High Motility of Triple-negative Breast Cancer Cells Is Due to Repression of Plakoglobin Gene by Metastasis Modulator Protein SLUG*

High motility of aggressive breast cancer cells is associated with high SLUG and low plakoglobin levels.  SLUG binds to plakoglobin gene promoter and represses its expression.  SLUG-induced increase in breast cancer cell motility is due to repression of plakoglobin by SLUG.  Management of SLUG level should diminish the motility and thus aggressiveness in breast cancer cells. One of highly pathogenic breast cancer cell types are the triple negative (negative in the expression of estrogen, progesterone, and ERBB2 receptors) breast cancer cells. These cells are highly motile and metastatic and are characterized by high levels of the metastasis regulator protein SLUG. Using isogenic breast cancer cell systems we have shown here that high motility of these cells is directly correlated with the levels of the SLUG in these cells. Because epithelial/mesenchymal cell motility is known to be negatively regulated by the catenin protein plakoglobin, we postulated that the transcriptional repressor protein SLUG increases the motility of the aggressive breast cancer cells through the knockdown of the transcription of the plakoglobin gene. We found that SLUG inhibits the expression of plakoglobin gene directly in these cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Blocking of SLUG function with a double-stranded DNA decoy that competes with the E2-box binding of SLUG also increased the levels of plakoglobin mRNA, protein, and promoter activity in the SLUG-high triple negative breast cancer cells. Overexpression of SLUG in the SLUG-deficient cells elevated the motility of these cells. Knockdown of plakoglobin in these low motility non-invasive breast cancer cells rearranged the actin filaments and increased the motility of these cells. Forced expression of plakoglobin in SLUG-high cells had the reverse effects on cellular motility. This study thus implicates SLUG-induced repression of plakoglobin as a motility determinant in highly disseminating breast cancer.

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title: Biology, metastatic patterns, and treatment of patients with triple-negative breast cancer.
abstract: Of the estimated 1 million cases of breast cancer diagnosed annually worldwide, it is estimated that over 170,000 will harbor the triple-negative (estrogen receptor/progesterone receptor/HER2-negative) phenotype. Most, though not all, triple-negative breast cancers will be basal-like on gene expression micorarrays. The basal-like molecular subtype exhibits a unique molecular profile and set of risk factors, aggressive and early pattern of metastasis, limited treatment options, and poor prognosis. Large population-based studies have identified a higher proportion of triple-negative breast tumors among premenopausal African American women, and a suggestion that increased parity, younger age at first-term pregnancy, shorter duration of breast feeding, and elevated hip-to-waist ratio might be particular risk factors. When BRCA1 mutation carriers develop breast cancer, it is usually basal-like; given the central role of BRCA1 in DNA repair, this could have profound therapeutic implications. When diagnosed, triple-negative breast cancers illustrate preferential relapse in visceral organs, including the central nervous system. Although initial response to chemotherapy might be more profound, relapse is early and common among triple-negative breast cancers compared with luminal breast cancers. The armamentarium of "targeted therapeutics" for triple-negative breast cancer is evolving and includes strategies to inhibit angiogenesis, epidermal growth factor receptor, and other kinases. Finally, the positive association between triple-negative breast cancer and BRCA mutations makes inhibition of poly(adenosine diphosphate-ribose) polymerase-1 an attractive therapeutic strategy that is in active study.

title: Higher population‐based incidence rates of triple‐negative breast cancer among young African‐American women
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title: The skinny on slug
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title: Role of the epithelial-mesenchymal transition regulator Slug in primary human cancers.
abstract: Epithelial-mesenchymal-transition (EMT) is a crucial process during morphogenesis of multi-cellular organisms. EMT not only is a normal developmental process but also plays a role in tumor invasion and metastasis. Indeed, molecules involved in EMT, such as the transcription factor and E-cadherin repressor Slug (SNAI2), have recently been demonstrated to be important for cancer cells to down-regulate epithelial markers and up-regulate mesenchymal markers in order to become motile and invasive. Here we summarize major studies focusing on Slug expression in human tumor samples. We review a total of 13 studies involving 1150 cases from 9 different types of tumors. It is becoming clear that this transcription factor plays a role in the progression of some tumor types, including breast and gastric cancer. Interestingly, Slug expression is not always associated with down-regulation of E-cadherin. The mode of action, the signaling pathways involved in its regulation, and the interplay with other EMT regulators need to be addressed in future studies in order to fully understand Slug's role in tumor progression.

