CD44 Variant Isoforms Research Articles

Identification, characterization and utilization of tumor cell selectin ligands in the design of colon cancer diagnostics

The efficiency of secondary tumor establishment is controlled by the ability of tumor cells to withstand a barrage of mechanical and immunological stresses during their passage through the circulatory system. Accumulating evidence suggests that the selectin-dependent interactions of circulating tumor cells with host cells promote their survival and extravasation from the vasculature, therefore representing a critical checkpoint for colonization of distant organs. These observations have motivated the identification and biochemical characterization of functional selectin ligands such as CD44 variant isoforms, carcinoembryonic antigen and podocalyxin-like protein, present on the surface of metastatic colon carcinoma cells. Understanding the molecular underpinnings of selectin-ligand interactions involved in heterotypic tumor cell-host cell adhesion events may provide guidelines for developing novel cancer diagnostics. Recent advancements in diagnostic device fabrication and design integrated with novel biomarkers exploiting the tissue specific biochemistry of malignant versus normal tissue-expressed selectin ligands may hold promise in providing effective alternatives to current cancer diagnostic technologies.

Podocalyxin-like protein is an E-/L-selectin ligand on colon carcinoma cells: comparative biochemical properties of selectin ligands in host and tumor cells

Selectins facilitate metastasis and tumor cell arrest in the microvasculature by mediating binding of selectin-expressing host cells to ligands on tumor cells. We recently identified CD44 variant isoforms as functional P-, but not E-/L-, selectin ligands on colon carcinoma cells. Furthermore, a approximately 180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify podocalyxin-like protein (PCLP) as an alternative selectin ligand. Blot rolling and cell-free flow-based adhesion assays disclose that PCLP on LS174T colon carcinoma cells possesses E-/L-, but not P-, selectin binding activity. The selectin-binding determinants on LS174T PCLP are non-MECA-79-reactive sialofucosylated structures displayed on O-linked glycans, distinct from the MECA-79-reactive O-glycans on PCLP expressed by high endothelial venules, which is an L-selectin ligand. PCLP on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than PCLP on wild-type cells, suggesting that PCLP functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactivity on PCLP from CD44-knockdown cells correlates with the increased avidity of PCLP for E- but not L-selectin. The novel finding that PCLP is an E-/L-selectin ligand on carcinoma cells offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell PCLP overexpression and the role of selectins in metastasis.

Genetic Modulation of CD44 Expression by Intragraft Fibroblasts

This study investigated the genetic composition and the functional implication of CD44 species expressed by intragraft fibroblasts. An LEW-to-F344 heart transplant model of chronic rejection was used. Intragraft fibroblasts recovered from the chronically rejecting allografts displayed a 4.5-fold increase in expression of CD44 mRNA when compared with that of the fibroblasts isolated from non-rejecting heart allografts (P < 0.01). The intragraft fibroblasts preferentially expressed CD44 variant isoforms containing v1 exon transcript. Automated nucleotide sequence analysis revealed that the majority (90.12%) of the CD44 v1 isoforms expressed by the rejecting graft fibroblasts were encoded by a mutated CD44 mRNA, which contained two point mutations and a codon deletion in the v1 coding region. Histochemistry demonstrated a massive deposition of extracellular HA in the rejecting heart allografts. Hyaluronic acid (HA) was able to promote in vitro fibroblast adhesion, migration in a CD44-dependent manner, and survival in a serum-free culture condition. The study concludes that up-regulation of CD44 v1 isoforms expressed by the intragraft fibroblasts is associated with an increase in the deposition of extracellular HA, the principal ligand for CD44, in the allografts, suggesting that CD44-HA interaction plays an important role in regulating fibroblast recruitment and growth in allografts developing chronic rejection.

