Interaction of Wnt-1 proteins with the binding protein BiP.

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.     

A novel integrin alpha5beta1 antagonistic peptide, A5-1, screened by Protein Chip system as a potent angiogenesis inhibitor

Integrin α5β1 immobilized on a ProteoChip was used to screen new antagonistic peptides from multiple hexapeptide sub-libraries of the positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin α5β1-Fibronectin interaction was demonstrated on the chip. A novel peptide ligand, A5-1 (VILVLF), with high affinity to integrin α5β1 was identified from the hexapeptide libraries with this chip-based screening method on the basis of a competitive inhibition assay. A5-1 inhibits the integrin-fibronectin interaction in a dose-dependent manner (IC₅₀; 1.56 ± 0.28 μM. In addition, it inhibits human umbilical vein endothelial cell proliferation, migration, adhesion, tubular network formation, and bFGF-induced neovascularization in a chick chorioallantoic membrane. These results suggest that A5-1 will be a potent inhibitor of neovascularization.     

Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer.     

Peptide tags for enhanced cellular and protein adhesion to single-crystalline sapphire

In order to facilitate a novel means for coupling proteins to metal oxides, peptides were identified from a dodecamer peptide yeast surface display library that bound a model metal oxide material, the C, A, and R crystalline faces of synthetic sapphire (alpha-Al(2)O(3)). Seven rounds of screening yielded peptides enriched in basic amino acids compared to the naive library. While the C-face had a high background of endogenous yeast cell binding, the A- and R faces displayed clear peptide-mediated cell adhesion. Cell detachment assays showed that cell adhesion strength correlated positively with increasing basicity of expressed peptides. Cell adhesion was also shown to be sensitive to buffer ionic strength as well as incubation with soluble peptide (with half maximal inhibition of cell binding at approximately 5 microM peptide). Next, dodecamer peptides cloned into yeast showed that lysine led to stronger interactions than arginine, and that charge distribution affected adhesion strength. We postulate binding to arise from peptide geometries that permit conformation alignment of the basic amino acids towards the surface so that the charged groups can undergo local electrostatic interactions with the surface oxide. Lastly, peptide K1 (-(GK)(6)) was cloned onto the c-terminus of maltose binding protein (MBP) and the resultant mutant protein showed a half-maximal binding at approximately 10(-7)-10(-6) M, which marked a approximately 500- to 1,000-fold binding improvement to sapphire's A-face as compared with wild-type MBP. Targeting proteins to metal oxide surfaces with peptide tags may provide a facile one-step alternative coupling chemistry for the formation of protein bioassays and biosensors.     

Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein

The lactating mammary gland utilizes free plasma amino acids as well as those derived by hydrolysis from circulating short-chain peptides for protein synthesis. Apart from the major route of amino acid nitrogen delivery to the gland by the various transporters for free amino acids, it has been suggested that dipeptides may also be taken up in intact form to serve as a source of amino acids. The identification of peptide transporters in the mammary gland may therefore provide new insights into protein metabolism and secretion by the gland. The expression and distribution of the high-affinity type proton-coupled peptide transporter PEPT2 were investigated in rat lactating mammary gland as well as in human epithelial cells derived from breast milk. By use of RT-PCR, PEPT2 mRNA was detected in rat mammary gland extracts and human milk epithelial cells. The expression pattern of PEPT2 mRNA revealed a localization in epithelial cells of ducts and glands by nonisotopic high resolution in situ hybridization. In addition, immunohistochemistry was carried out and showed transporter immunoreactivity in the same epithelial cells of the glands and ducts. In addition, two-electrode voltage clamp recordings using PEPT2-expressing Xenopus laevis oocytes demonstrated positive inward currents induced by selected dipeptides that may play a role in aminonitrogen handling in mammalian mammary gland. Taken together, these data suggest that PEPT2 is expressed in mammary gland epithelia, in which it may contribute to the reuptake of short-chain peptides derived from hydrolysis of milk proteins secreted into the lumen. Whereas PEPT2 also transports a variety of drugs, such as selected beta-lactams, angiotensin-converting enzyme inhibitors, and antiviral and anticancer metabolites, their efficient reabsorption via PEPT2 may reduce the burden of xenobiotics in milk.     

