KEAP1 E3 ligase-mediated downregulation of NF-kappaB signaling by targeting IKKbeta.

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

14-3-3 proteins interact with the beta-thymosin repeat protein Csp24.

Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.

Active water in protein-protein communication within the membrane: the case of SRII-HtrII signal relay.

We detect internal water molecules in a membrane-embedded receptor-transducer complex and demonstrate water structure changes during formation of the signaling state. Time-resolved FTIR spectroscopy reveals stimulus-induced repositioning of one or more structurally active water molecules to a significantly more hydrophobic environment in the signaling state of the sensory rhodopsin II (SRII)-transducer (HtrII) complex. These waters, distinct from bound water molecules within the SRII receptor, appear to be in the middle of the transmembrane interface region near the Tyr199(SRII)-Asn74(HtrII) hydrogen bond. We conclude that water potentially plays an important role in the SRII --> HtrII signal transfer mechanism in the membrane's hydrophobic core.

Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling.

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

Differential dynamic properties of scleroderma fibroblasts in response to perturbation of environmental stimuli.

Diseases are believed to arise from dysregulation of biological systems (pathways) perturbed by environmental triggers. Biological systems as a whole are not just the sum of their components, rather ever-changing, complex and dynamic systems over time in response to internal and external perturbation. In the past, biologists have mainly focused on studying either functions of isolated genes or steady-states of small biological pathways. However, it is systems dynamics that play an essential role in giving rise to cellular function/dysfunction which cause diseases, such as growth, differentiation, division and apoptosis. Biological phenomena of the entire organism are not only determined by steady-state characteristics of the biological systems, but also by intrinsic dynamic properties of biological systems, including stability, transient-response, and controllability, which determine how the systems maintain their functions and performance under a broad range of random internal and external perturbations. As a proof of principle, we examine signal transduction pathways and genetic regulatory pathways as biological systems. We employ widely used state-space equations in systems science to model biological systems, and use expectation-maximization (EM) algorithms and Kalman filter to estimate the parameters in the models. We apply the developed state-space models to human fibroblasts obtained from the autoimmune fibrosing disease, scleroderma, and then perform dynamic analysis of partial TGF-beta pathway in both normal and scleroderma fibroblasts stimulated by silica. We find that TGF-beta pathway under perturbation of silica shows significant differences in dynamic properties between normal and scleroderma fibroblasts. Our findings may open a new avenue in exploring the functions of cells and mechanism operative in disease development.

AUTOIMMUNE RESPONSES TO ATHEROSCLEROTIC

Atherosclerosis is a chronic, complex arterial disease characterized by intimal lipid accumulation and inflammation. A unique lipid-binding molecule, namely cluster of differentiation 1d (CD1d), may impact atherosclerosis. Structurally, CD1d acts as a nonpolymorphic cell-surface receptor, resembling the major histocompatibility complex-I (MHC-I). While MHC-I restricts peptide antigen presentation to T cells, CD1d presents lipid antigens to T cells named CD1d-restrictedd T cells. Although increased expression of CD1d has been found in human plaques, the exact nature of CD1d-recognized lipids in atherosclerosis remains to be determined. Three groups of lipids may undergo oxidation in atherosclerosis producing atherogenic lipids: phospholipids, fatty acids, and cholesterol. The central hypothesis is that CD1d recognizes and present oxidative lipids to activate CD1d-restricted T cells, and trigger proinflammatory signal transduction In the first part of this study, oxidative phospholipids were identified and characterized as potential autoantigen for CD1d-restricted T cells. Derived from phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine by oxidization, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) is commonly found in atherosclerotic plaques. Upon stimulation with PGPC, spleen-derived CD1d-restricted T cells produced higher levels of cytokines and proliferated at higher rates than those without PGPC stimulation. CD1d deficiency compromised the PGPC-triggered T cell activation, suggesting that PGPC may function as a potentially novel autoantigen for T cells in atherosclerosis. In the second part of this study, CD1d-mediated proinflammatory signaling was evaluated in murine models. Enhanced CD1 expression occurred in spleens of db/db mice with hyperlipidemia. Tumor necrosis factor-alpha (TNF-α) was increased in db/db spleen, while TNF-α receptor expression augmented in the db/db murine heart, in comparison with those in normal mice. The nuclear factor-κ B (NF-κB) expression was enhanced in the db/db heart, whereas CD1d-null mice showed lower NF-κB, implying the involvement of CD1d in inflammation of the spleen and heart tissues in the mice with hyperlipidemia. The current study has identified PGPC as a novel lipid antigen recognized by CD1d-restricted T cells in atherosclerosis. The animal study has also provided evidence that CD1d regulates NF-κB-mediated proinflammatory signaling. Hence, CD1d-restricted T cell responses to autolipid antigen and mediated inflammatory signal may represent a new molecular pathway that triggers cardiovascular tissue injury in atherosclerosis and hyperlipidemia.

