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.     

CD44v6 coordinates tumor matrix-triggered motility and apoptosis resistance

Tumor progression requires a crosstalk with the tumor surrounding, where the tumor matrix plays an essential role. We recently reported that only the matrix delivered by a CD44v6-competent (ASML(wt)), but not that of a CD44v6-deficient (ASML-CD44v(kd)) rat pancreatic adenocarcinoma line supports metastasis formation. We here describe that this matrix provides an important feedback toward the tumor cell and that CD44v6 accounts for orchestrating signals received from the matrix. ASML(wt) cells contain more hyaluronan synthase-3 and secrete higher amounts of >50 kDa HA than ASML-CD44v(kd) cells, which secrete more hyaluronidase. Only the ASML(wt)-matrix supports migration and apoptosis resistance, which both can be initiated via CD44v6, c-Met, and α6β4 ligand binding and proceed via FAK, PI3K/Akt, and MAPK activation, respectively. However, c-Met- and α6β4-initiated signaling are strongly augmented by the association with CD44v6 as only very weak effects are observed in CD44v6-deficient cells. The same CD44v6-dependent convergence of motility- and apoptosis resistance-related signals also accounts for human tumor lines. Thus, CD44v6 promotes motility and apoptosis resistance via its involvement in assembling a matrix that, in turn, triggers activation of signaling cascades, which proceeds, independent of the initiating receptor-ligand interaction, in a concerted action via CD44v6.     

Involvement of CD44v6 in InlB-dependent Listeria invasion

Listeria monocytogenes , a Gram-positive bacterium, is the causative agent for the disease called listeriosis. This pathogen utilizes host cell surface proteins such as E-cadherin or c-Met in order to invade eukaryotic cells. The invasion via c-Met depends on the bacterial protein InlB that activates c-Met phosphorylation and internalization mimicking in many regards HGF, the authentic c-Met ligand. In this paper, we demonstrate that the activation of c-Met induced by InlB is dependent on CD44v6, a member of the CD44 family of transmembrane glycoproteins. Inhibiting CD44v6 by means of a blocking peptide, a CD44v6 antibody or CD44v6-specific siRNA prevents the activation of c-Met induced by InlB. Subsequently, signalling, scattering and the entry of InlB-coated beads into host cells are also impaired by CD44v6 blocking reagents. For the entry process, ezrin, a protein that links the CD44v6 cytoplasmic domain to the cytoskeleton, is required as well. Most importantly, this collaboration between c-Met and CD44v6 contributes to the invasion of L. monocytogenes into target cells as demonstrated by a drastic decrease in bacterial invasion in the presence of blocking agents such as the CD44v6 peptide or antibody.     

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     

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.     

PDGF Suppresses the Sulfation of CD44v and Potentiates CD44v-Mediated Binding of Colon Carcinoma Cells to Fibrin under Flow

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen).     

Multisite and bidirectional exonic splicing enhancer in CD44 alternative exon v3

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.     

Role of CD44 in epithelial wound repair: migration of rat hepatic stellate cells utilizes hyaluronic acid and CD44v6

The hyaluronic acid receptor, CD44, exists as multiple splice variants that appear to have a role in migration of tumor cells. The role of this receptor and its variants in normal wound repair is poorly understood. A central feature of wound repair in the liver is activation and migration of perisinusoidal stellate cells. We have examined CD44 expression by stellate cells from normal or injured rat liver, finding that it increases with injury and involves a distinct set of CD44 splice variants. Among the latter, variants containing the v6 exon (CD44v6) are strikingly increased. Analysis of migration of primary cells on transwell filter inserts reveals that only cells isolated from injured liver are migratory. Also, they move more rapidly on hyaluronic acid than on collagen I or collagen IV. A polyclonal antibody to recombinant CD44v6 blocks migration by 50%, whereas antibody to CD44v4 has no effect. The inhibition is specific for cells migrating on hyaluronic acid and is reversed by synthetic peptide representing the N terminus of the v6 protein. In conclusion, activated stellate cells use CD44v6 and hyaluronic acid for migration. Given the evidence that migration is required for progression of injury with scar formation, blockers of CD44v6 expression or function are candidates for preventing the deleterious effects of chronic fibrosis.     

CD44v10: an antimetastatic membrane glycoprotein for pancreatic cancer

AIMS: The aims of this study were 1) to investigate the mRNA pattern of CD44 variants in three primary (MIA PaCa 2, PANC-1, PSN-1) and two metastatic (CAPAN-1, SUIT-2) pancreatic cancer (PC) cell lines; 2) to ascertain whether the genetic transfer of CD44s and CD44v10 modifies the adhesion of PC cells to the extracellular matrix (ECM) in vitro and their metastatic behavior in vivo. METHODS: CD44 mRNA analysis was done by means of RT-PCR. Adhesion to ECM the was assessed using coated microtiter plates. For the study of CD44v10 insertion in the CAPAN-1 line, liposome-mediated DNA transfer was used. SCID mice were employed for in vivo experiments. RESULTS: CD44v10 mRNA was not expressed by the CAPAN-1 nor by four of the six SUIT-2-derived clones. The stable expression of CD44v10 by modified CAPAN-1 significantly enhanced fibronectin adhesion. Mice without either liver or pancreatic metastases were more frequently found among the animals injected with modified (CD44v10 expressing) than with non-modified CAPAN-1. CONCLUSIONS: 1) It is possible to differentiate between metastatic and non-metastatic PC cells on the basis of CD44v10 expression; 2) CD44v10 seems to be involved in mediating fibronectin adhesion in vitro and in counteracting metastases in vivo.     

