CD44v6 cell surface expression is a common feature of macrophages and macrophage-like cells - implication for a natural macrophage extravasation mechanism mimicked by tumor cells

Soluble CD44standard (sCD44s) and CD44v6 (sCD44v6) cannot only be detected in sera of patients with pancreatic carcinoma but also of healthy blood donors. To investigate whether sCD44s and sCD44v6 are derived from white blood cells, we stimulated whole blood with phytohemagglutinin and interleukin-2, which induced expression of CD44v6 only on monocytes. For further investigations, we used the promyelocytic leukemia cell line Hl-60. Only Hl-60 cells differentiating along the macrophage pathway showed increased expression of CD44s and CD44v6. Furthermore, only macrophages showed increased secretion of sCD44s and sCD44v6. Our data suggest that CD44s and CD44v6 are common adhesion molecules on macrophages and macrophage-like cells.     

A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression

High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. Expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. Expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7-associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation.     

Apoptosis and release of CD44s in bleomycin-treated L132 cells

The anti-cancer drug bleomycin (BLM) induces lung injury and triggers apoptosis of alveolar epithelial cells. In epithelia, among other functions, the adhesion protein CD44 promotes the contact to components of the extracellular matrix like hyaluronate. A functional link between apoptosis and the loss of CD44 has been observed in colon carcinoma cells and involvement of CD44 in apoptosis of lung cells has been reported in several studies. The present in vitro study examined the expression of CD44s (CD44 standard) in two human epithelial lung cell lines, L132 and A549, during BLM-induced apoptosis. A loss of CD44s by lung epithelial cells and an increase of the soluble form of this adhesion protein in culture supernatants upon exposure to BLM were observed. Apoptosis was characterized by an activation of caspase-3 as well as by release of cytochrome C into the cytosol as shown for L132 cells. Inhibition of apoptosis by the broad-range caspase inhibitor Z-VAD-fmk reduced CD44 release by both cell lines demonstrating that CD44 release is a result of apoptotic processes. Kinetic experiments failed to discriminate between the initiation of apoptosis and CD44 release. Blocking experiments using antagonistic anti-CD95 receptor antibodies revealed that BLM may cause apoptosis and CD44 release in a CD95-independent manner.     

An immunohistochemical study of the expression of adhesion molecules in gallbladder lesions.

We investigated the expression of 10 adhesion molecules (alpha-catenin, beta-catenin, gamma-catenin, CD44, CD44v6, ICAM-1, CD56, CEA, E-cadherin, and CD99) in 46 gallbladder carcinomas, 14 adenomas, 15 low-grade dysplasias, nine intestinal metaplasias, and 20 samples of normal gallbladder epithelium by immunohistochemistry. The expression of adhesion molecules was altered in gallbladder carcinomas and adenomas. In gallbladder carcinomas, increased expression of ICAM-1, CEA, and CD44v6 was observed, together with decreased expression of alpha/beta/gamma-catenin and CD99. In adenomas, aberrant expression of CD44v6 and CD56, as well as reduced expression of alpha/beta/gamma- and E-cadherins, was noted. Expression of alpha/beta/gamma-catenin was reduced in low-grade dysplasia, whereas there was no change in the expression of these adhesion molecules in metaplasia. Expression of ICAM-1, CD99, E-cadherin, and CD56 was correlated with clinical stage. In addition a correlation was noted between expression of ICAM-1 and E-cadherin and lymph node metastasis (p<0.05). These results suggest that altered expression of these adhesion molecules is involved in the progression and metastasis of gallbladder carcinomas.     

Overexpression of c-Met and CD44v6 Receptors Contributes to Autocrine TGF-β1 Signaling in Interstitial Lung Disease

The hepatocyte growth factor (HGF) and the HGF receptor Met pathway are important in the pathogenesis of interstitial lung disease (ILD). Alternatively spliced isoforms of CD44 containing variable exon 6 (CD44v6) and its ligand hyaluronan (HA) alter cellular function in response to interaction between CD44v6 and HGF. TGF-β1 is the crucial cytokine that induces fibrotic action in ILD fibroblasts (ILDFbs). We have identified an autocrine TGF-β1 signaling that up-regulates both Met and CD44v6 mRNA and protein expression. Western blot analysis, flow cytometry, and immunostaining revealed that CD44v6 and Met colocalize in fibroblasts and in tissue sections from ILD patients and in lungs of bleomycin-treated mice. Interestingly, cell proliferation induced by TGF-β1 is mediated through Met and CD44v6. Further, cell proliferation mediated by TGF-β1/CD44v6 is ERK-dependent. In contrast, action of Met on ILDFb proliferation does not require ERK but does require p38^MAPK. ILDFbs were sorted into CD44v6⁺/Met⁺ and CD44v6⁻/Met⁺ subpopulations. HGF inhibited TGF-β1-stimulated collagen-1 and α-smooth muscle cell actin expression in both of these subpopulations by interfering with TGF-β1 signaling. HGF alone markedly stimulated CD44v6 expression, which in turn regulated collagen-1 synthesis. Our data with primary lung fibroblast cultures with respect to collagen-1, CD44v6, and Met expressions were supported by immunostaining of lung sections from bleomycin-treated mice and from ILD patients. These results define the relationships between CD44v6, Met, and autocrine TGF-β1 signaling and the potential modulating influence of HGF on TGF-β1-induced CD44v6-dependent fibroblast function in ILD fibrosis.     

Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable,but Shows Quantitative Changes during Tumour Progression

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 ‘fingerprint’. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression.     

Coexpression of EpCAM,CD44 Variant Isoforms and Claudin-7 in Anaplastic Thyroid Carcinoma

Background

Anaplastic thyroid cancer is considered to be one of the most aggressive human malignancies, and the mean survival time after diagnosis is approximately six months, regardless of treatments. This study aimed to examine how EpCAM and its related molecules are involved in the characteristics of anaplastic thyroid carcinoma.

Methodology/Principal Findings

Two differentiated thyroid cancer cell lines (TPC-1 and FTC-133), and two anaplastic thyroid cancer cell lines (FRO, ACT-1) were analyzed for expression of CD44 standard isoform (CD44s), CD44 variant isoforms, and EpCAM, and human aldehyde dehydrogenase-1 (ALDH1) enzymatic activity using flow cytometry. CD44s expression was higher in TPC-1 and FTC-133 than in the FRO and ACT-1, whereas ALDH1 activities were higher in FRO and ACT-1 than in TPC-1 and FTC-133. An inverse correlation between CD44s expression and ALDH1 activity was observed in all thyroid cancer cell lines. As for the expressions of CD44 variant isoforms, ACT-1 showed higher and FRO showed moderate CD44v6 expressions, whereas either TPC-1 or FTC-133 showed negative CD44v6 expression. EpCAM expressions in FRO and ACT-1 were higher than those in TPC-1 and FTC-133, and EpCAM expressions inversely correlated with those of CD44s. A positive correlation was observed between EpCAM expression and ALDH1 activity in thyroid cancer cell lines. In the RT-PCR analysis, the expression levels of EpCAM, caludin-7 and ALDH1 in FRO and ATC-1 cells were significantly higher than those in TPC-1 and FTC-133 cells. In clinical specimens of thyroid cancers, nuclear expression of EpCAM and high expression of CD44v6 were detected significantly more frequently in anaplastic carcinomas.

Conclusions/Significance

Our study suggests the possibility that EpCAM, together with CD44v6 and claudin-7 as well as ALDH1, may be involved in the development of the aggressive phenotype of anaplastic thyroid carcinoma. Our findings may suggest a novel therapeutic strategy for treatment of anaplastic thyroid carcinoma.     

Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44

Correlations have been noted between the expression of certain alternatively spliced CD44 isoforms and the metastatic propensity of various histologically distinct tumor cell types. The precise mechanism by which particular CD44 isoforms contribute to the metastatic process is, however, unclear. In the present study we demonstrate that CD44R2, a CD44 isoform highly expressed on activated and transformed hemopoietic cells, can recognize and bind a common determinant present on CD44H and CD44R1. Importantly, CD44H lacked this activity. Pretreatment of TIL1 cells expressing CD44H or CD44R1 with chondroitinase ABC inhibited adhesion to CD44R2, suggesting that the unique inserted region present within the CD44R2 molecule, encoded by exon v10, mediates cell adhesion by potentiating the recognition of chondroitin sulfate moieties presented in association with other CD44 molecules. These data help explain the differential involvement of v10-containing CD44 isoforms in tumor metastasis.     

Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development

 Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7–9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7–8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events. Accepted: 13 January 1998     

CD44 variant isoforms associate with tetraspanins and EpCAM

The metastasizing subline of the rat pancreatic adenocarcinoma BSp73 expresses a set of membrane molecules, the combination of which has not been detected on non-metastasizing tumor lines. Hence, it became of interest whether these molecules function independently or may associate and exert specialized functions as membrane complexes. Separation of CD44v4-v7 containing membrane complexes in mild detergent revealed an association with the alpha3 integrin, annexin I, EpCAM, and the tetraspanins D6.1A and CD9. EpCAM and the tetraspanins associate selectively with CD44 variant (CD44v), but not with the CD44 standard (CD44s) isoform. The complexes are found in glycolipid-enriched membrane (GEM) microdomains, which are dissolved by stringent detergents, but the complexes are not destroyed by methyl-beta-cyclodextrin (MbetaCD) treatment, which implies that complex formation does not depend on a lipid-rich microenvironment. However, a complex-associated impact on cell-matrix and cell-cell adhesion as well as on resistance towards apoptosis essentially depended on the location in GEMs. Thus, CD44v-specific functions may well be brought about by complex formation of CD44v with EpCAM, the tetraspanins, and the alpha3 integrin. Because CD44v4-v7-EpCAM complex-specific functions strictly depended on the GEM localization, linker or signal-transducing molecules associating with the complex are likely located in GEMs.     