Carcinoembryonic Antigen and CD44 Variant Isoforms Cooperate to Mediate Colon Carcinoma Cell Adhesion to E- and L-selectin in Shear Flow

Selectin-mediated adhesion of tumor cells to platelets, leukocytes, and endothelial cells may regulate their hematogenous dissemination in the microvasculature. We recently identified CD44 variant isoforms (CD44v) as functional P-, but not E- or L-, selectin ligands on colon carcinoma cells. Moreover, an approximately 180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify this glycoprotein as the carcinoembryonic antigen (CEA). Blot rolling assays and flow-based adhesion assays using microbeads coated with CEA immunopurified from LS174T colon carcinoma cells and selectins as substrate reveal that CEA possesses E- and L-, but not P-, selectin ligand activity. CEA on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than CEA on wild-type cells, suggesting that CEA functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactive epitopes on CEA from CD44-knockdown cells correlates with the increased CEA avidity for E- but not L-selectin. Through the generation of stable knockdown cell lines, we demonstrate that CEA serves as an auxiliary L-selectin ligand, which stabilizes L-selectin-dependent cell rolling against fluid shear. Moreover, CEA and CD44v cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin at elevated shear stresses. The novel finding that CEA is an E- and L-selectin ligand may explain the enhanced metastatic potential associated with tumor cell CEA overexpression and the supportive role of selectins in metastasis.

DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis

We have shown recently that cDNA vaccination, using a virtual lymph node, ameliorates experimental allergic encephalomyelitis. Successful cure from mammary tumor requires resolution of local tumor growth and metastases. We have examined whether targeting of CD44 cell surface adhesion molecule by cDNA vaccination plays a role in resolving mammary tumor development. We show here that CD44 cDNA vaccination decreases the tumor mass and metastatic potential in experimental mammary tumor of BALB/c mice. Vaccination of mice, inoculated with the mammary tumors, by cDNA of CD44 variant (CD44v) but not by cDNA of standard CD44, markedly reduced local tumor development and lung metastasis. Concomitantly, transfection of CD44 antisense into a highly metastatic mammary tumor cell line disrupted the CD44 expression of the cells and reduced their ability to establish local tumors as well as metastatic colonies in the lung. Moreover, when CD44v, but not standard CD44 sense cDNA, was transfected into the poorly metastatic cell line, tumor development was markedly enhanced. It is possible therefore that DNA vaccination with a specific CD44v construct could induce an immune resistance to mammary tumor progression.

Hyaluronic Acid Synthase-1 Expression Regulates Bladder Cancer Growth, Invasion, and Angiogenesis through CD44

Hyaluronic acid (HA) promotes tumor metastasis and is an accurate diagnostic marker for bladder cancer. HA is synthesized by HA synthases HAS1, HAS2, or HAS3. We have previously shown that HAS1 expression in tumor tissues is a predictor of bladder cancer recurrence and treatment failure. In this study, we stably transfected HT1376 bladder cancer cells with HAS1-sense (HAS1-S), HAS1-antisense (HAS1-AS), or vector cDNA constructs. Whereas HAS1-S transfectants produced approximately 1.7-fold more HA than vector transfectants, HA production was reduced by approximately 70% in HAS1-AS transfectants. HAS1-AS transfectants grew 5-fold slower and were approximately 60% less invasive than vector and HAS1-S transfectants. HAS1-AS transfectants were blocked in G(2)-M phase of the cell cycle due to down-regulation of cyclin B1, cdc25c, and cyclin-dependent kinase 1 levels. These transfectants were also 5- to 10-fold more apoptotic due to the activation of the Fas-Fas ligand-mediated extrinsic pathway. HAS1-AS transfectants showed a approximately 4-fold decrease in ErbB2 phosphorylation and down-regulation of CD44 variant isoforms (CD44-v3, CD44-v6, and CD44-E) both at the protein and mRNA levels. However, no decrease in RHAMM levels was observed. The decrease in CD44-v mRNA levels was not due to increased mRNA degradation. Whereas CD44 small interfering RNA (siRNA) transfection decreased cell growth and induced apoptosis in HT1376 cells, HA addition modestly increased CD44 expression and cell growth in HAS1-AS transfectants, which could be blocked by CD44 siRNA. In xenograft studies, HAS1-AS tumors grew 3- to 5-fold slower and had approximately 4-fold lower microvessel density. These results show that HAS1 regulates bladder cancer growth and progression by modulating HA synthesis and HA receptor levels.