title: The Snail Superfamily of Zinc-finger Transcription Factors
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title: The basal‐like breast carcinoma phenotype is regulated by SLUG gene expression
abstract: Basal‐like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up‐regulation of stem cell regulatory genes. We show here that tumour tissues expressing high levels of SLUG mRNA show a basal‐like breast carcinoma phenotype and that such tumours also express high levels of stem cell‐regulatory genes, ie CD133, Bmi1. Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal‐like breast carcinoma. We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX. In this regard, we report that the exposure of SLUG‐negative/luminal‐like MCF‐7 cells to a hypoxic environment promotes the onset of the basal‐like breast carcinoma phenotype, together with up‐regulation of the SLUG gene, which in turn blunts oestrogen receptor‐α and boosts carbonic anhydrase IX gene expression. Finally, we show that SLUG expression promotes the invasiveness of MCF‐7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia‐selected, MCF‐7‐derived cells in xenografts. These data indicate that SLUG gene expression is part of a hypoxia‐induced genetic programme which sets up a basal/stem cell‐like, aggressive phenotype in breast cancer cells.

title: The SLUG zinc-finger protein represses E-cadherin in breast cancer.
abstract: Loss of expression of the E-cadherin cell-cell adhesion molecule is important in carcinoma development and progression. Because previous data suggest that loss of E-cadherin expression in breast carcinoma may result from a dominant transcriptional repression pathway acting on the E-cadherin proximal promoter, we pursued studies of cis sequences and transcription factors regulating E-cadherin expression in breast cancer cells. E-box elements in the E-cadherin promoter were found to play a critical negative regulatory role in E-cadherin gene transcription in breast cancer cell lines lacking E-cadherin transcription. The E-box elements had a minimal role in E-cadherin transcription in breast cancer cell lines expressing E-cadherin. Two zinc-finger transcription factors known to bind E-box elements, SLUG and SNAIL, repressed E-cadherin-driven reporter gene constructs containing wild-type promoter sequences but not those with mutations in the E-box elements. Additionally, both SLUG and SNAIL repressed endogenous E-cadherin expression. These findings suggest SLUG and SNAIL are potential repressors of E-cadherin transcription in carcinomas lacking E-cadherin expression. Analysis of the expression patterns of SLUG, SNAIL, and E-cadherin in breast cancer cell lines demonstrated that expression of SLUG was strongly correlated with loss of E-cadherin transcripts. Taken together, the data indicate the E-box elements in the proximal E-cadherin promoter are critical in transcriptional repression of the E-cadherin gene, and SLUG is a likely in vivo repressor of E-cadherin in breast cancer.

title: SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination*
abstract: UbcH5c, a member of the UbcH5 family of protein ubiquitin conjugase E2 enzymes, is a critical component of biological processes in human cells, being the initial ubiquitinating enzyme of substrates like IκB, TP53, and cyclin D1. We report here that the metastasis regulator protein SLUG inhibits the expression of UbcH5c directly through chromatin remodeling and thus, among other downstream effects, elevates the level of cyclin D1, thus enhancing the growth rates of breast cancer cells. Overexpression of SLUG in the SLUG-deficient breast cancer cells significantly decreased the levels of mRNA and protein of UbcH5c but only elevated the protein levels of cyclin D1. On the contrary, knockdown of SLUG in SLUG-high breast cancer cells elevated the levels of UbcH5c while decreasing the level of cyclin D1 protein. SLUG is recruited at the E2-box sequence at the UbcH5c gene promoter along with the corepressor CtBP1 and the effector HDAC1 to silence the expression of this gene. Knockdown of UbcH5c in the SLUG-deficient human breast cells elevated the level of cyclin D1 as well as the rates of proliferation and invasiveness of these cells. Whereas the growth rates of the cells are enhanced due to overexpression of SLUG or knockdown of UbcH5c in the breast cancer cells tested, ER+ cells also acquire resistance to the anti-estrogen 4-hydroxytamoxifen due to the rise of cyclin D1 levels in these cells. This study thus implicates high levels of SLUG and low levels of UbcH5c as a determinant in the progression of metastatic breast cancer.

title: In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells.
abstract: The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628bp fragment (-613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling.

title: Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells.
abstract: Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process.

title: Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells*
abstract: The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the –701 to –921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.

title: Slug and Sox9 Cooperatively Determine the Mammary Stem Cell State
abstract: Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.

title: Plakoglobin regulates cell motility through Rho- and fibronectin-dependent Src signaling
abstract: We previously showed that the cell–cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PG−/− cells onto ECM deposited by PG+/− cells partially restored normal cell morphology and inhibited PG−/− cell motility. In over 70 adhesion molecules whose expression we previously showed to be altered in PG−/− cells, a substantial decrease in fibronectin (FN) in PG−/− cells stood out. Re-introduction of PG into PG−/− cells restored FN expression, and keratinocyte motility was reversed by plating PG−/− cells onto FN. Somewhat surprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not a decrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PG−/− cells exhibited an increase in activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PG−/− cells on ECM deposited by PG+/− keratinocytes. PG−/− cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG−/− keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM–Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability.

