Allelic heterogeneity of Crooked Tail Syndrome: result of balancing selection?

We report the identification of a second loss-of-function mutation (c.1906T>C) in the bovine MRC2 gene causing the Crooked Tail Syndrome in Belgian Blue Cattle. We demonstrate that the ensuing substitution of the highly conserved Cysteine 636 with Arginine causes illegitimate receptor oligomerization, which is predicted to impair function of the MRC2 encoded protein, Endo180. We propose that this second MRC2 mutation was selected by breeders as a result of its favourable effect on muscularity in heterozygotes.

Endoglin expression in breast tumor cells suppresses invasion and metastasis and correlates with improved clinical outcome.

Tumor growth factor-ß (TGF-ß) signaling in cancer has been implicated in growth suppression of early lesions and enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine this signaling output in individual tumors are still largely unknown. In endothelial cells, TGF-ß signaling is modulated by the TGF-ß co-receptor endoglin (CD105). Here we demonstrate that endoglin is expressed in a subset of invasive breast cancers and cell lines and is subject to epigenetic silencing by gene methylation. Endoglin downregulation in non-tumorigenic MCF10A breast cells leads to the formation of abnormal acini in 3D culture, but does not promote cell migration or transformation. In contrast, in the presence of activated ErbB2, endoglin downregulation in MCF10A cells leads to enhanced invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in MDA-MB-231 cells blocks TGF-ß-enhanced cell motility and invasion and reduces lung colonization in an in vivo metastasis model. Unlike endothelial cells, endoglin does not modulate Smad-mediated TGF-ß signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, in a large cohort of invasive breast cancers, lack of endoglin expression in the tumor cell compartment correlates with ENG gene methylation and poor clinical outcome.

Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance.

Endocrine therapy is the main therapeutic option for patients with estrogen receptor (ERalpha)-positive breast cancer. Resistance to this treatment is often associated with estrogen-independent activation of ERalpha. In this study, we show that in ERalpha-positive breast cancer cells, activation of the receptor tyrosine kinase RET (REarranged during Transfection) by its ligand GDNF results in increased ERalpha phosphorylation on Ser118 and Ser167 and estrogen-independent activation of ERalpha transcriptional activity. Further, we identify mTOR as a key component in this downstream signaling pathway. In tamoxifen response experiments, RET downregulation resulted in 6.2-fold increase in sensitivity of MCF7 cells to antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect against the drug. In tamoxifen-resistant (TAM(R)-1) MCF7 cells, targeting RET restored tamoxifen sensitivity. Finally, examination of two independent tissue microarrays of primary human breast cancers revealed that expression of RET protein was significantly associated with ERalpha-positive tumors and that in primary tumors from patients who subsequently developed invasive recurrence after adjuvant tamoxifen treatment, there was a twofold increase in the number of RET-positive tumors. Together these findings identify RET as a potentially important therapeutic target in ERalpha-positive breast cancers and in particular in tamoxifen-resistant tumors.

A comparative proteinomic analysis of nipple aspiration fluid from healthy women and women with breast cancer.

This pilot study examines the feasibility of nipple aspiration to distinguish women with breast cancer from healthy women using surface-enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF/MS). Nipple aspiration fluid (NAF) was collected from each breast in 21 women newly diagnosed with unilateral breast cancer and 44 healthy women. No differences were found when proteomic profiles of NAF from the cancer-bearing breast and the contralateral non-cancerous breast were compared. In contrast, 9 protein peaks were significantly different between the cancer-bearing breast compared with healthy women and 10 peaks were significantly different between the contralateral healthy breast and healthy women (P<0.05). These data suggest that invasive breast cancer may result in a field change across both breasts and that proteomic profiling of NAF may have more value in breast cancer risk assessment than as a diagnostic or screening tool.

Expression of Endo180 is spatially and temporally regulated during wound healing.