In Vivo Evidence for the Role of CD44s in Promoting Breast Cancer Metastasis to the Liver

The hyaluronan receptor CD44 plays an important role in facilitating invasion and metastasis of a variety of tumors, including breast carcinomas. CD44 functions as a bioactive signaling transmitter. Although a number of studies have implicated CD44 in breast tumor invasion, the evidence is still circumstantial. We have developed a tetracycline-regulated CD44s (standard form) system in the weakly metastatic breast cancer cell MCF7, which exhibits low endogenous expression of CD44 and generated a new cell line, MCF7F-B5. Induction of CD44s alone affected the growth characteristics of MCF7F-B5 cells by increasing their abilities to proliferate, migrate, and invade in vitro. In addition, we have identified and validated cortactin as a novel transcriptional target of hyaluronan/CD44s signaling in underpinning breast tumor invasion. To test these observations in vivo, we developed a doxycycline (DOX)-regulated CD44s breast cancer xenograft model. Induction of CD44s did not affect the growth rate or local invasion of the primary tumor. However, although no mice from the +DOX group developed metastasis, 8 of 11 mice from the -DOX group developed secondary tumors to the liver only. Interestingly, metastatic breast tumors expressed high levels of CD44. This study provides in vivo evidence for the role of the standard form of CD44 in promoting breast tumor invasion and metastasis to the liver.

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.

Selectin Ligand Expression Regulates the Initial Vascular Interactions of Colon Carcinoma Cells: THE ROLES OF CD44V AND ALTERNATIVE SIALOFUCOSYLATED SELECTIN LIGANDS

Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature. We recently reported that CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here we extended those findings by showing that T84 and Colo205 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion to selectins of T84 cell subpopulations sorted based on their CD44 expression levels and stable LS174T cell lines generated using CD44 short hairpin RNA. High versus low CD44-expressing T84 cells tethered more efficiently to P- and L-selectin, but not E-selectin, and rolled more slowly on P- and E-selectin. Knocking down CD44 expression on LS174T cells inhibited binding to P-selectin and increased rolling velocities over P- and L-selectin relative to control-transfected cells, without affecting tethering and rolling on E-selectin, however. Blot rolling analysis revealed the presence of alternative sialylated glycoproteins with molecular masses of approximately 170 and approximately 130 kDa, which can mediate selectin binding in CD44-knockdown cells. Heparin diminishes the avidity of colon carcinoma cells for P- and L-selectin, which may compromise integrin-mediated firm adhesion to host cells and mitigate metastasis. Our finding that CD44v is a functional P-selectin ligand on colon carcinoma provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v overexpression and the role of selectins in metastasis.

445: Preferential expression of CD44 v1 isoforms by intragraft fibroblasts from cardiac allografts with chronic rejection

Purpose: CD44 is a transmembrane glycoprotein primarily involved in cell-cell interactions and cell migration. This study investigated the genetic composition and the functional implication of CD44 species expressed by intragraft fibroblasts (IF). Methods and Materials: Intragraft fibroblasts recovered from the chronically rejecting heart allografts of rats were examined for CD44 gene expression using quantitative real time PCR. CD44 specific cDNA libraries were constructed and selected CD44 clones were analyzed in automate nucleotide sequencing. The role of CD44/hyaluronan interaction was studied using in vitro migration and proliferation assays. Results: IF displayed a 4.5-fold increase in expression of CD44 mRNA when compared with that of the fibroblasts isolated from non-rejecting heart allografts (p 0.01). The IF preferentially expressed CD44 variant isoforms containing v1 exon transcript. Automate nucleotide sequence analysis revealed that the majority (90.12%) of the CD44 v1 isoforms expressed by the rejecting graft fibroblasts were encoded by a mutated CD44 mRNA, which contained 2 point mutations and a codon deletion in the v1 coding region. Histochemistry demonstrated a massive deposition of extracellular hyaluronan in the rejecting heart allografts. Hyaluronan promoted in vitro fibroblast adhesion, migration in a CD44 dependent manner, and survival in a serum-free culture condition. Conclusions: A significant increase in CD44 expression by the rejecting graft fibroblasts was coupled with augmentation of extracellular hyaluronan synthesis in the heart allografts, indicating that CD44-HA interaction is critically involved in the pathogenesis of graft fibrosis. CD44v1 isoforms may play a role, in a way similar to other documented v exons such as v3 or v6, to provide additional signaling for enhancing or suppressing the biological activities of intragraft fibroblasts. CD44 antagonism may affect multiple cellular activities mediated by the CD44-HA pathway, leading to alleviation of allograft fibrosis and chronic inflammation.