title: Disparate effects of different mutations in plakoglobin on cell mechanical behavior.
abstract: Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of heart and skin diseases. This has led to the hypothesis that defective cell-cell adhesion is the underlying cause of injury in tissues that repeatedly bear high mechanical loads. In this study, we examined the effects of two different mutations in plakoglobin on cell migration, stiffness, and adhesion. One is a C-terminal mutation causing Naxos disease, a recessive syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal skin and hair. The other is an N-terminal mutation causing dominant inheritance of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin mutations on a broad range of cell mechanical behavior, we characterized a model system consisting of stably transfected HEK cells which are particularly well suited for analyses of cell migration and adhesion. Both mutations increased the speed of wound healing which appeared to be related to increased cell motility rather than increased cell proliferation. However, the C-terminal mutation led to dramatically decreased cell-cell adhesion, whereas the N-terminal mutation caused a decrease in cell stiffness. These results indicate that different mutations in plakoglobin have markedly disparate effects on cell mechanical behavior, suggesting complex biomechanical roles for this protein.

title: EBV latent membrane protein 1 effects on plakoglobin, cell growth, and migration.
abstract: Latent membrane protein 1 (LMP1), the major oncoprotein of EBV, is likely responsible for many of the altered cellular growth properties in EBV-associated cancers, including nasopharyngeal carcinoma (NPC). In this study, the effects of LMP1 on cell growth and migration were studied in the context of the EBV-positive C666-1 NPC cell line. In the soft agar transformation and Transwell metastasis assays, LMP1 enhanced cell growth and migration through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappaB (NF-kappaB) signaling. Inhibitors of PI3K, Akt, and NF-kappaB signaling dramatically reduced these enhanced properties. An IkappaBalpha super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-kappaB activation are required by LMP1. These enhanced effects required the full-length LMP1 encompassing both the PI3K/Akt-activating COOH-terminal activation region (CTAR) 1 and the nonredundant NF-kappaB-activating regions CTAR1 and CTAR2. LMP2A, a latent protein that is also frequently expressed in NPC, similarly activates the PI3K/Akt pathway; however, its overexpression in C666-1 cells did not affect cell growth or migration. LMP1 also decreased expression of the junctional protein plakoglobin, which was shown to be partially responsible for enhanced migration induced by LMP1. This study reveals that in epithelial cells the transforming properties of LMP1 require activation of both PI3K/Akt and NF-kappaB and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration.

title: Aberrant activation of γ-catenin promotes genomic instability and oncogenic effects during tumor progression
abstract: γ-catenin (plakoglobin) exists in cells either as a component of adherens junctions, along with β-catenin and α-catenin, or in association with desmoplakin in desmosomes, which are in turn coupled to the cytoskeleton linking to the plasma membrane. Although γ-catenin overexpression is observed in many cancers, the molecular basis of its contribution to tumor progression remains unclear. In this study, we examined γ-catenin overexpression-mediated effects leading to altered regulation of effector genes such as PTTG and c-Myc, as well as differential activation of signaling pathways. We found that overexpression of γ-catenin caused: (1) a reduction in E-cadherin and corresponding increase in vimentin levels concomitant with increased cell mobility and migration; (2) enhancement in the levels of phosphorylated Akt and Erk in the presence of EGF; and (3) an increase in PTTG and c-Myc protein levels, which are likely to accelerate chromosomal instability and uncontrolled proliferation, respectively, in the affected cells. These effects resulting from overexpression of γ-catenin were further validated in converse experiments with the aid of siRNA knockdown of the endogenous γ-catenin gene. In conclusion, our studies provide a molecular basis for the promotion of genomic instability and the oncogenic effects due to overexpression of γ-catenin in human cancer.