AIMS: Interactions of cells with the extracellular matrix are important for normal wound healing and may play a role in scar formation. Remarkably, wound healing in human gingiva does not result in scar formation and serves as a model for wound regeneration. Endo180 (CD280) is a cell surface receptor that has novel functions to regulate cell migration and bind and internalize collagens that are key processes in wound healing. The aim of this study was to examine the expression of Endo180 during gingival wound regeneration. METHODS AND RESULTS: Biopsies were collected from normal human gingiva and 1-60 days after wounding and expression of Endo180 was analysed by immunostaining. Expression of Endo180 by cultured fibroblasts and keratinocytes was studied by immunoblotting and semiquantitative reverse transcriptase-polymerase chain reaction. In normal gingiva, Endo180 was expressed by basal epithelial cells, fibroblasts, myofibroblasts, pericytes, macrophages and endothelial cells. In wounds, Endo180 expression was spatiotemporally increased in the migrating and differentiating wound epithelium, in subsets of myofibroblasts, pericytes, macrophages and endothelial cells. Growth factors involved in wound healing up-regulated the expression of Endo180 in keratinocytes and fibroblasts. CONCLUSIONS: The findings suggest that Endo180 plays a role in re-epithelialization and connective tissue remodelling during wound regeneration.

Identification of transmembrane proteins as potential prognostic markers and therapeutic targets in breast cancer by a screen for signal sequence encoding transcripts.

This study demonstrates, through a combination of stringent screening methods and thorough validation, that it is possible to identify transmembrane proteins preferentially expressed in primary breast tumour cells. mRNA was extracted from tumour cells isolated from invasive breast cancers and it was then subtracted against normal breast tissue mRNA prior to the generation of a signal sequence-trap library. Screening of the library identified 31 positive clones encoding 12 cell-surface and 12 secreted proteins. The expression of a subset of transmembrane genes was then interrogated using a high-throughput method (tissue microarray) coupled with cutting-edge in situ techniques in a large cohort of patients who had undergone uniform adjuvant chemotherapy. Expression of CD98 heavy chain (CD98HC) and low-level expression of the insulin-like growth factor 2 receptor/mannose-6-phosphate receptor (IGF2R/M6PR) correlated with poor patient prognosis in the whole cohort. Expression of bradykinin receptor B1 (BDKRB1) and testis enhanced gene transcript (TEGT) correlated with good prognosis in woman with oestrogen receptor (ER)-negative breast tumours. These results indicate that this combined approach of isolating primary tumour cells, generating a library to specifically isolate signal-sequence-containing transcripts, and in situ hybridization on tissue microarrays successfully identified novel prognostic markers (BDKRB1, CD98hc, and TEGT) and potential transmembrane therapeutic targets (CD98hc) in breast cancer.

Directional sensing of a phorbol ester gradient requires CD44 and is regulated by CD44 phosphorylation.

Cancer progression is associated with enhanced directional cell migration, both of the tumour cells invading into the stroma and stromal cells infiltrating the tumour site. In cell-based assays to study directional cell migration, phorbol esters are frequently used as a chemotactic agent. However, the molecular mechanism by which these activators of protein kinase C (PKC) result in the establishment of a polarized migratory phenotype is not known. Here we show that CD44 expression is essential for chemotaxis towards a phorbol ester gradient. In an investigation of CD44 phosphorylation kinetics in resting and stimulated cells, Ser316 was identified as a novel site of phosphorylation following activation of PKC. PKC does not phosphorylate Ser316 directly, but rather mediates the activation of downstream Ser316 kinase(s). In transfection studies, a phosphorylation-deficient Ser316 mutant was shown to act in a dominant-negative fashion to impair chemotaxis mediated by endogenous CD44 in response to a phorbol ester gradient. Importantly, this mutation had no effect on random cell motility or the ability of cells to migrate directionally towards a cocktail of chemoattractants. These studies demonstrate that CD44 functions to provide directional cues to migrating cells without affecting the motility apparatus.

Endothelial adhesion molecules in breast cancer invasion into the vascular and lymphatic systems.

AIMS: It is well recognised that intravasation of tumour cells into the vasculature and/or lymphatics is a key stage in the metastatic process. It is also clear that very little is known about the mechanisms underlying this event. In this review, we will focus on cell surface molecules that may be instrumental in mediating the attachment of tumour cells, and in particular breast carcinoma cells, to the lymphatic and microvascular endothelia and discuss the therapeutic and prognostic value in targeting these receptors in metastatic disease. METHODS: A literature search was carried out from PubMed for indexed articles and reviews. Websites containing information on gene expression profiles were located using standard web browser search functions. For articles containing gene expression data, relevant information was frequently located in supplementary tables or in associated websites. FINDINGS: The search yielded a very large number of indexed published articles and websites. Important major reports and studies were reviewed, screened and tracked for other relevant publications. The most important articles were analysed and discussed. CONCLUSIONS: The lack of knowledge as to the mechanism by which tumour cells intra-vasate into the vasculature and/or lymphatics is perhaps not surprising given the lack of suitable models with which to investigate tumour cell intravasation. However, recent advances in the identification of molecular markers of angiogenic and lymphangiogenic endothelium, the development of techniques to image tumour cells in vivo and a better understanding of the architecture of these vessels is beginning to offer hope that this least well understood event in the metastatic process is becoming more amenable to study.

