terminal kinase and p38 stress signaling resulting in apoptosis and senescence.

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 NH(2)-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.

Gadd45a functions as a promoter or suppressor of breast cancer dependent on the oncogenic stress.

Gadd45a plays a pivotal role as a stress sensor that modulates cellular responses to various stress stimuli including oncogenic stress. We reported that the stress sensor Gadd45a gene functions as a tumor suppressor in Ras-driven breast tumorigenesis via increasing JNK-mediated apoptosis and p38-mediated senescence. In contrast, here, we show that Gadd45a promotes Myc-driven breast cancer by negatively regulating MMP10 via GSK3 β/β-catenin signaling, resulting in increased tumor vascularization and growth. These novel findings indicate that Gadd45a functions as either tumor promoter or suppressor, is dependent on the oncogenic stress, and is mediated via distinct signaling pathways. Collectively, these novel findings highlight the significance of the type of oncogenic alteration on how stress response genes function during initiation and progression of tumorigenesis. Because Gadd45a is a target for BRCA1 and p53, these findings have implications regarding BRCA1/p53 tumor suppressor functions.

Gadd45a stress signaling regulates sFlt-1 expression in preeclampsia

Preeclampsia, which affects approximately 5–8% of all pregnancies and is one of the leading causes of maternal and fetal morbidity and mortality, is a pregnancy induced complex of multiple pathological changes, including elevated blood pressure, proteinuria and edema manifested after 20 weeks gestation. There is growing evidence that placental stresses during pregnancy, notably hypoxia, and an increase in circulating soluble Flt‐1 (sFlt‐1) are important in the etiopathogenesis of preeclampsia. How placental stress results in elevated sFlt‐1 expression is currently unknown. Here we provide novel data implicating the Gadd45a stress sensor protein as an upstream modulator of pathophysiological changes observed in preeclampsia. It is shown that Gadd45a expression and activation of its downstream effector p38 kinase are elevated in preeclamptic placentas compared to non‐preeclamptic controls, and correlate with elevated sFlt‐1. Furthermore, a regulatory loop is demonstrated where stress, including hypoxia, IL‐6 or hypertonic stress, caused induction of Gadd45a, leading to p38 activation and ultimately increasing sFlt‐1 secretion in endothelial cells. These data provide a compelling working frame to further test the role of Gadd45 stress sensors in the etiology of preeclampsia, and set the stage for considering novel therapeutic regimens, including p38 inhibitors, for treatment of preeclampsia. J. Cell. Physiol. 220: 632–639, 2009.

Gadd45a and Gadd45b modulate innate immune functions of granulocytes and macrophages by differential regulation of p38 and JNK signaling

Gadd45 proteins function as stress sensors in response to various physiological and environmental stressors, interacting with other cellular proteins implicated in cellular stress responses, including p38 and JNK. This study shows that mice lacking either Gadd45a or Gadd45b are defective in the recruitment of granulocytes and macrophages to the intra‐peritoneal cavity following intra‐peritoneal administration of the bacterial cell wall pathogen‐associated molecular pattern lipopolysaccharide (LPS). Bone marrow derived granulocytes and macrophages lacking either Gadd45a or Gadd45b are shown to be impaired in their chemotactic response to LPS, as well as other inflammatory stimuli such as N‐formyl‐methionine–leucine–phenylalanine and IL‐8. Evidence was obtained also implicating Gadd45a and Gadd45b in other myeloid innate immune functions, including reactive oxygen species production, phagocytosis, and adhesion. Gadd45a and Gadd45b activation of p38 kinase was implicated in the response of granulocytes to LPS mediated chemotaxis, whereas Gadd45a and Gadd45b curtailment of JNK activation was linked to chemotaxis of macrophages in response to LPS. Collectively, these data highlight a novel role for both Gadd45a and Gadd45b in myeloid innate immune functions by differential modulation of p38 and JNK signaling in granulocytes compared to macrophages. J. Cell. Physiol. 227: 3613–3620, 2012.