title: DNMT3A and DNMT3B mediate autocrine hGH repression of plakoglobin gene transcription and consequent phenotypic conversion of mammary carcinoma cells
abstract: Directed by microarray analyses, we report that autocrine human growth hormone (hGH) increased the mRNA and protein expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B in mammary carcinoma cells. Autocrine hGH stimulation of DNMT3A and DNMT3B expression was mediated by JAK2 and Src kinases, and treatment of mammary carcinoma cells with the DNMT inhibitor, 5′-aza-2′-deoxycytidine (AZA), abrogated autocrine hGH-stimulated cellular proliferation, apoptosis and anchorage-independent growth. AZA reversed the epitheliomesenchymal transition of mammary carcinoma cells induced by autocrine hGH, to an epithelioid morphology and abrogated cell migration stimulated by autocrine hGH. Autocrine hGH-stimulated hypermethylation of the first exon of the PLAKOGLOBIN gene and AZA abrogated the ability of autocrine hGH to repress plakoglobin gene transcription. Small interfering RNA (siRNA)-mediated depletion of the individual DNMT molecules did not release autocrine hGH repression of PLAKOGLOBIN promoter activity nor did individual DNMT depletion affect autocrine hGH-stimulated migration. However, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B abrogated hypermethylation of the PLAKOGLOBIN gene stimulated by autocrine hGH and subsequent repression of plakoglobin gene transcription and increased cell migration. Thus, the autocrine hGH-stimulated increases in DNMT3A and DNMT3B expression mediate repression of plakoglobin gene transcription by direct hypermethylation of its promoter and consequent phenotypic conversion of mammary carcinoma cells. Autocrine hGH, therefore, utilizes DNA methylation as a mechanism to exert its oncogenic effects in mammary carcinoma cells.

title: Restoration of plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and tumorigenic potential
abstract: The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with <1% of cells displaying detectable nuclear localisation of plakoglobin. siRNA knockdown experiments targeting plakoglobin, revealed enhanced migration in all cell lines in the presence and absence of E-cadherin expression. In bladder cell lines expressing low levels of plakoglobin and desmoglein-2, elevated levels of desmoglein-2 were detected following restoration of plakoglobin expression in transfected cell lines. Analysis of wnt signalling revealed no activation event associated with plakoglobin expression in the bladder model. These results show that plakoglobin acts as a tumour suppressor gene in bladder carcinoma cells and the silencing of plakoglobin gene expression in late-stage bladder cancer is a primary event in tumour progression.

title: Plakoglobin suppresses keratinocyte motility through both cell-cell adhesion-dependent and -independent mechanisms.
abstract: Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins and has been shown to play a critical role in the organization of desmosomes and tissue integrity. Because dissolution of intercellular junctions is frequently an initial step in the onset of epithelial cell migration, we examined whether loss of PG promotes cell motility by compromising adhesive strength. Keratinocyte cultures established from PG-/-mice exhibited weakened adhesion and increased motility in transwell migration assays; both were restored by reintroducing PG through adenoviral infection. Interestingly, single PG-/- cells also exhibited increased motility, which was suppressed by reintroducing PG, but not the closely related beta-catenin. Whereas both N- and C-terminally truncated PG deletion mutants restored adhesion, only N-terminally deleted PG, but not C-terminally deleted PG, suppressed single-cell migration. Furthermore, both the chemical inhibitor PP2 and dominant-negative Src tyrosine kinase inhibited single-cell motility in PG-/- cells, whereas constitutively active Src overcame the inhibitory effect of PG. These data demonstrate that PG strengthens adhesion and suppresses motility in mouse keratinocytes, through both intercellular adhesion-dependent and -independent mechanisms, the latter of which may involve suppression of Src signaling through a mechanism requiring the PG C terminus.

title: Armadillo repeat proteins: beyond the animal kingdom.
abstract: Armadillo (Arm) repeat proteins contain tandem copies of a degenerate protein sequence motif that forms a conserved three-dimensional structure. Animal Arm repeat proteins function in various processes, including intracellular signalling and cytoskeletal regulation. A subset of these proteins are conserved across eukaryotic kingdoms, and non-metazoa such as Dictyostelium and Chlamydomonas possess homologues of members of the animal Arm repeat family. Higher plants also possess Arm repeat proteins, which, like their animal counterparts, function in intracellular signalling. Notably, these plant Arm proteins have novel functions. In addition, genome sequencing has identified a plethora of Arm-related proteins in Arabidopsis.

title: Life at the Leading Edge
abstract: Cell migration requires sustained forward movement of the plasma membrane at the cell's front or "leading edge." To date, researchers have uncovered four distinct ways of extending the membrane at the leading edge. In lamellipodia and filopodia, actin polymerization directly pushes the plasma membrane forward, whereas in invadopodia, actin polymerization couples with the extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the extracellular matrix. Membrane blebs drive the plasma membrane forward using a combination of actomyosin-based contractility and reversible detachment of the membrane from the cortical actin cytoskeleton. Each protrusion type requires the coordination of a wide spectrum of signaling molecules and regulators of cytoskeletal dynamics. In addition, these different protrusion methods likely act in concert to move cells through complex environments in vivo.

title: The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function
abstract: Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to — and degradation of — the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott–Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.