Distribution of Endo180 receptor and ligand in developing articular cartilage.

OBJECTIVE: To investigate the expression of a novel member of the mannose receptor family, Endo180 (also known as uPARAP), and the distribution of Endo180 ligand(s) in the articular cartilage and growth plate of normal CBA mice and STR/ort mice, a well characterized model of spontaneous osteoarthritis. DESIGN: A polyclonal anti-Endo180 antibody was used to determine receptor expression. The Endo180 extracellular domain fused to a human immunoglobulin Fc tail was used to detect ligand. RESULTS: Endo180 receptor was strongly expressed in chondrocytes both in vitro and throughout the articular cartilage of young CBA and STR/ort mice. Expression decreased in older animals. In STR/ort mice with osteoarthritic lesions, no upregulation of Endo180 was detected. In the developing growth plate, Endo180 was expressed strongly by the proliferating chondrocytes. In contrast, Endo180 ligand was detected most strongly in hypertrophic zone of the growth plate and only at low levels in articular cartilage. In cultured chondrocytes, Endo180 was localized on the cell surface and in intracellular vesicles. CONCLUSION: Constitutively recycling endocytic receptors function to internalize ligand from the extracellular milieu and the ability of Endo180 to bind both glycosylated ligands and collagens suggests a role in extracellular matrix remodeling. Expression of Endo180 in articular cartilage chondrocytes of young, but not old, mice and the reciprocal expression of Endo180 and its ligands in the growth plate suggest that this receptor is involved in cartilage development but not in cartilage homeostasis. In addition, our data indicates that Endo180 does not appear to play a role in the development or progression of murine osteoarthritis.

Ca(2+)/calmodulin-dependent protein kinase mediates the phosphorylation of CD44 required for cell migration on hyaluronan.

CD44 is the principal cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and binding to this ligand underlies CD44-mediated cell attachment and migration. As would be expected for a widely expressed adhesion receptor, CD44 is subject to complex regulatory events, and mis-regulation of the receptor has been associated with a number of disease pathologies, including chronic inflammatory conditions and the progression of metastatic tumours. In previous studies we have demonstrated that a key control point for this receptor is the phosphorylation of CD44 on a conserved cytoplasmic serine residue, Ser(325). This modification is not required for efficient ligand binding, but is an essential component of CD44-dependent cell migration on a hyaluronan substratum. To understand better the mechanism regulating CD44 phosphorylation on Ser(325), we have generated a monoclonal antibody that specifically recognizes CD44 phosphorylated on Ser(325), and have developed assays to identify the Ser(325) kinase. We demonstrate here that CD44 is phosphorylated to high stoichiometry in resting cells and that Ca(2+)/calmodulin-dependent protein kinase II is a CD44 Ser(325) kinase.

Endo180, an endocytic recycling glycoprotein related to the macrophage mannose receptor is expressed on fibroblasts, endothelial cells and macrophages and functions as a lectin receptor.

Endo180 was previously characterized as a novel, cell type specific, recycling transmembrane glycoprotein. This manuscript describes the isolation of a full length human Endo180 cDNA clone which was shown to encode a fourth member of a family of proteins comprising the macrophage mannose receptor, the phospholipase A(2) receptor and the DEC-205/MR6 receptor. This receptor family is unusual in that they contain 8-10 C-type lectin carbohydrate recognition domains in a single polypeptide backbone, however, only the macrophage mannose receptor had been shown to function as a lectin. Sequence analysis of Endo180 reveals that the second carbohydrate recognition domain has retained key conserved amino acids found in other functional C-type lectins. Furthermore, it is demonstrated that this protein displays Ca(2+)-dependent binding to N-acetylglucosamine but not mannose affinity columns. In order to characterize the physiological function of Endo180, a series of biochemical and morphological studies were undertaken. Endo180 is found to be predominantly expressed in vivo and in vitro on fibroblasts, endothelial cells and macrophages, and the distribution and post-translational processing in these cells is consistent with Endo180 functioning to internalize glycosylated ligands from the extracellular milieu for release in an endosomal compartment.

Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44.

ERM (ezrin, radixin and moesin) proteins function as linkers between the actin cytoskeleton and the plasma membrane. In addition to this structural role, these proteins are highly regulatable making them ideal candidates to mediate important physiological events such as adhesion and membrane morphology and to control formation and breakdown of membrane-cytoskeletal junctions. Recently, a direct interaction in vitro has been demonstrated between ERM proteins and the hyaluronan receptor, CD44. We have mapped the ezrin-binding site to two clusters of basic amino acids in a membrane-proximal 9 amino-acid region within the CD44 cytoplasmic domain. To investigate the functional importance of this interaction in vivo, we created a number of mutations within full-length CD44 and expressed these mutants in human melanoma cells. We demonstrate here that mutations within the ezrin-binding site do not disrupt the plasma membrane localization of CD44 and, in addition, that this region is not required to mediate efficient hyaluronan binding. These studies suggest that ERM proteins mediate the outside-in, rather than inside-out, signalling of adhesion receptors.

Hyaluronan-dependent cell migration can be blocked by a CD44 cytoplasmic domain peptide containing a phosphoserine at position 325.

CD44 is the principle transmembrane receptor for the extracellular matrix glycosaminoglycan hyaluronan. This receptor:ligand interaction plays an essential role in a number of physiological events including tumour progression, lymphocyte homing into inflammatory sites and tissue morphogenesis during development. In previous studies we have shown that serine phosphorylation is a critical control mechanism for CD44-dependent cell migration. Here we have investigated the target phosphorylation residues by mutating them individually or in combination. These studies demonstrate that Ser325 is the principle CD44 phosphorylation site and that mutation of this residue blocks CD44-mediated cell migration but not hyaluronan binding. In addition, we show that an upstream Ser323 residue is required as part of the kinase consensus site. To further characterize the role of CD44 phosphorylation, phosphorylated and non-phosphorylated peptides spanning the Ser325 region were synthesised and linked to a 16 amino acid Penetratin sequence to mediate efficient plasma membrane translocation. Peptides containing a phosphoserine at residue 325 are efficient blockers of CD44-mediated cell migration but do not reduce CD44 expression or its ability to bind hyaluronan. These data strongly argue that CD44 adhesion and migration are regulated by distinct mechanisms and that migration requires the specific interaction of intracellular component(s) with phosphorylated CD44 receptors.

CD44 phosphorylation regulates melanoma cell and fibroblast migration on, but not attachment to, a hyaluronan substratum.

BACKGROUND: CD44 is a transmembrane receptor for the extracellular matrix glycosaminoglycan, hyaluronan. This receptor-ligand interaction plays an essential role in tumour progression, in embryonic tissue morphogenesis and in leukocyte migration during inflammation. It is well documented that the interaction between CD44 and hyaluronan is strictly regulated, but little is known about the relationship between hyaluronan-dependent cell adhesion and cell migration. RESULTS: In these studies we have used a CD44-negative human melanoma cell line and a murine fibroblast line which expresses low levels of endogenous CD44. Both cell lines were transfected with plasmids encoding wild-type human CD44 or CD44 phosphorylation mutants, in which the target serines had been mutated to small neutral amino acids or large acidic residues. We show that expression of wild-type CD44 enhances the ability of both cell lines to bind to, and migrate on, a hyaluronan-coated substratum. In contrast, the two CD44 phosphorylation mutants were as efficient as wild-type CD44 in mediating cell adhesion but were unable to support hyaluronan-dependent migration. CONCLUSIONS: These studies demonstrate a control mechanism specific for CD44-mediated cell motility and have implications for the regulation of metastatic progression by cell-adhesion receptors.

A di-hydrophobic Leu-Val motif regulates the basolateral localization of CD44 in polarized Madin-Darby canine kidney epithelial cells.