The involvement of osteopontin and its receptors in multiple myeloma cell survival, migration and invasion in the murine 5T33MM model

Multiple myeloma (MM) is a malignancy characterised by the accumulation of monoclonal plasma cells in the bone marrow. Different reports indicate the expression of CD44 variant isoforms (CD44v) by MM cells. Osteopontin (OPN), which is expressed by MM cells, is known to be a ligand for CD44v6. In this study, we investigated the role of OPN with emphasis on a functional correlation between OPN and CD44v in the 5T33MM model. Our group reported the expression of CD44v by 5T33MM cells. Using this model, we have demonstrated the secretion of OPN by 5T33MM cells. OPN affected 5T33MM cell survival by increasing proliferation and inhibiting apoptosis. OPN also stimulated 5T33MM cell migration, transendothelial migration and matrix metalloproteinase-9 activity. We confirmed the proliferative and migratory effects of OPN on human MM cells. By applying inhibiting anti-CD44v6 antibodies, we found that OPN stimulated cell proliferation by engaging this isoform. Anti-CD44v antibodies and RGD peptides both inhibited cell migration, suggesting an involvement of both, CD44v isoforms and integrins. In conclusion, OPN may act as a mediator of MM cell survival by engaging CD44v. The protein is further involved in migration and invasion of MM cells through the activation of either alphavbeta3 integrin or CD44v isoforms.

The influence of CD44v3-v10 on adhesion, invasion and MMP-14 expression in prostate cancer cells

The expression of certain CD44 variants has been linked with metastasis and tumour progression. In particular, high molecular weight forms of CD44 show restricted expression in tumours and may correlate with tumour development and metastasis. In this study, we examined the expression of CD44 variants in prostate cancer cell lines: the invasive PC-3 and DU-145, low invasive LNCaP, and two non-invasive prostate epithelial cell lines. PC-3 prostate cancer cells were transfected with a high molecular weight CD44 variant isoform, CD44v3-v10, isolated from non-invasive prostate epithelial cell lines. These transfected cells (PC-NIVO) were assessed using in vitro invasion, tumour-endothelial, growth, and migration assays. The expression of MMP-14 was examined using SDS-PAGE and Western blot analysis. Transfected PC-3 cells (PC-NIVO) were found to be less adherent to endothelial cells and had significantly reduced invasiveness compared to wild-type PC-3 or control cells. In addition, tumour cell adhesion to endothelial cells and invasiveness was increased after exposure to HGF/SF, and can be blocked by the presence of anti-CD44 antibodies. Further investigation revealed a reduction in the expression of MMP-14 in PC-NIVO cells, but not in PC-3 or control cells. In conclusion, non-invasive prostate epithelial cells express a high molecular weight CD44 isoform, CD44v3-v10, which may counteract the standard isoform function of CD44 by reducing adhesion and invasion of endothelium by prostate tumour cells through negation of the MMP-14 function.