title: Invadosome regulation by adhesion signaling.
abstract: Invadosomes are adhesive mechanosensory modules composed of a dense F-actin core surrounded by a ring of adhesion molecules and able to infiltrate compact tissue environment in physiological and pathological conditions. These structures comprise podosomes that are found in a variety of cells under physiological conditions and invadopodia in transformed or cancer cells. Invadosomes are regulated by extracellular matrix signals and are endowed with degradative machinery for extracellular matrix. The ability of extracellular matrix signals to orchestrate the building, dynamics, and function of invadosomes is based on mechano-chemical integrin outside-in signaling and requires integrin cross-talk. This review highlights recent findings that place Src as an inducer and PKC as an amplifier in the assembly of integrin stimulated invadosome through mechanotransduction and polarized endo/exocytic trafficking pathways for key proteolytic and enzymatic activities in a temporally and spatially confined manner.

title: b- and g-Catenin Mutations, but not E-Cadherin Inactivation, Underlie T-Cell Factor/Lymphoid Enhancer Factor Transcriptional Deregulation in Gastric and Pancreatic Cancer
abstract: Adenomatous polyposis coli (APC) mutations are present in >70% of colon cancers. The APC protein binds to b-catenin (b-cat), a protein first identified because of its role in E-cadherin (E-cad) cell adhesion. In some colon cancers lacking APC defects, mutations in presumptive glycogen synthase kinase 3b phosphorylation sites near the b-cat NH2 terminus appear to render b-cat resistant to regulation by APC and glycogen synthase kinase 3b. In cells with APC or b-cat defects, b-cat is stabilized and, in turn, binds to and activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef) transcription factors. To further explore the role of APC, b-cat, Tcf, and E-cad defects in gastrointestinal cancers, we assessed gastric and pancreatic cancers for constitutive Tcf transcriptional activity (CTTA). Two of four gastric and two of eight pancreatic cancer lines showed CTTA. One gastric and one pancreatic cancer had mutations in the NH2terminal phosphorylation sites of b-cat. The other gastric cancer with CTTA had a missense mutation at serine 28 of g-cat, a potential phosphorylation site in this b-cat-related protein. Although E-cad is an important binding partner for b-cat and g-cat, E-cad inactivation did not result in CTTA. The b-cat and g-cat mutant proteins identified in our studies strongly activated Tcf transcription in vitro, whereas b-cat mutant proteins with large NH2-terminal deletions had only modest effects on Tcf. Our results suggest a role for Tcf deregulation in gastric and pancreatic cancer, resulting from b-cat and g-cat mutations in some cases and, in others, from yet to be defined defects. Furthermore, these data imply that the consequences of APC and b-cat mutations are distinct from the effects of E-cad inactivation.  Germ-line inactivating mutations in the APC tumor suppressor gene are responsible for familial polyposis. Somatic mutations in APC are present in ;70% of colorectal adenomas and carcinomas but are rare in other cancers (reviewed in Ref. 1). The APC protein has been found to bind to a number of proteins, including b-cat (2, 3), g-cat (also known as plakoglobin; Refs. 4 and 5), GSK3b (6), EB1 (7), hDLG (8), microtubules (9, 10), and the related proteins axin and conductin (11–14). With the exception of b-cat, GSK3b, and the conductin and axin proteins, the significance and role of APC’s interactions with its various binding partners is not well understood. b-cat, initially identified as a coprecipitating protein with E-cad, was subsequently shown to link E-cad to a-cat, a vinculin-like protein, which, in turn, links the E-cad/cat cellcell adhesion complex to the cortical cytoskeleton (15–19). Molecular cloning revealed that b-cat is a member of the armadillo (arm) family. The prototype molecule Arm functions in the wingless signaling pathway in Drosophila, with b-cat functioning in the conserved wnt pathway in vertebrates (12, 15). The g-cat protein is closely related to b-cat and appears to have similar functions in cell-cell adhesion and the wnt pathway (16–19). The identification of Tcf/Lef transcriptional factor proteins as downstream targets of b-cat (20–22), together with the observation of CTTA in colorectal cancer cell lines with APC defects (23), supported the hypothesis that APC had a critical role in regulating b-cat (24). Further support was provided by data, indicating that a subset of the colorectal Received 1/19/99; revised 3/30/99; accepted 4/2/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the William and Catherine Weldon Donaghue Foundation for Medical Research, NIH Grants GM57604 and CA70097, United States Army Grant DAMD17-94-J4366, and Deutsche Forschungsgemeinschaft Grants CA207/1 and KO1826/1. 2 The first two authors contributed equally to this work. 3 Present address: Department of Internal Medicine, University of Leipzig, Leipzig 04103, Germany. 4 Present address: GI Division, Department of Internal Medicine, Peking Union Medical College Hospital, Beijing 100730, People’s Republic of China. 5 To whom requests for reprints should be addressed, at Division of Molecular Medicine and Genetics, University of Michigan Medical Center 4301 MSRB III, Box 0638, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0638.  (734) 764-1549; Fax: (734) 647-7979;  The abbreviations used are: APC, adenomatous polyposis coli; b-cat, b-catenin; g-cat, g-catenin; GSK3b, glycogen synthase kinase 3b; E-cad, E-cadherin; Tcf, T-cell factor; Lef, lymphoid enhancer factor; CTTA, constitutive Tcf transcriptional activity; GI, gastrointestinal; RT-PCR, reverse transcriptase-PCR ECL, enhanced chemiluminescence. 369 Vol. 10, 369–376, June 1999 Cell Growth & Differentiation

