Kimberly A Brown

Publications of Kimberly A Brown

• Identification of novel Smad2 and Smad3 associated proteins in response to TGF-beta1.

  Authors: Kimberly A Brown, Amy-Joan L Ham, Cara N Clark, Nahum Meller, Brian K Law, Anna Chytil, Nikki Cheng, Jennifer A Pietenpol, Harold L Moses

  Journal of cellular biochemistry. 09/2008;

  Transforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute toTransforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-beta1 signals through the TGF-beta type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A non-transformed human cell line was transduced with Myc-His(6)-Smad2 or Myc-His(6)-Smad3-expressing retrovirus and was treated with TGF-beta1. Myc-His(6)-Smad2 or Myc-His(6)-Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-beta1 signaling. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

• A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling.

  Authors: Kimberly A Brown, Jennifer A Pietenpol, Harold L Moses

  Journal of cellular biochemistry. 06/2007; 101(1):9-33.

  Transforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute toTransforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. Smad2 and Smad3 are direct mediators of TGF-beta signaling, however little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-beta target genes, suggest that Smad2 and Smad3 have distinct roles in TGF-beta signaling. The observation that TGF-beta inhibits proliferation of Smad3-null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that Smad3 has a different role in epithelial cells and fibroblasts. Herein, the current understanding of Smad2 and Smad3-mediated TGF-beta signaling and their relative roles are discussed, in addition to potential mechanisms for the selective activation of Smad2 versus Smad3. Since alterations in the TGF-beta signaling pathway play an important role in promoting tumorigenesis and cancer progression, methods for therapeutic targeting of the TGF-beta signaling pathway are being pursued. Determining how Smad2 or Smad3 differentially regulate the TGF-beta response may translate into developing more effective strategies for cancer therapy.

• Epidermal growth factor receptor plays a significant role in hepatocyte growth factor mediated biological responses in mammary epithelial cells.

  Authors: Alyssa R Bonine-Summers, Mary E Aakre, Kimberly A Brown, Carlos L Arteaga, Jennifer A Pietenpol, Harold L Moses, Nikki Cheng

  Cancer biology & therapy. 05/2007; 6(4):561-70.

  Breast cancers often have deregulated hepatocyte growth factor (HGF) and c-Met signaling that results in increased tumor growth and invasion. Elucidating the mechanism responsible for HGF/c-MetBreast cancers often have deregulated hepatocyte growth factor (HGF) and c-Met signaling that results in increased tumor growth and invasion. Elucidating the mechanism responsible for HGF/c-Met action in breast cancer progression has been difficult as c-Met communicates with a number of secondary receptors that can lead to various pathological outcomes. Understanding how these secondary receptors facilitate HGF/c-Met cellular responses will aid in the development of better therapeutic treatment options for breast cancer patients with elevated HGF signaling. In the present study it was shown that the epidermal growth factor receptor (EGFR) plays a significant role in HGF/c-Met mediated biological activities indicative of advanced tumor pathology, including enhanced proliferation and invasion. The clinically relevant EGFR inhibitor gefitinib was used to determine the role of EGFR in HGF-induced proliferation and motility in several mammary carcinoma cells including PyVmT, MDA-MB-231 and 4T1. Our analyses indicated that EGFR inhibition significantly blocked HGF activation of c-Met and EGFR and that inhibition of these pathways mitigated HGF induced proliferation and motility. The data indicate that this inhibition was not through a direct effect of gefitinib on c-Met, but that EGFR is necessary for c-Met activation in the assays performed. These results provide a novel mechanism of action for EGFR as a mediator of HGF signaling thereby linking EGFR to the oncogenic potential of c-Met in mammary carcinomas cells.

• Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis.

  Authors: Christopher E Barbieri, Luo Jia Tang, Kimberly A Brown, Jennifer A Pietenpol

  Cancer research. 09/2006; 66(15):7589-97.

  p63, a homologue of the tumor suppressor p53, is critical for the development and maintenance of squamous epithelia. p63 is specifically expressed in the basal layers of stratified epithelial tissuesp63, a homologue of the tumor suppressor p53, is critical for the development and maintenance of squamous epithelia. p63 is specifically expressed in the basal layers of stratified epithelial tissues and is considered a specific marker for cells of this type. The role of p63 in tumorigenesis remains poorly defined. Numerous studies have highlighted the oncogenic potential of the predominant p63 isoform DeltaNp63alpha; however, data suggest that other p63 proteins can act as tumor suppressors or alter the metastatic potential of tumors. DeltaNp63alpha can act as a transcriptional repressor, but the link between the transcriptional functions of p63 and its biological role is still unclear. In this study, we used a loss-of-function approach to investigate the transcriptional programs controlled by p63. Disruption of p63 in squamous cell lines resulted in down-regulation of transcripts specifically expressed in squamous tissues and a significant alteration of keratinocyte differentiation. Interestingly, we found that disruption of p63 led to up-regulation of markers of nonepithelial tissues (mesenchyme and neural tissue) in both primary and immortalized squamous cells. Many of these up-regulated genes are associated with increased capacity for invasion and metastasis in tumors. Furthermore, loss of p63 expression was accompanied by a shift toward mesenchymal morphology and an increase in motility in primary keratinocytes and squamous cell lines. We conclude that loss of endogenous p63 expression results in up-regulation of genes associated with invasion and metastasis, and predisposes to a loss of epithelial and acquisition of mesenchymal characteristics. These findings have implications for the role of p63 in both development and tumorigenesis.