Both in vivo and in vitro the distribution of the resident plasma membrane adhesion protein, CD44, is restricted to the basolateral domain of polarized epithelial cells, suggesting a role in interepithelial interactions. To determine how this localization might be regulated a range of CD44 cytoplasmic domain mutations were generated and a minimal 5 amino acid sequence, His330-Leu-Val-Asn-Lys334, was identified which when deleted results in expression of CD44 on the apical microvillal membrane. Further mutagenesis throughout this regions pinpointed a critical di-hydrophobic motif, Leu331/Val332. The ability of wild type but not mutant CD44 cytoplasmic domains to redirect an apically targeted protein, placental alkaline phosphatase, to the basolateral plasma membrane demonstrates that this sequence can function as a dominant localization signal. This His330-Lys334 sequence is spatially separate from other CD44 regulatory elements and as discussed here, a comparison with known basolateral sorting sequences identified in other transmembrane proteins suggests that a distinct mechanism operates to retain resident plasma membrane proteins in their correct plasma membrane subdomains.

CD44 exhibits a cell type dependent interaction with triton X-100 insoluble, lipid rich, plasma membrane domains.

CD44 is an abundant, widely expressed transmembrane glycoprotein which can act as a receptor for the extracellular matrix glycosaminoglycan, hyaluronan. Biochemical and morphological studies have demonstrated that in fibroblasts a significant of the CD44 population is resistant to Triton X-100 extraction and that the detergent insoluble protein is co-localized with components of the cortical cytoskeleton. Surprisingly, this distribution is not abrogated upon deletion of the CD44 cytoplasmic tail indicating that mechanisms other than a direct interaction with the cytoskeleton can regulate CD44. In this manuscript, the mechanisms underlying this detergent-insoluble association are further investigated. There was no evidence that the Triton X-100 insolubility of CD44 resulted from homotypic aggregation, an association with hyaluronan or from a direct, or indirect, association with the cytoskeleton. Instead, evidence is presented that the detergent insolubility of fibroblast CD44 at 4 degrees C results from an association of the CD44 transmembrane domain with Triton X-100 resistant, lipid rich, plasma membrane domains. The proportion of the CD44 found in these Triton X-100 insoluble structures is dependent upon cell type and cannot be altered by changing cell motility or extracellular matrix associations. These studies provide evidence for a novel mechanism regulating this adhesion protein in the plasma membrane.

Induction of a hyaluronan receptor, CD44, during embryonal carcinoma and embryonic stem cell differentiation.

This paper describes the expression profile of the CD44 glycoprotein during differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. We have recently shown that CD44 is expressed in discrete embryonic structures and, in view of this, we sought an in vitro differentiation model of development in which we could study more readily the structure and function of the CD44 molecule. The P19 EC and CGR8 ES cells were chosen as they have the capacity to develop down the cardiac muscle pathway and we have previously demonstrated that CD44 is expressed abundantly in the embryonic myocardium. The differentiation process in both cell types is accompanied by an induction of CD44 mRNA and protein. However, in differentiated cultures CD44 is not expressed in contractile cells, indicating that these P19 cells do not represent CD44-positive embryonic cardiomyocytes. Expression of CD44 is observed on fibroblast-like cells which appear to migrate over and out from the plated aggregates. Hyaluronan, the major ligand for CD44, is also associated with these CD44-positive fibroblast-like cells. It is suggested that expression of both receptor and ligand by the fibroblast cells is required for cell:matrix adhesion and cell motility. As CD44 is up-regulated in these cultures, P19 cells are now established as a useful model system to study the factors regulating expression of the CD44 gene.

Hyaluronan binding by CD44 is regulated by a phosphorylation-independent mechanism.

CD44 is an adhesion receptor for which the major characterized ligand is the extracellular matrix glycosaminoglycan, hyaluronan. This interaction underlies CD44-mediated cell attachment, cell migration, and matrix remodelling during development and wound healing. Truncation of the CD44 cytoplasmic domain does not prevent cell surface expression of this hyaluronan receptor but it dramatically impairs ligand binding. In this study we have examined the role of phosphorylation in regulating this function by mutating the target serine residues to either neutral amino acids with the aim of creating a phosphorylation-incompetent molecule, or to acidic residues to mimic a fully phosphorylated CD44. In transfected AKR1 cells the behavior of both the neutral and acidic mutants was indistinguishable from wild-type CD44, indicating that there is a phosphorylation-independent mechanism involved in regulating hyaluronan binding.