CD44 variant,but not standard CD44 isoforms,mediate disassembly of endothelial VE-cadherin junction on metastatic melanoma cells

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8-v10 induced junction disassembly and vascular endothelial (VE)-cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)-mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8-v10 promoted endothelial junction disruption. Overexpression of CD44v8-v10 but not of standard CD44 (CD44s) promoted gap formation in the non-metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE-cadherin mutant Y658E supported transmigration of CD44-silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.     

Hyaluronan-CD44 interactions as potential targets for cancer therapy

It is becoming increasingly clear that signals generated in tumor microenvironments are crucial to tumor cell behavior, such as survival, progression and metastasis. The establishment of these malignant behaviors requires that tumor cells acquire novel adhesion and migration properties to detach from their original sites and to localize to distant organs. CD44, an adhesion/homing molecule, is a major receptor for the glycosaminoglycan hyaluronan, which is one of the major components of the tumor extracellular matrix. CD44, a multistructural and multifunctional molecule, detects changes in extracellular matrix components, and thus is well positioned to provide appropriate responses to changes in the microenvironment, i.e. engagement in cell-cell and cell-extracellular matrix interactions, cell trafficking, lymph node homing and the presentation of growth factors/cytokines/chemokines to co-ordinate signaling events that enable the cell responses that change in the tissue environment. The potential involvement of CD44 variants (CD44v), especially CD44v4-v7 and CD44v6-v9, in tumor progression has been confirmed for many tumor types in numerous clinical studies. The downregulation of the standard CD44 isoform (CD44s) in colon cancer is postulated to result in increased tumorigenicity. CD44v-specific functions could be caused by their higher binding affinity than CD44s for hyaluronan. Alternatively, CD44v-specific functions could be caused by differences in associating molecules, which may bind selectively to the CD44v exon. This minireview summarizes how the interaction between hyaluronan and CD44v can serve as a potential target for cancer therapy, in particular how silencing CD44v can target multiple metastatic tumors.     

Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon

The CD44 cell surface glycoprotein is expressed on a broad range of different tissues as multiple isoforms containing from one to ten alternatively spliced exons v1-v10 inserted within the extracellular domain. Differential glycosylation generates still further variability, yielding both N- and O-glycan-modified forms of CD44 in addition to proteoglycan-like variants containing chondroitin sulphate and heparan sulphate. These high molecular mass proteoglycan-like variants, previously identified in lymphocytes, melanomas, and keratinocytes have been implicated in cell-matrix adhesion, cell motility, and invasiveness. More recently, monocyte CD44 molecules presumed to carry glycosaminoglycan chains were shown to bind the chemokine MIP-1 beta (Tanaka, Y.,D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. Nature (Lond). 361:79-82.) raising the intriguing possibility that proteoglycan-like CD44 variants might play a role in regulating inflammatory responses. Here we have investigated the molecular identity of these proteoglycan-like CD44 variants by generating a panel of recombinant CD44 isoforms using a novel cassette cloning strategy. We show that both chondroitin and heparan sulphate modifications are associated specifically with isoforms (CD44v3-10 and CD44v3,8-10) containing the v3 alternative exon which encodes a consensus motif SGXG for GAG addition. Other isoforms (CD44v10, CD44v8- 10, CD44v7-10, and CD44v6-10) are shown to lack these GAG chains but to carry extensive O-glycan modifications, most likely within the mucin- like alternative exon inserts. We also demonstrate that the majority of endogenous GAG-modified CD44 isoforms present in epithelial cells constitute v3 isoforms thus establishing that in these cells the majority of proteoglycan-like CD44 variants are generated by alternative splicing. Finally we present evidence using transfected B lymphoma cells that the GAG-modified CD44 isoforms CD44v3-10 and CD44v3,8-10, unlike CD44H, bind only weakly to hyaluronan. Together with the demonstration in the accompanying paper (Bennett, K., D. G. Jackson, J.C. Simon, E. Tanczos, R. Peach, B. Modrell, I. Stamenkovic, G. Plowman, and A. Aruffo. 1995. J. Cell Biol. 128:687-698.), that CD44 molecules containing the v3 exon bind growth factors, these results highlight a new and potentially important role for CD44 alternative splicing in the control of cell-surface proteoglycan expression.     

