Id-1 promotes TGF-beta1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-beta1 (transforming growth factor beta1). Here we demonstrate a novel role of Id-1 in promoting TGF-beta1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-beta1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-beta1-induced cell motility was mediated through activation of MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-beta1-treated cells through down-regulation of E-cadherin, redistribution of beta-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-beta1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-beta1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.     

G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.     

Evi5 promotes collective cell migration through its Rab-GAP activity

Membrane trafficking has well-defined roles during cell migration. However, its regulation is poorly characterized. In this paper, we describe the first screen for putative Rab-GTPase-activating proteins (GAPs) during collective cell migration of Drosophila melanogaster border cells (BCs), identify the uncharacterized Drosophila protein Evi5 as an essential membrane trafficking regulator, and describe the molecular mechanism by which Evi5 regulates BC migration. Evi5 requires its Rab-GAP activity to fulfill its functions during migration and acts as a GAP protein for Rab11. Both loss and gain of Evi5 function blocked BC migration by disrupting the Rab11-dependent polarization of active guidance receptors. Altogether, our findings deepen our understanding of the molecular machinery regulating endocytosis and subsequently cell signaling during migration.     

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.     

Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11-amino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKRS(727)L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1).Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged recombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1-ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.     

S-Allylcysteine reduces breast tumor cell adhesion and invasion

Previous studies show that aqueous garlic extract and its derivatives (e.g. S-allylcysteine [SAC]) prevent carcinogen-induced breast tumorigenesis. However, investigations testing the effect of SAC on later stages of breast tumorigenesis and/or metastasis have produced mixed results. Here we show that SAC significantly reduced anchorage-dependent and -independent growth of MDA-MB-231 breast tumor cells in a dose- and time-dependent fashion, and sub-lethal SAC-treatment altered mammary tumor cell adhesion and invasion through components of the extracellular matrix. We provide evidence to suggest increased expression of E-cadherin and reduced MMP-2 expression and activity are partially responsible for inhibition of mammary tumor cell invasion by SAC. Because E-cadherin and MMP-2 are important in cancer metastasis, these results suggest a link between SAC induction of E-cadherin and reduction of MMP2 activity with the inhibition of cell motility and invasion; thus providing evidence that events leading to breast cancer metastasis are repressed by sub-lethal SAC-treatment.     

Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation

Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane.     

Mta2 promotes Tipin-dependent maintenance of replication fork integrity

Orderly progression of S phase requires the action of replisome-associated Tipin and Tim1 proteins, whose molecular function is poorly understood. Here, we show that Tipin deficiency leads to the accumulation of aberrant replication intermediates known as reversed forks. We identified Mta2, a subunit of the NuRD chromatin remodeler complex, as a novel Tipin binding partner and mediator of its function. Mta2 is required for Tipin-dependent Polymerase α binding to replicating chromatin, and this function is essential to prevent the accumulation of reversed forks. Given the role of the Mta2–NuRD complex in the maintenance of heterochromatin, which is usually associated with hard-to-replicate DNA sequences, we tested the role of Tipin in the replication of such regions. Using a novel assay we developed to monitor replication of specific genomic loci in Xenopus laevis egg extract we demonstrated that Tipin is directly required for efficient replication of vertebrate centromeric DNA. Overall these results suggest that Mta2 and Tipin cooperate to maintain replication fork integrity, especially on regions that are intrinsically difficult to duplicate.     

Associated proteins of lens adherens junction

Cytoplasmic proteins associated with adherens junctions were identified in the chicken ocular lens. The catenins, alpha, beta, and gamma, were present in epithelial and fiber cells, although their pattern of distribution changed with fiber cell differentiation. The sharp decline in alpha-catenin with fiber cell formation and the increasing Triton-insolubility of N-cadherin suggests that another subtype of alpha-catenin exists in the lens.     

Molecular components of the adherens junction

Adherens junctions serve to couple individual cells into various arrangements required for tissue structure and function. The central structural components of adherens junctions are transmembrane adhesion receptors, and their associated actin-binding/regulatory proteins. The molecular machineries that organize these adhesion receptor complexes into higher order junction structures, and the functional consequences of this junctional organization will be discussed.     