• Loss of TGF-beta type II receptor in fibroblasts promotes mammary carcinoma growth and invasion through upregulation of TGF-alpha-, MSP- and HGF-mediated signaling networks.

  Authors: Nikki Cheng, Neil A Bhowmick, Anna Chytil, Agnieszka E Gorksa, Kimberly A Brown, Rebecca Muraoka, Carlos L Arteaga, Eric G Neilson, Simon W Hayward, Harold L Moses

  Oncogene. 08/2005; 24(32):5053-68.

  Stromal fibroblasts regulate epithelial cell behavior through direct and indirect cell-cell interactions. To clarify the role of TGF-beta signaling in stromal fibroblasts during mammary developmentStromal fibroblasts regulate epithelial cell behavior through direct and indirect cell-cell interactions. To clarify the role of TGF-beta signaling in stromal fibroblasts during mammary development and tumorigenesis, we conditionally knocked out the TGF-beta type II receptor gene in mouse mammary fibroblasts (Tgfbr2(fspKO)). Tgfbr2(fspKO) mice exhibit defective mammary ductal development, characterized in part by increased ductal epithelial cell turnover associated with an increase in stromal fibroblast abundance. Tgfbr2(fspKO) mammary fibroblasts transplanted with mammary carcinoma cells promote growth and invasion, which is associated with increased activating phosphorylation of the receptors: erbB1, erbB2, RON, and c-Met. Furthermore, the increased receptor phosphorylation correlates with increased secretion of the cognate ligands by Tgfbr2(fspKO) fibroblasts. Treatment of tumor cells with fibroblast-conditioned medium leads to increased tumor cell proliferation and motility, which are blocked by addition of pharmacologic inhibitors of TGF-alpha signaling or neutralizing antibodies to macrophage-stimulating protein (MSP), HGF, or c-Met. These studies characterize a significant role for stromal TGF-beta signaling in mammary tissue homeostasis and mammary tumor progression via regulation of TGF-alpha, MSP, and HGF signaling pathways.

• Transforming growth factor-beta induces Cdk2 relocalization to the cytoplasm coincident with dephosphorylation of retinoblastoma tumor suppressor protein.

  Authors: Kimberly A Brown, Richard L Roberts, Carlos L Arteaga, Brian K Law

  Breast cancer research : BCR. 02/2004; 6(2):R130-9.

  BACKGROUND: The transforming growth factor-beta (TGF-beta) signaling pathway functions to prevent tumorigenesis, and loss of sensitivity to TGF-beta-mediated cell cycle arrest is nearly ubiquitousBACKGROUND: The transforming growth factor-beta (TGF-beta) signaling pathway functions to prevent tumorigenesis, and loss of sensitivity to TGF-beta-mediated cell cycle arrest is nearly ubiquitous among human cancers. Our previous studies demonstrated that rapamycin potentiates TGF-beta-induced cell cycle arrest in nontransformed epithelial cells and partially restores TGF-beta-induced growth arrest of some human cancer cell lines. Growth arrest correlated with increased binding of p21 and p27 to cyclin-dependent kinase-2 (Cdk2), and inhibition of Cdk2 kinase activity. However, it was unclear how TGF-beta caused increased binding of p21 and p27 to Cdk2. METHODS: Cell fractionation and immunofluorescence microscopy experiments were performed to examine the effect of TGF-beta on the intracellular localization of Cdk2, p21, and p27. Kinase assays were performed on cytoplasmic and nuclear extracts to determine how TGF-beta altered Cdk2 activity in both subcellular compartments. RESULTS: In breast epithelial cells treatment with TGF-beta induced a decrease in nuclear Cdk2 concentrations and relocalization of Cdk2 to the cytoplasm. Cdk2 relocalization to the cytoplasm correlated with dephosphorylation of nuclear retinoblastoma tumor suppressor protein and decreased nuclear Cdk2 activity. In these epithelial cell lines, p21 and p27 were localized primarily in the cytoplasm. Decreases in nuclear Cdk2 concentrations correlated with increased binding of Cdk2 to cytoplasmic p21 and p27. CONCLUSION: Cooperative growth arrest induced by treatment with TGF-beta + rapamycin causes inhibition of nuclear Cdk2 activity through multiple mechanisms, including Cdk2 relocalization to the cytoplasm, increased p27 and p21 binding to Cdk2, and increased phosphorylation of nuclear Cdk2 on its inhibitory site, Tyr15.