Variant isoforms of CD44 are P‐ and L‐selectin ligands on colon carcinoma cells

The initial selectin-dependent events that mediate tumor cell tethering to platelets, leukocytes, and vascular endothelium can regulate the extravasation and colonization of metastatic cells into distant tissues. Little is known, however, about the identity of selectin counter-receptors on tumor cells, which facilitate the metastatic process. To address this issue, we performed SDS-PAGE analysis of membrane proteins, metabolic inhibition studies, blot rolling assays, and cell-free flow-based adhesion experiments using microbeads coated with CD44 immunoprecipitated from carcinomas and purified selectins as substrate. Here, we demonstrate that variant isoforms of CD44 (CD44v) on LS174T colon carcinoma cells possess P-/L-/E-selectin binding activity, in contrast to the standard isoform of CD44 (CD44s) on hematopoietic-progenitor cells (HPCs), which is primarily an L-/E-selectin ligand. Moreover, the selectin-binding determinants on CD44v from LS174T cells are sialofucosylated structures displayed on O-linked glycans, akin to those on P-selectin glycoprotein ligand-1, but distinct from the HECA-452-reactive N-glycans on CD44s expressed on HPCs. Using flow-based adhesion assays, we systematically characterize shear-dependent LS174T CD44 vs. HL60 CD44s adhesion to E-/P-/L-selectin. The novel finding that CD44v are selectin ligands offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell CD44v overexpression and the critical role of selectins in metastasis.

Expression and Glycosylation with Polylactosamine of CD44 Antigen on Macrophages During Follicular Atresia in Pig Ovaries1

Macrophages are essential in cleaning up apoptotic debris during follicular atresia. However, the key factors of this process are still unclear. In the present study, we evaluated CD44 mRNA, CD44 protein, and CD44 antigen glycosylation on macrophages during follicular atresia in the pig. Atresia was classified into five stages: stage I, healthy follicles; stage II, early atretic follicles having apoptotic granulosa cells with an unclear basement membrane; stage III, progressing atretic follicles having apoptotic granulosa cells completely diffused from the basement membrane; stage IV, late atretic follicles with increasing lysosomal activity; and stage V, disintegrated atretic follicles having collapsed theca cells and strong lysosomal activity. Immunohistological analysis showed that macrophages expressing CD44 invaded the inside of stage III follicles, accompanied by a collapse of basement membrane. Semiquantitative RT-PCR showed that only mRNA of the CD44 standard isoform (CD44s) was present in inner cells of follicles, and not any CD44 variant isoform (CD44v) mRNAs. The amount of CD44s mRNA was increased at stage III. Western blot and lectin blot analyses showed that CD44 was markedly expressed at stage III and glycosylated with polylactosamine at the same time. After macrophages invaded atretic follicles at stages III-V, the CD44 expressed on macrophages was glycosylated with polylactosamine. The lysosomal activity began to increase at stage IV, and reached the highest level at stage V. Increased CD44s protein and posttranslational modification of CD44 with polylactosamine on macrophages from stage III could be involved in the cleaning up apoptotic granulosa cells.

Upregulation and dephosphorylation of cofilin: Modulation by CD44 variant isoform in human colon cancer cells

Colon cancer is among the leading causes of cancer death in North America. Dysregulation of the crypt homeostasis is evident in the early stage of colon cancer. Moreover, cytoskeletal rearrangement of actin also plays a crucial role in morphological changes during apoptosis. CD44, an adhesion and anti-apoptotic molecule is overexpressed in colon cancer and is known to interact with certain cytoskeletal proteins. In this study, we used the human colon cancer cell line SW620, which does not express CD44 but stably transfected with standard, 3–10v and 8–10v variant isoforms of CD44. By two-dimensional isoelectric focusing (2DIEF), we found an increasing concentration of a 21-kDa protein in SW620 colon cancer cells transfected with CD44 3–10v, as compared to cells transfected with an empty vector. Mass spectrometry (MS) and proteomic analysis of this protein identified the peptide fragment (YALYDATYETK) of 11 amino acids in length spanning residue 82 to 92 of cofilin, a widely distributed 21-kDa actin-modulating protein. Western blot analysis of lysates from cells expressing CD44 variant isoforms 3–10v had increased level of expression of cofilin compared to vector control consistent with our finding by 2DIEF. Also, immunocytochemistry showed that cofilin expression in colonic epithelial cells was greater in cells transfected with CD44 3–10v, as compared to vector controls. We observed that the phosphorylated form of cofilin is downregulated in cells expressing the 3–10v isoform of CD44 both by Western blot and immunocytochemistry. Cofilin expression is thus mechanistically associated with CD44 expression and its 3–10v isoform. Dephosphorylation of cofilin could bring about directional motility of cells that could have important implications to the proliferation and motility of colonic epithelial cells in cancer.

