Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml−1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Silencing of CD44 Gene Expression in Human 143-B Osteosarcoma Cells Promotes Metastasis of Intratibial Tumors in SCID Mice

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells'' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.     

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.     

CD44 in Cancer Progression: Adhesion, Migration and Growth Regulation

It is well established that the large array of functions that a tumour cell has to fulfil to settle as a metastasis in a distant organ requires cooperative activities between the tumour and the surrounding tissue and that several classes of molecules are involved, such as cell-cell and cell-matrix adhesion molecules and matrix degrading enzymes, to name only a few. Furthermore, metastasis formation requires concerted activities between tumour cells and surrounding cells as well as matrix elements and possibly concerted activities between individual molecules of the tumour cell itself. Adhesion molecules have originally been thought to be essential for the formation of multicellular organisms and to tether cells to the extracellular matrix or to neighbouring cells. CD44 transmembrane glycoproteins belong to the families of adhesion molecules and have originally been described to mediate lymphocyte homing to peripheral lymphoid tissues. It was soon recognized that the molecules, under selective conditions, may suffice to initiate metastatic spread of tumour cells. The question remained as to how a single adhesion molecule can fulfil that task. This review outlines that adhesion is by no means a passive task. Rather, ligand binding, as exemplified for CD44 and other similar adhesion molecules, initiates a cascade of events that can be started by adherence to the extracellular matrix. This leads to activation of the molecule itself, binding to additional ligands, such as growth factors and matrix degrading enzymes, complex formation with additional transmembrane molecules and association with cytoskeletal elements and signal transducing molecules. Thus, through the interplay of CD44 with its ligands and associating molecules CD44 modulates adhesiveness, motility, matrix degradation, proliferation and cell survival, features that together may well allow a tumour cell to proceed through all steps of the metastatic cascade.     

E-cadherin negatively regulates CD44-hyaluronan interaction and CD44-mediated tumor invasion and branching morphogenesis

CD44 is a principal cell-surface receptor for hyaluronan (HA). Up-regulation of CD44 is often associated with morphogenesis and tumor invasion. On the contrary, reduction of cell-cell adhesion due to down-regulation of E-cadherin is associated with the invasive and metastatic phenotype of carcinomas. In our current study, we investigated the functional relationship between CD44 and E-cadherin. We established an inverse correlation between CD44 and E-cadherin indicating that the cells expressing higher levels of E-cadherin display weaker binding affinity between CD44 and HA. By using TA3 murine mammary carcinoma (TA3) cells, which display CD44-dependent HA binding, branching morphogenesis, and invasion, we demonstrated an inverse functional relationship between CD44 and E-cadherin by transfecting exogenous E-cadherin into the cells. Our results showed that increased expression of E-cadherin in TA3 cells, but not ICAM-1, weakens the binding between CD44 and HA and blocks spreading of the cells on HA substratum and CD44-mediated branching morphogenesis and tumor cell invasion. The results reported here demonstrated for the first time that E-cadherin negatively regulated CD44-HA interaction and CD44 function and suggested that balanced function of CD44 and E-cadherin may be essential for normal epithelial cell functions, and imbalanced up-regulation of CD44 function and/or down-regulation of E-cadherin function likely contributes to tumor progression.     

SHAP potentiates the CD44-mediated leukocyte adhesion to the hyaluronan substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.     

Tumour-expressed CD43 (sialophorin) mediates tumourmesothelial cell adhesion

Mesothelial cell intercellular adhesion molecule-1 (ICAM-1) has recently been shown to play a role in tumour cell adherence to the peritoneum. However, solid tumours poorly express its most ubiquitous ligand, beta2 integrin. The aim of this study was to investigate the role of the beta2 integrin subunit and CD43, a known ligand for ICAM-1, in the development of peritoneal metastases. beta2 Integrin subunit and CD43 expression was assessed on a number of tumour cell lines. Adhesion of SW1222 and PSN-1 cells to human peritoneal mesothelial cells was investigated using a fluorometric assay incorporating an inhibitory antibody to beta2 integrin and CD43. beta2 Integrin expression was not inducible on these tumour cell lines, but Western blotting demonstrated CD43 expression in all the cancer cell lines examined and cell surface expression was confirmed by flow cytometry. The anti-CD43 antibody significantly reduced adhesion of PSN-1 and SW1222 cells to HPMC, however beta2 integrin inhibition did not reduce tumour cell adhesion. CD43 is expressed by a variety of carcinoma cell lines, and plays a role in tumour cell-peritoneal adhesion probably via interactions with its putative ligand ICAM-1.     

