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1.  Nrf2 amplifies oxidative stress via induction of Klf9 
Molecular cell  2014;53(6):916-928.
Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feed-forward regulation of ROS and identify Klf9 as a novel ubiquitous regulator of oxidative stress and lung injury.
PMCID: PMC4049522  PMID: 24613345
2.  Integrin-β5 and zyxin mediate formation of ventral stress fibers in response to transforming growth factor β 
Cell Cycle  2013;12(21):3377-3389.
Cell adhesion to the extracellular matrix is an essential element of various biological processes. TGF-β cytokines regulate the matrix components and cell–matrix adhesions. The present study investigates the molecular organization of TGF-β-induced matrix adhesions. The study demonstrates that in various mouse and human epithelial cells TGF-β induces cellular structures containing 2 matrix adhesions bridged by a stretch of actin fibers. These structures are similar to ventral stress fibers (VSFs). Suppression of integrin-β5 by RNA interference reduces VSFs in majority of cells (>75%), while overexpression of integrin-β5 fragments revealed a critical role of a distinct sequence in the cytoplasmic domain of integrin-β5 in the VSF structures. In addition, the integrity of actin fibers and Src kinase activity contribute to integrin-β5-mediated signaling and VSF formation. TGF-β-Smad signaling upregulates actin-regulatory proteins, such as caldesmon, zyxin, and zyxin-binding protein Csrp1 in mouse and human epithelial cells. Suppression of zyxin markedly inhibits formation of VSFs in response to TGF-β and integrin-β5. Zyxin is localized at actin fibers and matrix adhesions of VSFs and might bridge integrin-β5-mediated adhesions to actin fibers. These findings provide a platform for defining the molecular mechanism regulating the organization and activities of VSFs in response to TGF-β.
PMCID: PMC3895427  PMID: 24036928
TGF-beta; adhesion; epithelial-mesenchymal transition; integrin; ventral stress fibers; zyxin
3.  Androgen-responsive Serum Response Factor target genes regulate prostate cancer cell migration 
Carcinogenesis  2013;34(8):1737-1746.
Progression of prostate cancer (CaP) relies on androgen receptor (AR) signaling, but AR-dependent events that underlie the lethal phenotype remain unknown. Recently, an indirect mechanism of androgen action in which effects of AR on CaP cells are mediated by Serum Response Factor (SRF) has been identified. This is the first mode of androgen action to be associated with aggressive CaP and disease recurrence. The manner in which androgen-responsive SRF activity controls aggressive CaP cell behavior is unknown. Here, the contribution of two representative SRF effector genes that are underexpressed, calponin 2 (CNN2), or overexpressed, sidekick-homolog 1 (SDK1), in clinical CaP specimens is studied. AR- and SRF- dependency of CNN2 and SDK1 expression was verified using synthetic and natural androgens, antiandrogens, and small interfering RNAs targeting AR or SRF, and evaluating the kinetics of androgen induction and SRF binding to endogenously and exogenously expressed regulatory gene regions in AR-positive CaP model systems that mimic the transition from androgen-stimulated to castration-recurrent disease. Small interfering RNA-mediated deregulation of CNN2 or SDK1 expression did not affect CaP cell proliferation or apoptosis but had marked effects on CaP cell morphology and actin cytoskeleton organization. Loss of CNN2 induced cellular protrusions and increased CaP cell migration, whereas silencing of SDK1 led to cell rounding and blunted CaP cell migration. Changes in cell migration did not involve epithelial-mesenchymal transition but correlated with altered β1-integrin expression. Taken together, individual androgen-responsive SRF target genes affect CaP cell behavior by modulating cell migration, which may have implications for therapeutic intervention downstream of AR and SRF.
PMCID: PMC3731805  PMID: 23576568
4.  Integrin β5 contributes to the tumorigenic potential of breast cancer cells through Src-FAK and MEK-ERK signaling pathways 
Oncogene  2012;32(25):3049-3058.