Quantitative proteomics profiling of murine mammary gland cells unravels impact of annexin-1 on DNA damage response, cell adhesion, and migration

Annexin 1 (ANXA1), the first characterized member of the annexin superfamily, is known to bind or annex to cellular membranes in a calcium-dependent manner. Besides mediating inflammation, ANXA1 has also been reported to be involved in important physiopathological implications including cell proliferation, differentiation, apoptosis, cancer, and metastasis. However, with controversies in ANXA1 expression in breast carcinomas, its role in breast cancer initiation and progression remains unclear. To elucidate how ANXA1 plays a role in breast cancer initiation, we performed stable isotope labeling of amino acids in cell culture analysis on normal mammary gland epithelial cells from ANXA1-heterozygous (ANXA1(+/-)) and ANXA1-null (ANXA1(-/-)) mice. Among over 4000 quantified proteins, we observed 214 up-regulated and 169 down-regulated with ANXA1(-/-). Bioinformatics analysis of the down-regulated proteins revealed that ANXA1 is potentially implicated in DNA damage response, whereas the analysis of up-regulated proteins showed the possible roles of ANXA1 in cell adhesion and migration pathways. These observations were supported by relevant functional assays. The assays for DNA damage response demonstrated an accumulation of more DNA damage with slower recovery on heat stress and an impaired oxidative damage response in ANXA1(-/-) cells in comparison with ANXA1(+/-) cells. Overexpressing Yes-associated protein 1 or Yap1, the most down-regulated protein in DNA damage response pathway cluster, rescued the proliferative response in ANXA1(-/-) cells exposed to oxidative damage. Both migration and wound healing assays showed that ANXA1(+/-) cells possess higher motility with better wound closure capability than ANXA1(-/-) cells. Knocking down of β-parvin, the protein with the highest fold change in the cell adhesion protein cluster, indicated an increased cell migration in ANXA1(-/-) cells. Altogether our quantitative proteomics study on ANXA1 suggests that ANXA1 plays a protective role in DNA damage and modulates cell adhesion and motility, indicating its potential role in cancer initiation as well as progression in breast carcinoma.     

Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of alpha3 chain

The basement membrane protein laminin-5 promotes cell adhesion and migration. The carboxyl-terminal G3 domain in the alpha3 chain is essential for the unique activity of laminin-5. To investigate the function of the G3 domain, we prepared various recombinant laminin-5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl-terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ-1 cells. This change was attributed to the loss of Lys-Arg-Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin-5 receptors showed that integrins alpha3beta1, alpha6beta1, and alpha6beta4 had different but synergistic effects on cell adhesion and migration on laminin-5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin-5 plays a central role to produce different biological effects on cells.     

Characterization of a synthetic peptide from type IV collagen that promotes melanoma cell adhesion, spreading, and motility

The adhesion and motility of tumor cells on basement membranes is a central consideration in tumor cell invasion and metastasis. Basement membrane type IV collagen directly promotes the adhesion and migration of various tumor cell types in vitro. Our previous studies demonstrated that tumor cells adhered and spread on surfaces coated with intact type IV collagen or either of the two major enzymatically purified domains of this protein. Only one of these major domains, the pepsin-generated major triple helical fragment, also supported tumor cell motility in vitro, implicating the involvement of the major triple helical region in type IV collagen-mediated tumor cell invasion in vivo. The present studies extend our previous observations using a synthetic peptide approach. A peptide, designated IV-H1, was derived from a continuous collagenous region of the major triple helical domain of the human alpha 1(IV) chain. This peptide, which has the sequence GVKGDKGNPGWPGAP, directly supported the adhesion, spreading, and motility of the highly metastatic K1735 M4 murine melanoma cell line, as well as the adhesion and spreading of other cell types, in a concentration-dependent manner in vitro. Furthermore, excess soluble peptide IV-H1, or polyclonal antibodies directed against peptide IV-H1, inhibited type IV collagen-mediated melanoma cell adhesion, spreading, and motility, but had no effect on these cellular responses to type I collagen. The full complement of cell adhesion, spreading, and motility promoting activities was dependent upon the preservation of the three prolyl residues in the peptide IV-H1 sequence. These studies indicate that peptide IV-H1 represents a cell-specific adhesion, spreading, and motility promoting domain that is active within the type IV collagen molecule.     