Sequence variation in the monoclonal-antibody-U36-defined CD44v6 epitope

 Monoclonal antibody (mAb) U36 was developed for the treatment of minimal residual disease of head and neck squamous cell carcinoma (HNSCC). The mAb-U36-defined antigen was characterized by cDNA cloning, and was shown to be identical to the keratinocyte-specific CD44 splice variant epican. The epitope recognized by mAb U36 was shown to be located in the v6 domain. Two amino acids within the epitope appeared to differ from the sequences that have been described in literature. The sequence of the epitope appeared to contain glutamic acid at position 367 and lysine at position 374, while valine and arginine respectively have been described before. Interestingly, another anti-CD44v6 antibody with possible clinical application, VFF18, recognizes an epitope in the same area. With respect to the applicability of these antibodies for tumor targeting, this variation might have an influence on antibody-antigen interaction and mAb accumulation in the tumor. Furthermore, this observation raised the question whether the different epitopes are related to the malignant behavior of tumor cells. In this paper we determine the relative affinity of mAb U36 for the variant epitope sequences by tumor cell binding assays using synthetic peptides for competition. The presence of glutamic acid instead of valine at position 367 caused strong competition. Further evaluation showed that the published valine variant does not exist in vivo, and is the result of a sequencing artefact. The effect of substitution of lysine for arginine at position 374 had no effect on the binding of mAb U36 to the cells. This amino acid variation was shown to be due to allelic polymorphism. There was no trend towards allelic imbalance in tumor cells as compared to normal cells. Received: 5 June 1997 / Accepted: 14 August 1997     

CD44v6 promotes proliferation by persisting activation of MAP kinases

CD44v6 is transiently expressed during T cell activation, and constitutively CD44v4-v7 expressing transgenic T cells show accelerated responses towards nominal antigens. The underlying mechanism is unknown. The mouse thymoma EL4 was transfected with CD44 standard isoform (CD44s) or CD44v6 cDNA (EL4-s, EL4-v6). Only EL4-v6 cells proliferated at an over 10-fold higher rate than untransfected cells, displayed up-regulated expression of CD69, CD25, and IL-2, and were protected from apoptosis by CD44v6 cross-linking. In the absence of any stimulus, ERK1/2 was partly phosphorylated, and phosphorylation was significantly increased by CD44v6 cross-linking. The same accounted for JNK, c-jun, and IkappaBalpha. Moreover, NF-kappaB was partly translocated into the nucleus. Instead, CD44s cross-linking induced ERK1/2, JNK, c-jun, and IkappaBalpha phosphorylation only in the context of TCR engagement. No selectively CD44v6 associated transmembrane proteins were uncovered in EL4 cells. However, CD44v6, as opposed to CD44s, did not colocalise with the TCR/CD3 complex after CD3 cross-linking. Furthermore, a CD44-associated 85-kDa protein became hypophosphorylated only after CD44v6 cross-linking. Threonine hypophosphorylation of this protein coincided with the activation of MAP and SAP kinases, which was prohibited in the presence of a phosphatase inhibitor. Thus, CD44v6, distinct to CD44s, stimulates autonomously growth and IL-2 secretion of a thymoma line and rescues cells from apoptosis.     

Coexpression of CD44 variant (v10/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis

CD44 is the major hyaluronan cell surface receptor and functions as an adhesion molecule in many different cell types, including human breast epithelial cells. The coexpression of certain CD44 variants (CD44v), such as CD44v (v10/ex14), with CD44s (standard form) appears to be closely associated with human breast tumor metastasis. In this study we have established a stable transfection of CD44v (v10/ex14) cDNA into nontumorigenic human breast epithelial cells (HBL100) which contain endogenous CD44s. Our results indicate that coexpression of both CD44v (v10/ex14) and CD44s alters the following important biological properties of these cells: 1) there is a significant reduction in hyaluronic acid (HA)-mediated cell adhesion; 2) there is an increased migration capability in collagen-matrix gel; and 3) these cells constitutively produce certain angiogenic factors and effectively promote tumorigenesis in athymic nude mice. These findings suggest that coexpression of CD44v (v10/ex14) and CD44s may trigger the onset of cell transformation required for breast cancer development. J. Cell. Physiol. 171:152–160, 1997. © 1997 Wiley-Liss, Inc.     