Cancer progression, response to therapy and metastasis depend on tumor microenvironment. Integrins are cell adhesion receptors that mediate interactions of cells with extracellular matrix (ECM). The αv-β-family of integrins contributes to tumorigenesis, response to therapy and cancer-stem cell biology. Thus, understanding the function of specific integrins in cancer is critical for development of therapeutic approaches targeting integrins. The study investigated the role of integrin β5 in breast carcinomas by depleting integrin β5 using RNA interference and re-expression of integrin β5. Depletion of integrin β5 in triple-negative breast carcinoma cells markedly reduced tumor take, growth, and tumor angiogenesis, while re-expression of integrin β5 rescued this phenotype. Reduction in tumor angiogenesis is associated with lower expression of VEGF-A in integrin β5-depleted tumors. Tumor cells deficient for integrin β5 have lower migration and proliferative capacities. Biochemical assays revealed that integrin β5 mediates Src-FAK and MEK-ERK signaling events that operate independently, and inhibition of these pathways phenocopies integrin β5-deficiency. Breast carcinoma cells express high levels of integrin β5, whereas expression of integrin β3 is limited to stromal compartments and integrin β6 is lost in metastatic cells. Together these findings show a critical role for integrin β5 in the tumorigenic potential of breast carcinoma cells and therapeutic targeting of integrin β5 is especially attractive for triple-negative breast carcinomas, which are refractory to most of the current therapies.
PMCID: PMC3481019  PMID: 22824793
Integrin; focal adhesion kinase (FAK); extracellular signal-regulated kinase (ERK); anchorage-independent growth; transforming growth factor β (TGF-β)
5.  JunB contributes to Id2 repression and the epithelial–mesenchymal transition in response to transforming growth factor–β 
The Journal of Cell Biology  2012;196(5):589-603.
JunB helps set in motion the transcriptional program necessary for the epithelial–mesenchymal transition and tissue fibrosis in response to TGF-β.
The process of epithelial–mesenchymal transition (EMT) in response to transforming growth factor–β (TGF-β) contributes to tissue fibrosis, wound healing, and cancer via a mechanism that is not fully understood. This study identifies a critical role of JunB in the EMT and profibrotic responses to TGF-β. Depletion of JunB by small interfering ribonucleic acid abrogates TGF-β–induced disruption of cell–cell junctions, formation of actin fibers, focal adhesions, and expression of fibrotic proteins. JunB contributes to Smad-mediated repression of inhibitor of differentiation 2 through interaction with transcription repressor activating transcription factor 3. Importantly, JunB mediates the TGF-β induction of profibrotic response factors, fibronectin, fibulin-2, tropomyosin (Tpm1), and integrin-β3, which play critical roles in matrix deposition, cell–matrix adhesion, and actin stress fibers. In summary, JunB provides important input in setting the transcriptional program of the EMT and profibrotic responses to TGF-β. Thus, JunB represents an important target in diseases associated with EMT, including cancer and fibrosis.
PMCID: PMC3307698  PMID: 22391036
6.  TAK1-TAB2 signaling contributes to bone destruction by breast carcinoma cells 
Molecular cancer research : MCR  2011;9(8):1042-1053.
Advanced-stage breast cancers frequently metastasize to the bones and cause bone destruction, but the underlying mechanism is not fully understood. This study presents evidence that TGF-β-activated protein kinase 1 (TAK1) signaling in tumor cells promotes bone destruction by metastatic breast carcinoma cells, controlling expression of pro-metastatic factors, including MMP-9 and COX2. Suppression of TAK1 signaling by dominant-negative (dn) TAK1 in breast carcinoma MDA-MB-231 cells impairs bone colonization by carcinoma cells and bone osteolysis in the intra-cardiac injection model. Mechanistic studies showed that inhibition of TAK1 by dn-TAK1 or siRNA blocked expression of factors implicated in bone metastasis, such as MMP-9, COX2/PTGS2, PTHrP, and IL8, but did not affect activation of p38MAPK by TGF-β. TAK1 signaling is mediated by TAK1-binding partners TAB1, TAB2 and TAB3. Carcinoma cells express elevated mRNA levels of TAB2 and TAB3, whereas the TAB1 expression is noticeably low. Accordingly, depletion of TAB2 by siRNA reduced expression of MMP-9 and COX2. Together, these studies demonstrate that the TAK1-TAB2/TAB3 signaling axis is critical for carcinoma-induced bone lesions, mediating expression of pro-invasive and osteolytic factors. These findings identify the TAK1-TAB2 axis as a potential therapeutic target in bone metastasis.
PMCID: PMC3157546  PMID: 21700681
Invasion; metastasis; bone metastasis; TAK1; TGF-beta
7.  TGF-β autocrine pathway and MAPK signaling promote cell invasiveness and in vivo mammary adenocarcinoma tumor progression 
Oncology Reports  2012;28(2):567-575.