title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells
abstract: BackgroundBRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study.ResultsHuman BRCA2 gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G2), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm.ConclusionsBRCA2 gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter in vivo and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.

title: Slug Antagonizes p53-Mediated Apoptosis of Hematopoietic Progenitors by Repressing puma
abstract: In response to DNA damage, the p53 tumor suppressor can elicit either apoptosis or cell-cycle arrest and repair, but how this critical decision is made in specific cell types remains largely undefined. We investigated the mechanism by which the transcriptional repressor Slug specifically rescues hematopoietic progenitor cells from lethal doses of gamma radiation. We show that Slug is transcriptionally induced by p53 upon irradiation and then protects the damaged cell from apoptosis by directly repressing p53-mediated transcription of puma, a key BH3-only antagonist of the antiapoptotic Bcl-2 proteins. We established the physiologic significance of Slug-mediated repression of puma by demonstrating that mice deficient in both genes survive doses of total-body irradiation that lethally deplete hematopoietic progenitor populations in mice lacking only slug. Thus, Slug functions downstream of p53 in developing blood cells as a critical switch that prevents their apoptosis by antagonizing the trans-activation of puma by p53.

title: Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin
abstract: Leong et al. 2007. 

[OpenUrl][1][Abstract/FREE Full Text][2]

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title: Role of epithelial-to-mesenchymal transition (EMT) in drug sensitivity and metastasis in bladder cancer
abstract: Epithelial-to-mesenchymal transition (EMT) is a process that plays essential roles in development and wound healing that is characterized by loss of homotypic adhesion and cell polarity and increased invasion and migration. At the molecular level, EMT is characterized by loss of E-cadherin and increased expression of several transcriptional repressors of E-cadherin expression (Zeb-1, Zeb-2, Twist, Snail, and Slug). Early work established that loss of E-cadherin and increased expression of MMP-9 was associated with a poor clinical outcome in patients with urothelial tumors, suggesting that EMT might also be associated with bladder cancer progression and metastasis. More recently, we have used global gene expression profiling to characterize the molecular heterogeneity in human urothelial cancer cell lines (n = 20) and primary patient tumors, and unsupervised clustering analyses revealed that the cells naturally segregate into two discrete “epithelial” and “mesenchymal” subsets, the latter consisting entirely of muscle-invasive tumors. Importantly, sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) or type-3 fibroblast growth factor receptor (FGFR3) was confined to the “epithelial” subset, and sensitivity to EGFR inhibitors could be reestablished by micro-RNA-mediated molecular reversal of EMT. The results suggest that EMT coordinately regulates drug resistance and muscle invasion/metastasis in urothelial cancer and is a dominant feature of overall cancer biology.

title: Human cancer cells express Slug-based epithelial-mesenchymal transition gene expression signature obtained in vivo
abstract: BackgroundThe biological mechanisms underlying cancer cell motility and invasiveness remain unclear, although it has been hypothesized that they involve some type of epithelial-mesenchymal transition (EMT).MethodsWe used xenograft models of human cancer cells in immunocompromised mice, profiling the harvested tumors separately with species-specific probes and computationally analyzing the results.ResultsHere we show that human cancer cells express in vivo a precise multi-cancer invasion-associated gene expression signature that prominently includes many EMT markers, among them the transcription factor Slug, fibronectin, and α-SMA. We found that human, but not mouse, cells express the signature and Slug is the only upregulated EMT-inducing transcription factor. The signature is also present in samples from many publicly available cancer gene expression datasets, suggesting that it is produced by the cancer cells themselves in multiple cancer types, including nonepithelial cancers such as neuroblastoma. Furthermore, we found that the presence of the signature in human xenografted cells was associated with a downregulation of adipocyte markers in the mouse tissue adjacent to the invasive tumor, suggesting that the signature is triggered by contextual microenvironmental interactions when the cancer cells encounter adipocytes, as previously reported.ConclusionsThe known, precise and consistent gene composition of this cancer mesenchymal transition signature, particularly when combined with simultaneous analysis of the adjacent microenvironment, provides unique opportunities for shedding light on the underlying mechanisms of cancer invasiveness as well as identifying potential diagnostic markers and targets for metastasis-inhibiting therapeutics.