CD44v6和MMP-2 在子宫内膜异位症组织中的表达及相关性

目的:探讨粘附分子CD44拼构变异体6(CD44v6)和基质金属蛋白酶-2(MMP-2)在子宫内膜异位症(EMs)组织中的表达及相关性。方法:选取40例异位内膜组织标本、40例在位内膜组织标本及40例正常子宫内膜标本,用免疫组织化学方法检测CD44v6和MMP-2的表达,并分析其相关性。结果:CD44v6在异位内膜组的表达明显高于在位内膜组和对照组,且对照组明显高于在位内膜组,差异具有统计学意义(P0.05);CD44v6在在位内膜组和对照组中分泌期的表达明显高于同组增生期,差异具有统计学意义(P0.05)。MMP-2在异位内膜组和在位内膜组的表达明显高于对照组,差异具有统计学意义(P0.01);MMP-2在各组增生期和分泌期表达不规律。异位内膜组中,CD44v6和MMP-2在Ⅲ-Ⅳ期的表达明显高于Ⅰ-Ⅱ期,差异具有统计学意义(P0.01)。Spearman相关性分析结果显示:EMs组织中CD44v6和MMP-2之间呈现正相关性(r=0.724,P0.05);EMs不同分期组织中CD44v6和MMP-2之间亦呈现正相关性(r=0.623,P0.05)。结论:CD44v6和MMP-2在EMs异位内膜中高表达,且有正协同作用,二者可能与EMs的发生发展有关。     

Differential expression of CD44s and CD4v10 proteins and syndecan in normal and irradiated mouse epidermis

 The role of the CD44s adhesion molecule, its epithelial isoforms and its relationship to epidermal proteoglycans such as syndecan was studied in normal and irradiated mouse skin. In normal mouse skin, only 10% of basal cells are strongly CD44s-immunopositive, with a cytoplasmic expression pattern. Double-label experiments with the basal cell marker keratin 14 confirmed the epithelial nature of the strongly CD44s-positive cell type in the basal layer. Some spinous keratinocytes and the majority of the remaining basal cells exhibited a weak membranous staining pattern. In contrast, the epithelial isoform, CD44v10, was strongly present in all basal and suprabasal epithelial cells of the epidermis, with a membranous staining pattern. Syndecan was found in the granular layer of the normal epidermis only. After 1 week of daily irradiation, the entire basal cell layer of the epidermis expressed CD44s in the membrane, but with a varying degree of staining intensity. This reactivity spread to the upper spinous layer after 3 weeks of treatment. In hyperproliferative epidermis, there was no difference in the staining patterns between CD44s and CD44v10. The expression of syndecan switched from the granular layer to the basal and lower spinous layers after 2 weeks of daily irradiation. Immunoreactivity for syndecan was also strongly enhanced in the dermis of irradiated samples. The results suggest an important role for syndecan and CD44 in proliferative processes during radiation-induced accelerated repopulation. Accepted: 30 September 1996     

CD44v10 expression in the mouse and functional activity in delayed type hypersensitivity

We have described recently that expression of CD44 exon v10 (CD44v10) is down-regulated upon metastasis of squamous cell carcinoma, whereas it is up-regulated in skin metastases of malignant melanoma. The striking regulation of CD44v10 prompted us to generate a murine CD44v10-specific monoclonal antibody to define expression and possible functions of this particular CD44 variant isoform. In the mouse, expression of exon v10 was restricted to basal layers of the epidermis and squamous epithelium of the oral cavity, the esophagus, the omasum, glandular epithelium of the submandibular and the uterine gland, as well as subpopulations of bone marrow cells and activated lymphocytes. Expression started late during development, e.g., was not observed before day 16 of gestation and there was no evidence for developmental regulation of CD44v10 expression. Functional in vivo studies revealed that anti-CD44v10 had no effect on wound healing but inhibited edema and granuloma formation in delayed type hypersensitivity (DTH). Furthermore, lymphocyte-monocyte interactions could be inhibited by anti-CD44v10. Because a CD44v10 transfected tumour line did not show any distinct pattern of cell-matrix or cell-cell adhesion, the data point toward an involvement of CD44v10 in cell migration, possibly by acting as a target structure for cytokines/chemokines provided by the contacted partner cell. J. Cell. Physiol. 171:305–317, 1997. © 1997 Wiley-Liss, Inc.     

CD44v6和PTEN在胃印戒细胞癌中的表达意义

目的:探讨CD44v6和PTEN在胃印戒细胞癌组织中的表达与临床病理特征及预后之间的关系.方法:应用免疫组织化学SP二步法,检测73例胃印戒细胞癌组织中CD44v6及PTEN的表达,分析CD44v6、PTEN与胃印戒细胞癌浸润深度、淋巴结转移、器官转移、临床分期和三年生存率的关系.结果:73例胃印戒细胞癌组织中CD44v6阳性率为78.1%(57/73),PTEN阳性表达率为34.2%(25/73),PTEN表达与器官转移、临床分期有差异(P＜0.05),CD44v6、PTEN表达均与胃印戒细胞癌浸润深度、淋巴结转移有差异(P＜0.05).两者之间Spearman等级表达呈负相关(r=-0.214,P＜0.05);Kaplan-Meier生存分析显示PTEN阴性组三年生存率16.1%显著低于PTEN阳性组66.9%(P＜0.01).结论:进行CD44v6和PTEN免疫组织化学检测对评估胃印戒细胞癌的预后具有一定意义.