Regulation of CD44-hyaluronan interactions in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid B cells by PMA and interleukin-4.

In this study, we investigated the regulation of CD44-hyaluronan (HA) interactions in a panel of EBV+ Burkitt's lymphoma (BL) and lymphoblastoid B cell lines (B-LCL) generated by in vitro EBV transformation of normal human B cells. The results show that among B cell mitogens, phorbol 12-myristate 13-acetate (PMA) alone induced strong HA recognition in EBV+ BL-30/B95-8 cells. Among the cytokines that affect B cell growth and differentiation, IL-4 alone induced HA recognition in BL-30/B95-8 cells. Attempts to delineate the molecular mechanism for this increased HA adhesion in BL-30/B95-8 cells revealed an enhanced expression of CD44 H, isoforms containing V3, V6, and V9 exons, alterations in the splicing pattern of the V4 exon, and the increased electrophoretic mobility of the CD44 H protein. In contrast, the ability to recognize HA was not observed in B-LCL cells stimulated with either PMA or IL-4, even though these cells respond to IL-4, as observed by upregulation of CD23 expression. The molecular pathways that regulate CD44 expression and CD44-mediated HA binding may be selectively inactivated in B-LCL cells. These results may have implications with respect to the generation and spread of B cell tumors.     

反义RNA抑制人黑色素瘤细胞表面粘附分子CD44的表达及抑瘤效应

将细胞表面粘附分子ＣＤ４４Ｓ的ｃＤＮＡ反向插入到真核细胞表达载体ｐＭＡＭｎｅｏ－ＣＡＴ和ＭＭＴＶ－ＬＴＲ启动子下游,构成ＣＤ４４Ｓ的反义ＲＮＡ载体．将其用电击法导入ＣＤ４４＋的人黑色素瘤细胞系ＨＭＭ２３９,转录出的反义ＲＮＡ能不同程度地抑制ＨＭＭ２３９表面ＣＤ４４的表达．ＣＤ４４的表达被抑制后,瘤细胞与透明质酸的结合力下降,细胞的体外生长速率不受影响．将其接种裸鼠皮下,发现其致瘤性明显降低     

CD44 in inflammation and metastasis

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered. Abbreviations: aa, amino acid(s); CS, chondroitin sulfate; CSPG, chondroitin sulfate containing proteoglycan; CD44H, ‘hematopoietic’, also called ‘standard’, isoform of CD44 which contains none of the alternatively spliced variant exons; CD44-Rg, CD44 receptor globulin, a secreted chimaeric protein composed of the external domain of the adhesion receptor CD44 and the hinge, CH2 and CH3 regions of human immunoglobulin-G heavy chain; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate; PB, peripheral blood; PBL, peripheral blood lymphocytes This revised version was published online in November 2006 with corrections to the Cover Date.     

Role of the extracellular and cytoplasmic domains of CD44 in the rolling interaction of lymphoid cells with hyaluronan under physiologic flow

CD44 can function as an adhesion receptor that mediates leukocyte rolling on hyaluronan (HA). To study the contributions of different domains of the standard isoform of CD44 to cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected with wild type (WT) or mutated cDNA constructs. A parallel flow chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that rolled and the rolling velocity was inversely proportional to the amount of cell surface CD44. When the cytoplasmic domain distal to Gly(305) or sequences that serve as binding sites for cytoskeletal linker proteins, were deleted or replaced with foreign sequences, no significant changes in the rolling behavior of mutant cells, compared with the transfectant expressing CD44WT, were observed. Transfectants lacking 64 amino acids of the cytoplasmic tail distal to Cys(295) adhered to HA but showed enhanced rolling at low shear forces. When 83 amino acids from the "non-conserved" membrane-proximal region of the CD44 extracellular domain were deleted, cells adhered firmly to the HA substrate and did not roll at any fluid shear force tested. Unlike wild type cells that exhibited a nearly homogeneous distribution of CD44 on a smooth cell surface, cells expressing the non-conserved region deletion mutant accumulated CD44 in membrane protrusions. Disruption of the actin cytoskeleton with cytochalasin B precluded the formation of membrane protrusions, however, treated cells still adhered firmly to HA and did not roll. We conclude that interaction between the cytoplasmic domain of CD44 and the cytoskeleton is not required for cell rolling on immobilized ligand. The strong effect of deletion of the non-conserved region of the extracellular domain argues for a critical role of this region in CD44-dependent rolling and adhesion interactions with HA under flow.     

Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition

We previously found that the CD44 glycoprotein on some lymphocytes can mediate adhesion to hyaluronate (HA) bearing cells. However, many questions remain about the molecular heterogeneity of CD44 and mechanisms which control its recognition of this ligand. In vitro mutagenesis and DNA sequencing have now been used to investigate the importance of the membrane proximal region of murine CD44 for recognition of soluble or cell surface HA. CD44 with an 83 amino acid deletion in this region mediated binding to soluble ligand and the apparent avidity increased markedly in the presence of a particular antibody to CD44, IRAWB14. The shortened CD44 was however inefficient in mediating adhesion of transfected cells to HA immobilized on cell surfaces. Four new murine isoforms of CD44 were isolated from a carcinoma line by use of the polymerase chain reaction. Only two of them correspond to ones recently discovered in rat and human cells. The longest variant nearly doubled the length of the extracellular portion of the molecule and introduced an additional 20 potential sites for glycosylation. When expressed on T lymphoma cells, all four of the new murine CD44 isoforms were capable of mediating adhesion to HA bearing cells. This result contrasts with a report that a related human CD44 isoform lacks this ability when expressed on B lineage lymphoma cells. The new murine isoforms also conferred the ability to recognize soluble HA and were very responsive to the IRAWB14 antibody. A brief survey of normal murine cell lines and tissues revealed that the hemopoietic isoform was the most abundant species. These findings indicate that the NH2-terminal portion of CD44 is sufficient for HA recognition and that this function is not necessarily abrogated by variations which occur in the membrane proximal domain. They add to the known molecular diversity of CD44 and provide another experimental model in which isoform specific functions can be investigated.     

Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells

In this study we have examined the interaction between CD44 (a hyaluronan (HA) receptor) and the transforming growth factor beta (TGF-beta) receptors (a family of serine/threonine kinase membrane receptors) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunological data indicate that both CD44 and TGF-beta receptors are expressed in MDA-MB-231 cells and that CD44 is physically linked to the TGF-beta receptor I (TGF-betaRI) (and to a lesser extent to the TGF-beta receptor II (TGF-betaRII)) as a complex in vivo. Scatchard plot analyses and in vitro binding experiments show that the cytoplasmic domain of CD44 binds to TGF-betaRI at a single site with high affinity (an apparent dissociation constant (K(d)) of approximately 1.78 nm). These findings indicate that TGF-betaRI contains a CD44-binding site. Furthermore, we have found that the binding of HA to CD44 in MDA-MB-231 cells stimulates TGF-betaRI serine/threonine kinase activity which, in turn, increases Smad2/Smad3 phosphorylation and parathyroid hormone-related protein (PTH-rP) production (well known downstream effector functions of TGF-beta signaling). Most importantly, TGF-betaRI kinase activated by HA phosphorylates CD44, which enhances its binding interaction with the cytoskeletal protein, ankyrin, leading to HA-mediated breast tumor cell migration. Overexpression of TGF-betaRI by transfection of MDA-MB-231 cells with TGF-betaRIcDNA stimulates formation of the CD44.TGF-betaRI complex, the association of ankyrin with membranes, and HA-dependent/CD44-specific breast tumor migration. Taken together, these findings strongly suggest that CD44 interaction with the TGF-betaRI kinase promotes activation of multiple signaling pathways required for ankyrin-membrane interaction, tumor cell migration, and important oncogenic events (e.g. Smad2/Smad3 phosphorylation and PTH-rP production) during HA and TGF-beta-mediated metastatic breast tumor progression.     

Role of Integrins and Evidence for two Distinct Mechanisms Mediating Human Colorectal Carcinoma Cell Interaction with Peritoneal Mesothelial Cells and Extracellular Matrix

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Col 15 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-β1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell—mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.     

Role of Integrins and Evidence for two Distinct Mechanisms Mediating Human Colorectal Carcinoma Cell Interaction with Peritoneal Mesothelial Cells and Extracellular Matrix

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Col 15 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-β1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell—mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.