title: Slug (SNAI2) expression in oral SCC cells results in altered cell-cell adhesion and increased motility
abstract: The Snail family of zinc finger transcription factors plays an important role in epithelial to mesenchymal transition (EMT) in a variety of tissues and systems. Slug (SNAI2) expression has been shown to directly contribute to a subset of events required for EMT in events such as re-epithelialization during wound healing and neural crest cell migration. In addition, slug expression was shown to correlate with disease recurrence in head and neck squamous cell carcinoma (HNSCC) patients. Based on this association we chose to specifically examine the effects of exogenous slug expression in HNSCC cells and specifically assess adhesive junction assembly and the motility characteristics in these cells. Slug expression led to changes in adherens junction and desmosome assembly characterized by a classical cadherin switch and loss of desmosome assembly. Additionally, we performed gene expression profiling to identify novel slug dependent gene expression changes in a HNSCC cell line. In addition to genes known to be altered during EMT, we identified a novel set of Slug responsive genes that will provide a better understanding of slug overexpression during EMT and HNSCC progression.

title: Transcriptional activation of ZEB1 by Slug leads to cooperative regulation of the epithelial-mesenchymal transition-like phenotype in melanoma.
abstract: The E-box-binding zinc finger transcription factors Slug and ZEB1 are important repressors of E-cadherin, contributing to epithelial-mesenchymal transition (EMT) in primary epithelial cancers. Activator or repressor status of EMT transcription factors defines consequences for tumorigenesis. We show that changes in expression levels of Slug in melanoma cell lines lead to concomitant alterations of ZEB1 expression. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays identified Slug as a direct transcriptional activator at E-boxes of the ZEB1 promoter. Transcriptional activation of ZEB1 was demonstrated to be specific for Slug, as EMT regulators Snail and Twist failed to influence ZEB1 expression. Slug and ZEB1 cooperatively repressed E-cadherin expression resulting in decreased adhesion to human keratinocytes, but promoted migration of melanoma cells. Our results show that the transcriptional activity of ZEB1 is increased by Slug, suggesting a hierarchical organized expression of EMT transcription factors through directed activation, triggering an EMT-like process in melanoma.

title: Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells.
abstract: Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

title: FMNL2 Enhances Invasion of Colorectal Carcinoma by Inducing Epithelial-Mesenchymal Transition
abstract: FMNL2 is a member of diaphanous-related formins that control actin-dependent processes such as cell motility and invasion. Its overexpression in metastatic cell lines and tissues of colorectal carcinoma has been associated with aggressive tumor development in our previous study. But its specific role in cancer is largely unknown. Here we report that FMNL2 is involved in epithelial-mesenchymal transition (EMT) maintenance in human colorectal carcinoma cells. A positive correlation between FMNL2 and vimentin expression and an inverse correlation between FMNL2 and E-cadherin expression were found in colorectal carcinoma cell lines and cancer tissues. Specific knockdown of FMNL2 led to an epithelial-state transition, confirmed by the cobblestone-like phenotype, upregulation of E-cadherin, α-catenin, and γ-catenin, and downregulation of vimentin, snail, slug. Loss of FMNL2 expression lowered the ability of TGF-β to induce cell invasion and EMT, as shown by morphology and the expression levels. Upregulation of vimentin, slug, snail, downregulation of E-cadherin and activation of receptor-Smad3 phosphorylation were observed in M5 and MDCK cells induced by TGF-β, whereas altered expression of these markers was not obvious in FMNL2-depleting M5 cells. High levels of activation of p-MAPK and p-MEK, but not p-PI3K and p-AKT, were observed in SW480/FMNL2+ cells compared with control cells. Treatment with U0126 could abrogate the activation of p-MAPK and p-MEK, whereas LY294002 treatment had no effect on the PI3K/AKT pathway. In conclusion, these findings identify a novel EMT and tumor promoting function for FMNL2, which is involved in TGF-β–induced EMT and colorectal carcinoma cell invasion via Smad3 effectors, or in collaboration with MAPK/MEK pathway.

title: The transcriptional corepressor CtBP: a foe of multiple tumor suppressors.
abstract: CtBP1 and CtBP2 are closely related and evolutionarily conserved transcriptional corepressors. There is strong evidence linking CtBPs to tumorigenesis and tumor progression. CtBPs promote epithelial-mesenchymal transition and function as apoptosis antagonists. Also, CtBPs mediate repression of several tumor suppressor genes. Certain tumor suppressors also target CtBPs to restrain their tumor-promoting activity. Down-regulation of CtBPs mediated by some tumor suppressors results in p53-independent apoptosis and reduced tumor cell migration and invasion. The role of CtBPs in modulating the activities of different tumor suppressors is reviewed here. The results discussed here suggest that CtBPs may constitute a novel p53-independent anticancer target.