Breast cancer progression and metastasis have been linked to abnormal signaling by transforming growth factor-β (TGF-β) cytokines. In early-stage breast cancers, TGF-β exhibits tumor suppressor activity by repressing cell proliferation and inducing cell death, whereas in advanced-stage tumors, TGF-β promotes invasion and metastatic dissemination. The molecular mechanisms underlying pro-oncogenic activities of TGF-β are not fully understood. The present study validates the role of TGF-β signaling in cancer progression and explores mediators of pro-oncogenic TGF-β activities using the LM3 mammary adenocarcinoma cell line, derived from a spontaneous murine mammary adenocarcinoma. Expression of kinase-inactive TGF-β receptors decreased both basal and TGF-β-induced invasion. Analysis of signal transduction mediators showed that p38MAPK and MEK contribute to TGF-β stimulation of cell motility and invasion. TGF-β disrupted the epithelial actin structures supporting cell-cell adhesions, and increased linear actin filaments. Moreover, MEK and p38MAPK pathways showed opposite effects on actin remodeling in response to TGF-β. Blockade of Raf-MEK signaling enhanced TGF-β induction of actin stress-fibers whereas p38MAPK inhibitors blocked this effect. A novel observation was made that TGF-β rapidly activates the actin nucleation Arp2/3 complex. In addition, TGF-β stimulated matrix metalloproteinase MMP-9 secretion via a MAPK-independent pathway. Experiments using syngeneic mice showed that kinase-inactive TGF-β receptors inhibit the first stages of LM3 tumor growth in vivo. Our studies demonstrate that autocrine TGF-β signaling contributes to the invasive behavior of mammary carcinoma cells. Moreover, we show that both MAPK-dependent and -independent pathways are necessary for TGF-β-induced effects. Therefore, MEK-ERK and p38 MAPK pathways are potential venues for therapeutic intervention in pro-oncogenic TGF-β signaling.
PMCID: PMC3981025  PMID: 22614218
transforming growth factor-β; migration; invasion; matrix metalloproteinase-9; actin cytoskeleton; mammary adenocarcinoma
8.  Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin 
PLoS ONE  2011;6(9):e24792.
The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho0) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho0 derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho0 breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2.
PMCID: PMC3174207  PMID: 21935467
9.  Tumorigenic transformation of human breast epithelial cells induced by mitochondrial DNA depletion 
Cancer biology & therapy  2008;7(11):1732-1743.
Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (ρ0 cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and ρ0 epithelial cells. The focal proteins in these networks include (i) FN1 (fibronectin) and (ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 3 of 6 members of peroxiredoxin whose expression were altered in ρ0 epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect chromosomal stability. Consistent with above finding our study revealed an increase in DNA double strand breaks and unique chromosomal rearrangements in ρ0 breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that (i) claudin-1 and 7 were indeed downregulated in ρ0 breast epithelial cells, (ii) downregulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and (iii) claudin-1 and -7 were also downregulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells.
PMCID: PMC2783327  PMID: 19151587
mitochondria; breast tumorigenesis; mtDNA depletion; claudin-1; claudin-7; ρ0 cells; mitochondrial DNA
10.  A Critical Role of Tropomyosins in TGF-β Regulation of the Actin Cytoskeleton and Cell Motility in Epithelial Cells 
Molecular Biology of the Cell  2004;15(10):4682-4694.
We have investigated transforming growth factor beta (TGF-β)–mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-β via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, α-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-β induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-β induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-β induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-β induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-β control of cell motility, and the loss of this TGF-β response is a critical step in the acquisition of metastatic phenotype by tumor cells.
PMCID: PMC519159  PMID: 15317845
11.  Transforming Growth Factor-β1 Mediates Epithelial to Mesenchymal Transdifferentiation through a RhoA-dependent Mechanism 
Molecular Biology of the Cell  2001;12(1):27-36.
Transforming growth factor-β1 (TGF-β) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-β are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-β–induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-β do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-β rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160ROCK, by the expression of dominant-negative mutants, inhibited TGF-β–mediated EMT. The data suggest that TGF-β rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.
PMCID: PMC30565  PMID: 11160820
12.  Transforming Growth Factor β Enhances Epithelial Cell Survival via Akt-dependent Regulation of FKHRL1 
Molecular Biology of the Cell  2001;12(11):3328-3339.
The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor β (TGFβ) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGFβ-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGFβ. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGFβ-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGFβ. Serum starvation-induced apoptosis was also inhibited by TGFβ in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGFβ. Taken together, these data suggest that TGFβ may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.
PMCID: PMC60258  PMID: 11694570

Results 1-12 (12)