A novel antiapoptotic mechanism based on interference of Fas signaling by CD44 variant isoforms

There is growing evidence that one of the central common characteristics of tumor and inflammatory cells is their resistance to programmed cell death. This feature results in the accumulation of harmful cells, which are mostly refractory to Fas (FAS, APO-1)-mediated apoptosis. A molecule found on these cells is the transmembrane receptor CD44 with its variant isoforms (CD44v). The establishment of transfectants expressing different CD44v isoforms allowed us to demonstrate that the CD44v6 and CD44v9 isoforms exhibit an antiapoptotic effect and can block Fas-mediated apoptosis. Moreover, we observed that CD44v6 and CD44v9 colocalize and interact with Fas. Importantly, an anti-CD44v6 antibody can abolish the antiapoptotic effect of CD44v6. These results are the first to show that CD44v isoforms interfere with Fas signaling. Our findings improve the understanding of the pathogenesis of cancer and autoimmunity and open new strategies to treat such disorders.

Short-term treatment with anti-CD44v7 antibody, but not CD44v4, restores the gut mucosa in established chronic dextran sulphate sodium (DSS)-induced colitis in mice

Increased expression of CD44 variant isoforms have been shown on the inflammatory infiltrates in human and mouse colitis and blockade or deletion of CD44 isoforms inhibit experimental colitis. The objective of this study was to find out if short-term treatment of CD44 antibodies specific to CD44v7, but not to other variant isoforms, suppresses leucocyte-endothelial interaction in chronic dextran sodium sulphate (DSS)-induced colitis in mice. Chronic colitis was induced by oral administration of four cycles of 5% DSS in BALB/c mice. Expression of CD44 was investigated on isolated mononuclear cells of the gut immune system. In established colitis, mice were treated with antibodies against CD44v7 or CD44v4 three times in 7 days. Intravital microscopy was used to study leucocyte-endothelial interactions and leucocyte extravasation. As a marker of inflammatory infiltrates myeloperoxidase was quantified in gut tissue. CD44-induced apoptosis was determined by fluorescence staining of hypodiploidic cell nuclei. In chronic DSS-induced colitis both CD44 variant isoforms, v4 and v7 were significantly up-regulated on mononuclear cells. However, whereas anti-CD44v7 antibody treatment induced a marked restoration of the gut mucosa and significantly reduced endothelial sticking and extravasation of circulating leucocyte in vivo (P < 0.01), application of anti-CD44v4 or an isotype control antibody had no anti-inflammatory effect. A significant reduction of myeloperoxidase activity was detected after blockade of CD44v7, but not v4. Short-term treatment with anti-CD44v7 antibody blocks T cell extravasation and recruitment to the intestinal mucosa and cures established experimental colitis.

CD44 splicing pattern is associated with disease progression in pulmonary adenocarcinoma.