Cloning and genetic characterization of Skb1: A novel regulator of Shk1 and mitosis in Schizosaccharomyces pombe

A partial skb1 gene was originally isolated in a yeast two-hybrid screen for Shk1-interacting polypeptides. Shk1 is one of two Schizosaccharomyces pombe p21Cdc42/Rac-activated kinases (PAKs) and is an essential component of the Ras1-dependent signal transduction pathways regulating cell morphology and mating responses in fission yeast. After cloning the skb1 gene we found the Skb1 gene product to be a novel, nonessential protein lacking homology to previously characterized proteins. However the identification of Skb1 homologs in C. elegans, S. cerevisiae, and H. sapiens reveals evolution has conserved the skb1 gene. Fission yeast cells carrying a deletion of skb1 exhibit a defect in cell size but not mating abilities. This defect is suppressed by high copy shk1. Fission yeast overexpressing skb1 were found to undergo cell division at a length 1.5X greater than normal. In the two-hybrid system, Skb1 interacts with a subdomain of the Shk1 regulatory region distinct from that with which Cdc42 interacts, and forms a ternary complex with Shk1 and Cdc42. By use of yeast genetics, we have established a role for Skb1 as a positive regulator of Shk1. Co-overexpression of shk1 with skb1 was found to suppress the morphology defect, but not the sterility, of ras1Δ fission yeast. Thus, the function of Skb1 is restricted to a morphology control pathway. We determined that Skb1 functions as a negative regulator of mitosis and does this through a Shk1-dependent mechanism. The mitotic regulatory function of Skb1 and Shk1 was also partially dependent upon Wee1, a direct negative regulator of the cyclin-dependent kinase Cdc2. The role for Skb1 and Shk1 as mitotic regulators is the first connection from a PAK protein to control of the cell cycle. Furthermore, Skb1 is the first non-Cdc42/Rac PAK modulator to be identified. ^

Role of transducer proteins in photoaxis signaling of Halobacterium salinarum

In Halobacterium salinarum phototaxis is mediated by the visual pigment-like photoreceptors sensory rhodopsin I (SRI) and II (SRII). SRI is a receptor for attractant orange and repellent UV-blue light, and SRII is a receptor for repellent blue-green light, and transmit signals through the membrane-bound transducer proteins HtrI and HtrII, respectively. ^ The primary sequences of HtrI and HtrII predict 2 transmembrane helices (TM1 and TM2) followed by a hydrophilic cytoplasmic domain. HtrII shows an additional large periplasmic domain for chemotactic ligand binding. The cytoplasmic regions are homologous to the adaptation and signaling domains of eubacterial chemotaxis receptors and, like their eubacterial homologs, modulate the transfer of phosphate groups from the histidine protein kinase CheA to the response regulator CheY that in turn controls flagellar motor rotation and the cell's swimming behavior. HtrII and Htrl are dimeric proteins which were predicted to contain carboxylmethylation sites in a 4-helix bundle in their cytoplasmic regions, like eubacterial chemotaxis receptors. ^ The phototaxis transducers of H. salinarum have provided a model for studying receptor/tranducer interaction, adaptation in sensory systems, and the role of membrane molecular complexes in signal transduction. ^ Interaction between the transducer HtrI and the photoreceptor SRI was explored by creating six deletion constructs of HtrI, with progressively shorter cytoplasmic domains. This study confirmed a putative chaperone-like function of HtrI, facilitating membrane insertion or stability of the SRI protein, a phenomenon previously observed in the laboratory, and identified the smallest HtrI fragment containing interaction sites for both the chaperone-like function and SRI photocycle control. The active fragment consisted of the N-terminal 147 residues of the 536-residue HtrI protein, a portion of the molecule predicted to contain the two transmembrane helices and the first ∼20% of the cytoplasmic portion of the protein. ^ Phototaxis and chemotaxis sensory systems adapt to stimuli, thereby signaling only in response to changes in environmental conditions. Observations made in our and in other laboratories and homologies between the halobacterial transducers with the chemoreceptors of enteric bacteria anticipated a role for methylation in adaptation to chemo- and photostimuli. By site directed mutagenesis we identified the methylation sites to be the glutamate pairs E265–E266 in HtrI and E513–E514 in HtrII. Cells containing the unmethylatable transducers are still able to perform phototaxis and adapt to light stimuli. By pulse-chase analysis we found that methanol production from carboxylmethyl group hydrolysis occurs upon specific photo stimulation of unmethylatable HtrI and HtrII and is due to turnover of methyl groups on other transducers. We demonstrated that the turnover in wild-type H. salinarum cells that follows a positive stimulus is CheY-dependent. The CheY-feedback pathway does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell. ^ Assembly of signaling molecules into architecturally defined complexes is considered essential in transmission of the signals. The spectroscopic characteristics of SRI were exploited to study the stoichiometric composition in the phototaxis complex SRI-HtrI. A molar ratio of 2.1 HtrI: 1 SRI was obtained, suggesting that only 1 SRI binding site is occupied on the HtrI homodimer. We used gold-immunoelectron microscopy and light fluorescence microscopy to investigate the structural organization and the distribution of other halobacterial transducers. We detected clusters of transducers, usually near the cell's poles, providing a ultrastructural basis for the global effects and intertransducer communication we observe. ^

Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

beta-catenin interacts with and inhibits NF-kappaB in colon cancer

The β-catenin pathway plays an important role in the progression of colon cancer as well as many other cancer types. Almost all colorectal tumors show an upregulation of β-catenin activity either through mutations in the β-catenin regulator APC or through mutations in β-catenin itself. Upregulation of β-catenin leads to the transcription of many target genes involved in tumorigenesis. NF-κB is a transcription factor which activates many target genes, including both anti-apoptotic and pro-apoptotic molecules. Recently, it has been shown that GSK-3β, a negative regulator of β-catenin, is involved in the activation of NF-κB. However, the mechanism of this regulation of NF-κB by GSK-3β is unclear. As GSK-3β inhibits β-catenin we hypothesized that β-catenin may be responsible for the regulation of NF-κB by GSK-3β; i.e. β-catenin may inhibit NF-κB activity. In this study we show that β-catenin physically interacts with NF-κB leading to the inhibition of NF-κB transcriptional and DNA-binding activities. We also show that in colon cancer cells with high β-catenin expression there is a suppressed NF-κB activity and depletion of β-catenin increases NF-κB activity. Similarly, in colon cancer cells that have a low level of β-catenin NF-κB activity is high and introduction of β-catenin reduces NF-κB activity. Importantly, we show that this suppression of NF-κB by β-catenin leads to a reduction of NF-κB target gene Fas expression. Also Fas-mediated apoptosis is reduced in β-catenin overexpressing cells, which can be reversed upon depletion of β-catenin. Introduction of the NF-κB subunit p65 can restore Fas expression indicating that the effect of β-catenin on Fas is through NF-κB. Furthermore, β-catenin expression was found to inversely correlate with Fas expression in human colon and breast primary tumor tissues. As Fas downregulation is important for tumors to evade immune surveillance, β-catenin inhibition of NF-κB and Fas downregulation likely plays and important role for colon cancer progression. Additionally, we found that phosphoinositide 3-kinase plays a role in the regulation of β-catenin inhibition of NF-κB through the disruption of the β-catenin/NF-κB complex. This study provides a link between two important signal transduction pathways as well as another mechanism of β-catenin oncogenesis. ^

14-3-3zeta overexpression in multiple human cancers plays a critical role in cancer progression