cDNA cloning and expression of ghrelin in giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. It plays an important role in stimulating growth hormone secretion, food intake, body weight gain and gastric motility. cDNA sequences coding for ghrelin precursor protein (prepro-ghrelin) were isolated from the stomach of a giant panda. Two different mRNA sequences of ghrelin were obtained. The long open reading frame of ghrelin (354 bp) encodes a precursor protein of 117 amino acids with a 23 amino acid signal peptide. The short one (351 bp) encodes a precursor protein of 116 amino acids with the same 23 amino acid signal peptide. The presumed giant panda mature ghrelin proteins also had two forms. Comparative analysis showed that the first and the fourth amino acids (Gly and Phe) were completely conserved and the third amino acid (Ser) was also highly conserved in the mature ghrelin. RT-PCR analysis of giant panda ghrelin mRNA in various tissues revealed high level of expression in stomach, relative lower levels of expression in small intestine, liver and kidney, and no expression in thymus, spleen and heart.     

MicroRNA-7 Inhibits Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Cells via Targeting FAK Expression

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix integrin signaling, cell motility, cell proliferation and cell survival. Increased FAK expression is observed in a variety of solid human tumors and increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Herein we identify miR-7 as a direct regulator of FAK expression. miR-7 expression is decreased in malignant versus normal breast tissue and its expression correlates inversely with metastasis in human breast cancer patients. Forced expression of miR-7 produced increased E-CADHERIN and decreased FIBRONECTIN and VIMENTIN expression in breast cancer cells. The levels of miR-7 expression was positively correlated with E-CADHERIN mRNA and negatively correlated with VIMENTIN mRNA levels in breast cancer samples. Forced expression of miR-7 in aggressive breast cancer cell lines suppressed tumor cell monolayer proliferation, anchorage independent growth, three-dimensional growth in Matrigel, migration and invasion. Conversely, inhibition of miR-7 in the HBL-100 mammary epithelial cell line promoted cell proliferation and anchorage independent growth. Rescue of FAK expression reversed miR-7 suppression of migration and invasion. miR-7 also inhibited primary breast tumor development, local invasion and metastatic colonization of breast cancer xenografts. Thus, miR-7 expression is decreased in metastatic breast cancer, correlates with the level of epithelial differentiation of the tumor and inhibits metastatic progression.     

Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland.

The mouse Wnt family comprises at least 10 members sharing substantial amino acid identity with the secreted glycoprotein Wnt-1/int-1. Two of these, Wnt-1 and Wnt-3, are implicated in mouse mammary tumor virus-associated adenocarcinomas, although neither member is normally expressed in the mammary gland. These results suggest the presence of active cellular pathways which mediate the action of Wnt-1 and Wnt-3 signals. An understanding of the normal role of these signalling pathways is clearly necessary to comprehend the involvement of Wnt-1 and Wnt-3 in mammary tumorigenesis. We demonstrate here that five Wnt family members are expressed and differentially regulated in the normal mouse mammary gland. In addition, some of these genes are also expressed in both Wnt-1-responsive and nonresponsive mammary epithelial cell lines. We propose that Wnt-mediated signalling is involved in normal regulation of mammary development and that inappropriate expression of Wnt-1, Wnt-3, and possibly other family members can interfere with these signalling pathways.     

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.     