Certain alternatively spliced exons of CD44 gene have been associated with specific functions. However, these functions may have come from inclusion of a central array of alternatively spliced exons, rather than a single one. The goals of this study were to analyze all of the variant exons included by alternative splicing, the entire population of CD44 mRNA transcripts, and the prognostic implications of CD44 mRNA and protein isoforms expressed by non-small cell lung cancer (NSCLC). Using a polymerase chain reaction protocol with short reaction times, we amplified, sequenced and quantified CD44 mRNA transcripts from 52 samples of NSCLC to determine the splicing patterns of alternatively included exons and the proportion of each CD44 mRNA transcript. The expression of CD44 standard form and variant isoforms CD44v3 and CD44v6 were also analyzed by immunohistochemistry (IHC). Normal lung and NSCLC expressed CD44 mRNA transcripts containing variant exons v10, v8-10, v6-10, v3-10 and v2-10. In squamous cell carcinoma, the expression rates of these mRNA transcripts were equal to or higher than those of the normal lung, and the splicing pattern was not associated with disease progression. In adenocarcinoma, the expression rates of CD44v6-10, v3-10 and v2-10 mRNA were lower than in normal lung. The down-regulation of CD44v6-10, CD44v3-10 mRNA and CD44v6 protein paralleled the progression of adenocarcinoma. Recurrence of adenocarcinoma was associated with negative expression of CD44v6-10 or CD44v3-10 mRNA, and with low-level expression of CD44v6 or CD44v3 by IHC. Negative expression of CD44v6-10 mRNA and reduced expression of CD44 v6 protein were associated with a shorter disease-free and overall survival in the univariate but not the multivariate analysis. Our data suggest that CD44 splicing pattern is associated with disease progression in adenocarcinoma.

The cell–cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7

We recently described that in the metastasizing rat pancreatic carcinoma line BSp73ASML the cell-cell adhesion molecule EpCAM, CD44 variant isoforms and the tetraspanins D6.1A and CD9 form a complex that is located in glycolipid-enriched membrane microdomains. This complex contains, in addition, an undefined 20 kDa protein. As such complex formation influenced cell-cell adhesion and apoptosis resistance, it became of interest to identify the 20 kDa polypeptide. This 20 kDa protein, which co-precipitated with EpCAM in BSp73ASML lysates, was identified as the tight junction protein claudin-7. Correspondingly, an association between EpCAM and claudin-7 was noted in rat and human tumors and in non-transformed tissues of the gastrointestinal tract. Co-localization of the two molecules was most pronounced at basolateral membranes, but was also observed in tight junctions. Evidence for direct protein-protein interactions between EpCAM and claudin-7 was obtained by co-immunoprecipitation after treatment of tumor cells with a membrane-permeable chemical cross-linker. The complex, which is located in glycolipid-enriched membrane microdomains, is not disrupted by partial cholesterol depletion, but claudin-7 phosphorylation is restricted to the localization in glycolipid-enriched membrane microdomains. This is the first report on an association between EpCAM and claudins in both non-transformed tissues and metastasizing tumor cell lines.

CD44 on LS174T Colon Carcinoma Cells Possesses E-Selectin Ligand Activity

Metastasis of circulating tumor cells requires a multistep cascade of events initiated by adhesion of tumor cells to the vascular endothelium of involved tissues. This process occurs under the forces of blood flow and is promoted by adhesion molecules specialized to interact under shear conditions. The endothelial molecule E-selectin is a major mediator of these adhesive events, and there is strong evidence that E-selectin receptor-ligand interactions contribute to the formation of metastasis. However, little is known about the identity of E-selectin ligand(s) expressed on cancer cells. To address this issue, we did SDS-PAGE analysis of membrane proteins, metabolic inhibition studies, and blot rolling assays of LS174T, a colon carcinoma cell line known to interact with E-selectin under physiologic flow conditions. Our studies show that LS174T cells express the hematopoietic cell E/L-selectin (HCELL) glycoform of CD44, which functions as a high-affinity E-selectin glycoprotein ligand on these cells. However, in contrast to the HCELL glycoform on human hematopoietic progenitor cells, which expresses carbohydrate-binding determinant(s) for E-selectin primarily on N-glycans of standard CD44, the relevant determinant(s) on LS174T cells is expressed on O-glycans and is predominantly found on variant isoforms of CD44 (CD44v). Our finding that tumor-associated CD44 splice variant(s) express E-selectin ligand activity provides novel perspectives on the biology of CD44 in cancer metastasis.