14-3-3 is a family of highly conserved and ubiquitously expressed proteins in eukaryotic organisms. 14-3-3 isoforms bind in a phospho-serine/threonine-dependent manner to a host of proteins involved in essential cellular processes including cell cycle, signal transduction and apoptosis. We fortuitously discovered 14-3-3 zeta overexpression in many human primary cancers, such as breast, lung, and sarcoma, and in a majority of cancer cell lines. To determine 14-3-3 zeta involvement in breast cancer progression, we used immunohistochemical analysis to examine 14-3-3 zeta expression in human primary invasive breast carcinomas. High 14-3-3 zeta expression was significantly correlated with poor prognosis of breast cancer patients. Increased expression of 14-3-3 zeta was also significantly correlated with elevated PKB/Akt activation in patient samples. Thus, 14-3-3 zeta is a marker of poor prognosis in breast cancers. Furthermore, up-regulation of 14-3-3 zeta enhanced malignant transformation of cancer cells in vitro. ^ To determine the biological significance of 14-3-3 zeta in human cancers, small interfering RNAs (siRNA) were used to specifically block 14-3-3 zeta expression in cancer cells. 14-3-3 zeta siRNA inhibited cellular proliferation by inducing a G1 arrest associated with up-regulation of p27 KIP1 and p21CIP1 cyclin dependent kinase inhibitors. Reduced 14-3-3 zeta inhibited PKB/Akt activation while stimulating the p38 signaling pathway. Silencing 14-3-3 zeta expression also increased stress-induced apoptosis by caspase activation. Notably, 14-3-3 zeta siRNA inhibited transformation related properties of breast cancer cells in vitro and inhibited tumor progression of breast cancer cells in vivo. 14-3-3 zeta may be a key regulatory factor controlling multiple signaling pathways leading to tumor progression. ^ The data indicate 14-3-3 zeta is a major regulator of cell growth and apoptosis and may play a critical role in the development of multiple cancer types. Hence, blocking 14-3-3 zeta may be a promising therapeutic approach for numerous cancers. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

A mechanism of insulin-like growth factor binding protein 2-induced cell mobility in glioma

Insulin-like growth factor binding protein 2 (IGFBP2) is a protein known to be overexpressed in a majority of glioblastoma multiforme (GBM) tumors. While it is known the IGFBP2 is involved in promoting GBM tumor cell invasion, no mechanism exists for how the protein is involved in signal transduction pathways leading to enhanced cell invasion. ^ We follow up on preliminary microarray data on IGFBP2-overexpressing GBM cells and protein sequence analysis of IGFBP2 in generating the hypothesis that IGFBP2 interacts with integnn α5 in regulating cell mobility. Microarray data showing upregulation of integrin α5 by IGFBP2 is validated and evidence of protein-protein interaction between IGFBP2 and integrin α5 is found. The exact binding domain on IGFBP2 responsible for its interaction with integrin α5 is also determined, confirming our initial findings and reaffirming that the IGFBP2/integrin α5 interaction is specific. Disruption of this interaction resulted in attenuation of IGFBP2-enhanced cell mobility. Further, we found that cell mobility is only enhanced when IGFBP2 and integrin α5 are both overexpressed and able to interact with each other. ^ We also determined fibronectin to be a critical player in the activation of the IGFBP2/integrin α5 pathway. The activation of this pathway appears to be progressive and initiates once GBM cells have sufficiently established anchorage. ^

14-3-3 zeta overexpression in early stage breast disease and tumor progression

Recent progress in diagnostic tools allows many breast cancers to be detected at an early pre-invasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. 14-3-3 is a family of highly conserved and ubiquitously expressed proteins that are expressed in all eukaryotic organisms. In mammals there are seven isoforms, which bind to phosphor-serine/threonine residues regulating essential cellular processes such as signal transduction, cell cycle progression, and apoptosis. Our laboratory has discovered that a particular 14-3-3 family member, Zeta, is overexpressed in over 40% of breast tumor tissues. Furthermore, I examined the stage of breast disease in which 14-3-3ζ overexpression occurs and found that increased expression of 14-3-3ζ begins at the stage of atypical ductal hyperplasia, a very early stage of breast disease that confers increased risk for progress toward breast cancer. To determine whether 14-3-3ζ overexpression is a decisive early event in breast cancer, I overexpressed 14-3-3ζ in MCF10A cells, a non-transformed mammary epithelial cell (MEC) line and examined its impact on acini formation in a three dimensional (3D) culture model which simulates a basic unit of structure in the mammary gland. I discovered that 14-3-3ζ overexpression severely disrupted the acini architecture resulting in the disruption of polarity and luminal filling. Both are critical morphological events in the pre-neoplastic breast disease. This thesis focuses on the molecular mechanism of luminal filling. Proper lumen formation is a result of anoikis, a specific type apoptosis of cells not attached to the basement membrane. I found that 14-3-3ζ overexpression conferred a resistance to anoikis. Additionally, 14-3-3ζ overexpression in MCF10A cells and in MECs from 14-3-3ζ transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3ζ induced hyperactivation of the PI3K/Akt pathway which led to phosphorylation and translocation of the MDM2 to the nucleus resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3ζ overexpressing MCF10A acini in 3D cultures. These data suggest that 14-3-3ζ overexpression is a critical event in early breast disease and down-regulation of p53 is one of the mechanisms by which 14-3-3ζ alters MEC acini structure and may increase the risk of progression to breast cancer. ^