title: Transcriptional regulation by C-terminal binding proteins.
abstract: C-terminal binding protein family members function predominantly as transcriptional corepressors in association with sequence specific DNA-binding transcriptional repressors. The vertebrates have two CtBP genes while the invertebrates contain a single gene. Genetic studies indicate that the CtBP genes play pivotal roles in animal development. The vertebrate C-terminal binding proteins (CtBP1 and CtBP2) are highly related and are functionally redundant for certain developmental processes and non-redundant for others. The animal C-terminal binding proteins exhibit structural and functional similarity to d-isomer-specific 2-hydroxy acid dehydrogenases (D2-HDH). They function as dimers, recruiting transcriptional regulators through two protein-binding interfaces in each monomer. The corepressor complex of CtBP1 contains enzymatic constituents that mediate coordinated histone modification by deacetylation and methylation of histone H3-Lysine 9 and demethylation of histone H3-Lysine 4. CtBP also recruits the small ubiquitin-related modifier (SUMO) conjugating E2 enzyme UBC9 and a SUMO E3 ligase (HPC2), suggesting that CtBP-mediated transcriptional regulation may also involve SUMOylation of transcription factors. In addition to gene-specific transcriptional repression, CtBP1 appears to antagonize the activity of the global transcriptional coactivators, p300/CBP. Genetic evidence also suggests that the fly CtBP (dCtBP) and the vertebrate CtBP2 might activate transcription in a context-dependent manner. The transcriptional regulatory activity of CtBP is modulated by the nuclear NADH/NAD+ ratio and hence appears to be influenced by the metabolic status of the cell. The nuclear dinucleotide ratio may differentially influence the repression activities of factors that recruit CtBP through PLDLS-like motifs and those through non-PLDLS-motifs.

title: Human SLUG does not directly bind to CtBP1.
abstract: SLUG is a transcriptional repressor protein implicated to have major role in the oncogenesis and metastasis of human breast cells. We previously have shown by chromatin immunoprecipitation assay that human SLUG (hSLUG) is co-localized with the co-repressor protein CtBP1 as bound to the BRCA2 gene silencer [M.K. Tripathi, S. Misra, S.V. Khedkar, N. Hamilton, C. Irvin-Wilson,, C. Sharan, L. Sealy, G. Chaudhuri,  (2005) 17163-17171]. hSLUG was predicted to be binding directly to CtBP1 because of an apparent presence of CtBP1 binding site in its amino acid sequences. Here, we provide evidence through yeast two-hybrid and in vitro co-immunoprecipitation analyses that hSLUG does not directly interacts with hCtBP1. This observation will help in the study of the mode of action of hSLUG in human cells.

title: Extracellular signal-regulated kinase signaling pathway regulates breast cancer cell migration by maintaining slug expression.
abstract: Cell migration is a critical step in cancer cell invasion. Recent studies have implicated the importance of the extracellular signal-regulated kinase (ERK) signaling pathway in cancer cell migration. However, the mechanism associated with ERK-regulated cell migration is poorly understood. Using a panel of breast cancer cell lines, we detected an excellent correlation between ERK activity and cell migration. Interestingly, we noticed that a 48-hour treatment with U0126 [specific mitogen-activated protein/ERK kinase (MEK)-1/2 inhibitor] was needed to significantly inhibit breast cancer cell migration, whereas this inhibitor blocked ERK activity within 1 hour. This observation suggests that ERK-dependent gene expression, rather than direct ERK signaling, is essential for cell migration. With further study, we found that ERK activity promoted the expression of the activator protein-1 (AP1) components Fra-1 and c-Jun, both of which were necessary for cell migration. Combination of U0126 treatment and Fra-1/c-Jun knockdown did not yield further reduction in cell migration than either alone, indicating that ERKs and Fra-1/c-Jun act by the same mechanism to facilitate cell migration. In an attempt to investigate the role of Fra-1/c-Jun in cell migration, we found that the ERK-Fra-1/c-Jun axis regulated slug expression in an AP1-dependent manner. Moreover, the occurrence of U0126-induced migratory inhibition coincided with slug reduction, and silencing slug expression abrogated breast cancer cell migration. These results suggest an association between ERK-regulated cell migration and slug expression. Indeed, cell migration was not significantly inhibited by U0126 treatment or Fra-1/c-Jun silencing in cells expressing slug transgene. Our study suggests that the ERK pathway regulates breast cancer cell migration by maintaining slug expression.