• Induction by transforming growth factor-beta1 of epithelial to mesenchymal transition is a rare event in vitro.

  Authors: Kimberly A Brown, Mary E Aakre, Agnieska E Gorska, James O Price, Sakina E Eltom, Jennifer A Pietenpol, Harold L Moses

  Breast cancer research : BCR. 02/2004; 6(3):R215-31.

  INTRODUCTION: Transforming growth factor (TGF)-beta1 is proposed to inhibit the growth of epithelial cells in early tumorigenesis, and to promote tumor cell motility and invasion in the later stagesINTRODUCTION: Transforming growth factor (TGF)-beta1 is proposed to inhibit the growth of epithelial cells in early tumorigenesis, and to promote tumor cell motility and invasion in the later stages of carcinogenesis through the induction of an epithelial to mesenchymal transition (EMT). EMT is a multistep process that is characterized by changes in cell morphology and dissociation of cell-cell contacts. Although there is growing interest in TGF-beta1-mediated EMT, the phenotype is limited to only a few murine cell lines and mouse models. METHODS: To identify alternative cell systems in which to study TGF-beta1-induced EMT, 18 human and mouse established cell lines and cultures of two human primary epithelial cell types were screened for TGF-beta1-induced EMT by analysis of cell morphology, and localization of zonula occludens-1, E-cadherin, and F-actin. Sensitivity to TGF-beta1 was also determined by [3H]thymidine incorporation, flow cytometry, phosphorylation of Smad2, and total levels of Smad2 and Smad3 in these cell lines and in six additional cancer cell lines. RESULTS: TGF-beta1 inhibited the growth of most nontransformed cells screened, but many of the cancer cell lines were insensitive to the growth inhibitory effects of TGF-beta1. In contrast, TGF-beta1 induced Smad2 phosphorylation in the majority of cell lines, including cell lines resistant to TGF-beta1-mediated cell cycle arrest. Of the cell lines screened only two underwent TGF-beta1-induced EMT. CONCLUSION: The results presented herein show that, although many cancer cell lines have lost sensitivity to the growth inhibitory effect of TGF-beta1, most show evidence of TGF-beta1 signal transduction, but only a few cell lines undergo TGF-beta1-mediated EMT.

• Activation of the Erk pathway is required for TGF-beta1-induced EMT in vitro.

  Authors: Lu Xie, Brian K Law, Anna M Chytil, Kimberly A Brown, Mary E Aakre, Harold L Moses

  Neoplasia (New York, N.Y.). 6(5):603-10.

  Transforming growth factor-beta1 (TGF-beta1) can be tumor-suppressive through the activation of the Smad-mediated signaling pathway. TGF-beta1 can also enhance tumor progression by stimulatingTransforming growth factor-beta1 (TGF-beta1) can be tumor-suppressive through the activation of the Smad-mediated signaling pathway. TGF-beta1 can also enhance tumor progression by stimulating epithelial-to-mesenchymal transition (EMT) through additional pathways. EMT is characterized by the acquisition of a fibroblast-like cell morphology, dissolution of tight junctions, disruption of adherence junctions, and formation of actin stress fibers. There is evidence linking the activation of mitogen-activated protein kinase pathways to the induction of TGF-beta1-mediated EMT. However, the role of Erk in the induction of TGF-beta1-mediated EMT remains unclear. TGF-beta1 treatment of normal murine mammary gland (NMuMG) epithelial cells resulted in increased gene expression of Ras, Raf, MEK1/2, and Erk1/2, as shown by microarray analysis and real-time polymerase chain reaction. Upon 24 and 48 hours of treatment with TGF-beta1, NMuMG and mouse cortical tubule (MCT) epithelial cells underwent EMT as shown by changes in cell morphology, delocalization of zonula occludens-1 and E-cadherin from cell-cell junctions, and formation of actin stress fibers. TGF-beta1 treatment also resulted in increased levels of phosphorylated Erk and Erk kinase activity. Treatment with an MEK inhibitor, U0126, inhibited increased Erk phosphorylation and kinase activity, and blocked TGF-beta1-induced EMT in both cell lines. These data show that TGF-beta1 induces the activation of the Erk signaling pathway, which is required for TGF-beta1-mediated EMT in vitro.