Gadd45a levels in human breast cancer are hormone receptor dependent

BackgroundGadd45a is a member of the Gadd45 family of genes that are known stress sensors. Gadd45a has been shown to serve as an effector in oncogenic stress in breast carcinogenesis in murine models. The present study was aimed at clarifying the expression of Gadd45a in human breast cancer and its correlation with clinicopathologic features.MethodsThe expression levels of Gadd45a in breast tissue samples of female breast surgery cases were examined by immunohistochemistry (IHC) using a Gadd45a antibody. Percent staining was determined and statistical analyses were applied to determine prognostic correlations.Results56 female breast surgery cases were studied: Normal (11), Luminal A (9), Luminal B (11), HER2+ (10), Triple Negative (15). There was a highly significant difference in percent Gadd45a staining between groups [Mean]: Normal 16.3%; Luminal A 65.3%; Luminal B 80.7%; HER2+ 40.5%; TN 32%, P < 0.001, ANOVA. Gadd45a IHC levels for Normal cases found 82% negative/low. Luminal A breast cancer cases were found to be 67% high. Luminal B breast cancers were 100% high. Her2+ cases were 50% negative/low. Triple Negative cases were 67% negative/low. This difference in distribution of Gadd45a levels across breast cancer receptor subtypes was significant, P = 0.0009.ConclusionsGadd45a levels are significantly associated with hormone receptor status in human breast cancer. Normal breast tissue displays low Gadd45a levels. High Gadd45a levels are associated with Luminal A and Luminal B subtypes. Absence of hormone receptors in Triple Negative subtype is associated with Negative/Low levels of Gadd45a. Further studies are indicated to elucidate the role of Gadd45a in breast cancer as a potential prognosticator or target for treatment.

Abstract 1697: Gadd45A expression is high in luminal A and her2 human breast cancers, but frequently absent in triple negative breast cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Gadd45a is a stress sensor, playing an important role in tumorigenesis. Generation and side by side analysis of breast cancer prone MMTV-myc and MMTV-ras mice highlight a unique role for Gadd45a as either a suppressor or promoter of breast cancer development, employing distinct signaling pathways in response to distinct oncogenic stress stimuli. Thus, it appears that gadd45a can function to either promote or suppress breast tumor development in mice via engagement of different signaling pathways depending on the molecular nature of the activated oncogene. Extending the work to human breast cancer has provided initial, novel data, showing that Gadd45a, which is not expressed in normal breast tissue, is expressed at high levels in less aggressive breast cancers and is low or absent in more aggressive subtypes. Notably, Gadd45a is expressed at very high levels in luminal A and her2 positive breast cancers, but frequently is absent in triple negative breast cancers. Experiments are currently underway to elucidate the functional role of Gadd45a in human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1697. doi:1538-7445.AM2012-1697

Abstract 5079: Gadd45a differentially modulates breast tumorigenesis by promoting or suppressing tumor growth in response to distinct oncogenic stress

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Gadd45a plays a pivotal role as a stress sensor that modulates cellular responses to various stress stimuli, including oncogenic stress. Generating novel MMTV-ras and MMTV-myc mouse models which are either wildtype or null for Gadd45a, we identified Gadd45a as a tumor suppressor in ras-mediated breast tumorigenesis. In sharp contrast, recent findings have shown that Gadd45a promotes myc-driven breast cancer. Furthermore, the function of Gadd45a as either a tumor promoter or suppressor was observed to be mediated through distinct, mutually exclusive, signaling pathways. Whereas Gadd45a tumor suppressor function is mediated via activation of JNK and p38 stress kinases promoting ras-induced apoptosis and senescence, Gadd45a function as a tumor promoter is exerted by negatively regulating MMP10 expression via the GSK3b/Beta-catenin signaling cascade, resulting in increased tumor vascularization. Analyzing specimens obtained from human breast cancer patients has shown relatively high levels of Gadd45a expression in low to moderate grade tumors compared to normal breast tissue, and no Gadd45a expression in high grade tumors. Correlating the status of Gadd45a expression in human breast cancer to activated oncogenes and Gadd45a signaling, as well as to patient survival following treatment is in progress. Collectively, these observations highlight the significance of the type of oncogenic alteration on how stress response genes function during initiation and progression of tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5079.