Evidence for a direct interaction between the tumor suppressor serpin, maspin, and types I and III collagen.

Maspin (mammary serine protease inhibitor) was originally identified as a tumor suppressor protein in human breast epithelial cells and is a member of the serine proteases inhibitor (serpin) superfamily. It inhibits tumor cell motility and angiogenesis, and although predominantly cytoplasmic, it is also localized to the cell surface. In this study we have investigated the use of the yeast two-hybrid interaction trap to identify novel maspin targets. A target human fibroblast cDNA library was screened, and the alpha-2 chain of type I collagen was identified as a potential interactant. Binding studies with isolated proteins showed interaction between recombinant maspin and types I and III collagen but not other collagen subtypes, a profile strikingly similar to mouse pigment epithelium-derived factor (caspin), which is similarly down-regulated in murine adenocarcinoma tumors and is a potent inhibitor of angiogenesis. Kinetic analysis using an IAsys resonant mirror biosensor determined the dissociation constant of maspin for collagen type I to be 0.63 microm. Further two-hybrid interactions with maspin truncation constructs suggest that collagen binding is localized to amino acids 84-112 of maspin, which aligns with the collagen-binding region of colligin. A direct interaction between exogenous or cell surface maspin and extracellular matrix collagen may contribute to a cell adhesion role in the prevention of tumor cell migration and angiogenesis.     

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion,Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs'' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.     

Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells

Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of beta1,6GlcNAc-branched N-glycans, products of Golgi beta1,6-acetylglucosaminyltransferase V (Mgat5) and the favored ligand for galectins. Herein we report that fibronectin fibrillogenesis and fibronectin-dependent cell spreading are deficient in Mgat5(-/-) mammary epithelial tumor cells and inhibited in Mgat5(+/+) cells by blocking Golgi N-glycan processing with swainsonine or by competitive inhibition of galectin binding. At an optimum dosage, exogenous galectin-3 added to Mgat5(+/+) cells activates focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), recruits conformationally active alpha5beta1-integrin to fibrillar adhesions, and increases F-actin turnover. RGD peptide inhibits PI3K-dependent fibronectin matrix remodeling and fibronectin-dependent cell motility, while galectin-3 stimulates and overrides the inhibitory effects of RGD. Antibodies to the galectin-3 N-terminal oligomerization domain stimulate alpha5beta1 activation and recruitment to fibrillar adhesions in Mgat5(+/+) cells, an effect that is blocked by disrupting galectin-glycan binding. Our results demonstrate that fibronectin polymerization and tumor cell motility are regulated by galectin-3 binding to branched N-glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local F-actin instability, and alpha5beta1 translocation to fibrillar adhesions.     

Biochemical and mechanical extracellular matrix properties dictate mammary epithelial cell motility and assembly

Biochemical and mechanical cues of the extracellular matrix have been shown to play important roles in cell-matrix and cell-cell interactions. We have experimentally tested the combined influence of these cues to better understand cell motility, force generation, cell-cell interaction, and assembly in an in vitro breast cancer model. MCF-10A non-tumorigenic mammary epithelial cells were observed on surfaces with varying fibronectin ligand concentration and polyacrylamide gel rigidity. Our data show that cell velocity is biphasic in both matrix rigidity and adhesiveness. The maximum cell migration velocity occurs only at specific combination of substrate stiffness and ligand density. We found cell-cell interactions reduce migration velocity. However, the traction forces cells exert onto the substrate increase linearly with both cues, with cells in pairs exerting higher maximum tractions observed over single cells. A relationship between force and motility shows a maximum in single cell velocity not observed in cell pairs. Cell-cell adhesion becomes strongly favored on softer gels with elasticity ≤ 1250 Pascals (Pa), implying the existence of a compliance threshold that promotes cell-cell over cell-matrix adhesion. Finally on gels with stiffness similar to pre-malignant breast tissue, 400 Pa, cells undergo multicellular assembly and division into 3D spherical aggregates on a 2D surface.