CD44s and CD44v6 expression in head and neck epithelia

Background

CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.

Methods

Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.

Results

In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.

Conclusion

CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.     

CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin

The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.     

Quantitative immunohistochemical analyses of the expression of E-cadherin, thrombomodulin, CD44H and CD44v6 in primary tumours of pharynx/larynx squamous cell carcinoma and their lymph node metastases.

The quantitative expression of E-cadherin, thrombomodulin, CD44H and CD44v6 in 32 specimens of primary tumours of pharynx/larynx squamous cell carcinoma and their lymph node metastases was studied by immunohistochemistry. With the aim of obtaining comparative and objective data, image acquisition conditions were kept unaltered for all the measurements and the immunostaining intensity was quantified by applying an image processing system. On the one hand, correlations were only observed between CD44H and CD44v6, both in primary tumours and metastases, and between E-cadherin and TM in metastases. On the other hand, statistical analyses of paired data did not show significant differences in the expression of these markers between the two tumour sites. In agreement with previous reports, E-cadherin expression was rather low or negative in primary tumours and metastases of the three poorly differentiated specimens we studied, as well as that of TM, but otherwise some of these samples showed intermediate immunostaining levels of CD44H/CD44v6. It may be concluded from the present study that the quantitative expression of these adhesion molecules in well established lymph node metastases of pharynx/larynx squamous cell carcinoma is essentially unaltered in relation to their primary sites.     

CD44v6: a target for antibody-based cancer therapy

The human CD44 gene encodes type 1 transmembrane glycoproteins involved in cell-cell and cell-matrix interactions. The structural heterogeneity of the gene products is caused primarily by alternative splicing of at least 10 out of 20 exons. Certain CD44 variant isoforms, in particular those containing CD44 variant domain 6 (CD44v6), have been implicated in tumourigenesis, tumour cell invasion and metastasis. Here we will give an overview of immunohistochemically determined CD44v6 expression in human malignancies (primary epithelial and nonepithelial tumours as well as metastases) and normal tissues, and review several examples of the clinical use of CD44v6-specific antibodies. In nonmalignant tissues, CD44v6 expression is essentially restricted to a subset of epithelia. Intense and homogeneous expression of CD44v6 was reported for the majority of squamous cell carcinomas and a proportion of adenocarcinomas of differing origin, but was rarely seen in nonepithelial tumours. This expression pattern has made CD44v6 an attractive target for antibody-guided therapy of various types of epithelium-derived cancers.Abbreviations CD44 type 1 transmembrane glycoprotein, cell surface receptor for hyaluronate - CD44s (CD44H) standard form of CD44 - CD44v6 splice variant exon 6 of CD44 - CTC common toxicity criteria - 2F10, VFF4, VFF7, VFF18 (BIWA 1), U36, V6B3, HB-256, Var 3.1 monoclonal antibodies targeting the CD44v6 antigen - SCC squamous cell carcinoma     

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion.     

Kunitz-type protease inhibitor bikunin disrupts phorbol ester-induced oligomerization of CD44 variant isoforms containing epitope v9 and subsequently suppresses expression of urokinase-type plasminogen activator in human chondrosarcoma cells.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses phorbol ester (PMA)-stimulated expression of urokinase-type plasminogen activator (uPA). In the present study, we tried to answer this mechanism using human chondrosarcoma HCS-2/8 cells. Our results showed the following novel findings: (a) the standard form of CD44 (CD44s; 85 kDa) is expressed in both unstimulated and PMA-stimulated cells, while CD44v isoforms containing epitope v9 (110 kDa) are strongly up-regulated in response to treatment with PMA; (b) CD44v isoforms containing epitope v9 present on the same cell exclusively form aggregates in stimulated cells; (c) induction of uPA mRNA expression could be achieved by using a second cross-linker antibody to cross-link Fab monomers of anti-CD44; (d) co-treatment of stimulated cells with anti-CD44 mAb alone or anti-CD44v9 mAb alone suppresses PMA-induced clustering of CD44, which results in inhibition of uPA overexpression; (e) bikunin efficiently disrupts PMA-induced clustering of CD44, but does not prevent PMA-induced up-regulation of CD44v isoforms containing epitope v9; and (f) after exposure to bik, approximately 150-kDa band is mainly detected with immunoprecipitation and this band is shown to be a heterodimer composed of the 110-kDa v9-containing CD44v isoforms and a 45-kDa bik receptor (bik-R). In conclusion, we provide, for the first time, evidence that the bik-R can physically interact with the CD44v isoforms containing epitope v9 and function as a repressor to down-regulate PMA-stimulated uPA expression, at least in part, by preventing clustering of CD44v isoforms containing epitope v9.