Cyclic AMP-dependent protein kinase A negatively modulates adherens junction integrity and differentiation of intestinal epithelial cells

Intestinal epithelial cell differentiation is a complex process in which many different signaling pathways are likely involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to inhibit enterocyte differentiation; however, the mechanisms through which cAMP/PKA signaling modulates differentiation of human intestinal epithelial cells are still not well understood. Herein, we report that: (1) treatment of Caco-2/15 cells with 8Br-cAMP repressed sucrase-isomaltase and villin protein expression and strongly attenuated morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush border formation; (2) treatment of confluent Caco-2/15 cells with 8Br-cAMP led to a strong decrease in F-actin localized at cell-cell contact sites along with a reduced amount of E-cadherin and catenins, but not of ZO-1, at cell-cell interfaces concomitant with a decreased association of these proteins with the actin cytoskeleton; (3) inhibition of PKA by H89 prevented disruption of adherens junctions by extracellular calcium depletion; (4) treatment of Caco-2/15 cells with 8Br-cAMP prevented the recruitment and activation of p85/PI-3K to E-cadherin-mediated cell-cell contacts, an important event in the assembly of adherens junctions and differentiation of these cells; (5) E-cadherin appears to be phosphorylated on serine in vivo in a PKA-dependent mechanism. Conclusion: Our studies show that cAMP/PKA signaling negatively regulates adherens junction integrity as well as morphological and functional differentiation of intestinal epithelial cells.     

Persistent signaling by dysregulated thrombin receptor trafficking promotes breast carcinoma cell invasion

Increased expression of protease-activated receptor 1 (PAR1), a G protein-coupled receptor for thrombin, has previously been correlated with breast carcinoma cell invasion. PAR1 is irreversibly proteolytically activated, internalized, and sorted directly to lysosomes, a critical process for the termination of signaling. We determined that activated PAR1 trafficking is severely altered in metastatic breast carcinoma cells but not in nonmetastatic or normal breast epithelial cells. Consequently, the proteolytically activated receptor is not sorted to lysosomes and degraded. Altered trafficking of proteolytically activated PAR1 caused sustained activation of phosphoinositide hydrolysis and extracellular signal-regulated kinase signaling, even after thrombin withdrawal, and enhanced cellular invasion. Thus, our results reveal that a novel alteration in trafficking of activated PAR1 causes persistent signaling and, in addition to other processes and proteins, contributes to breast carcinoma cell invasion.     

Molecular components of the adherens junction

Adherens junctions serve to couple individual cells into various arrangements required for tissue structure and function. The central structural components of adherens junctions are transmembrane adhesion receptors, and their associated actin-binding/regulatory proteins. The molecular machineries that organize these adhesion receptor complexes into higher order junction structures, and the functional consequences of this junctional organization will be discussed.     

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and β5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.     

CCDC12 promotes tumor development and invasion through the Snail pathway in colon adenocarcinoma

Integrative expression Quantitative Trait Loci (eQTL) analysis found that rs8180040 was significantly associated with Coiled-coil domain containing 12 (CCDC12) in colon adenocarcinoma (COAD) patients. Immunohistochemical staining and western blotting confirmed CCDC12 was highly expressed in COAD tissues, which was consistent with RNA-Seq data from the TCGA database. Knockdown of CCDC12 could significantly reduce proliferation, migration, invasion, and tumorigenicity of colon cancer cells, while exogenous overexpression of CCDC12 had the opposite effect. Four plex Isobaric Tags for Relative and Absolute Quantitation assays were performed to determine its function and potential regulatory mechanism and demonstrated that overexpression of CCDC12 would change proteins on the adherens junction pathway. Overexpressed Snail and knocked down CCDC12 subsequently in SW480 cells, and we found that overexpression of Snail did not significantly change CCDC12 levels in SW480 cells, while knockdown of CCDC12 reduced that of Snail. CCDC12 plays a significant role in tumorigenesis, development, and invasion of COAD and may affect the epithelial to mesenchymal transformation process of colon cancer cells by regulating the Snail pathway.Subject terms: Diagnostic markers, Colon cancer