Peanut lectin stimulates proliferation of colon cancer cells by interaction with glycosylated CD44v6 isoforms and consequential activation of c-Met and MAPK: functional implications for disease-associated glycosylation changes

Peanut agglutinin lectin (PNA) binds the Thomsen-Friedenreich (TF) oncofetal carbohydrate antigen (galactose beta1-3N-acetylgalactosamine alpha) that shows increased expression in colon cancer, adenomas, and inflammatory bowel disease. PNA is mitogenic, both in vitro and in vivo, for colon epithelial cells. In these cells, PNA binds predominantly to cell-surface TF antigen expressed by high molecular weight isoforms of the transmembrane glycoprotein CD44 that are generated in inflamed and neoplastic colonic epithelia by altered RNA splicing. Our aim was to identify the signaling mechanism underlying the proliferative response to PNA. This was investigated in HT29, T84, and Caco2 colon cancer cells. Parallel lectin and immunoblotting of PNA affinity-purified HT29 cell membrane extracts showed PNA binding to high molecular weight CD44v6 isoforms. Within 5 min, PNA (25 microg/mL) caused a 6-fold increase in phosphorylation of hepatocyte growth factor receptor c-Met, known to co-associate with CD44v6. This was followed by the downstream activation of p44/p42 mitogen-activated protein kinase (MAPK) over 15-20 min. The presence of 100 microg/mL asialofetuin, a TF antigen-expressing glycoprotein, blocked both PNA-induced c-Met and MAPK activation. A similar PNA-induced c-Met and MAPK phosphorylation was also seen in T84 cells that express CD44v6 but not in Caco2 cells that lack CD44v6. PNA-induced cell proliferation was completely blocked by 1 microM PD98059, an inhibitor of MAPK activation (p < 0.0001). The expression of TF antigen by CD44 isoforms in colonic epithelial cells allows lectin-induced mitogenesis that is mediated by phosphorylation of c-Met and MAPK. It provides a mechanism by which dietary, microbial, or endogenous galactose-binding lectins could affect epithelial proliferation in the cancerous and precancerous colon.     

The TF-antigen binding lectin from Sclerotium rolfsii inhibits growth of human colon cancer cells by inducing apoptosis in vitro and suppresses tumor growth in vivo

Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galβ1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer.     

Suppression of Core 1 Gal-Transferase Is Associated with Reduction of TF and Reciprocal Increase of Tn,sialyl-Tn and Core 3 Glycans in Human Colon Cancer Cells

It has long been presumed, though with surprisingly little evidence, a competition between Core 1 Gal-transferase (C1GalT), Core 3 GlcNAc-transferase (C3GnT) and sialyl-transferase (ST6GalNAc-T) for elongation of O-linked mucin-type glycans initiated with GalNAcα-Ser/Thr. This study tested this presumption by selective suppression of one of these glycosyltransferases and then analysed the expressions of the enzymatic products of the other three glycosyltransferases. It was found that siRNA suppression of C1GalT markedly reduced the expression of Galβ1,3GalNAcα- (Core 1) and in the meantime increased the expressions of sialyl-GalNAcα- (sialyl-Tn), GalNAcα- (Tn) and GlcNAcβ1,3GalNAcα- (Core 3)-associated glycans in human colon cancer HT29 and SW620 cells. This supports a competitive modification of the GalNAcα-Ser/Thr between C1GalT, C3GnT and ST6GalNAc-T in O-glycan biosynthesis. As Tn, TF and sialyl-Tn are oncofetal antigens and are over-expressed in most human cancers, this information is useful for the development of glycosyltransferase-targeted therapeutic strategies for cancer treatment.     

Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

CD44 Variant Isoforms are Essential for the Function of Epidermal Langerhans Cells and Dendritic Cells

Upon antigen encounter epidermal Langerhans cells (LC) and dendritic cells (DC) emigrate from peripheral organs and invade lymph nodes through the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Part of this process is mimicked by metastasizing tumor cells. Since splice variants of CD44 promote metastasis to lymph nodes we explored the expression of CD44 proteins on migrating LC and DC. We show that following antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6 and v9. Antibodies against CD44 epitopes arrest LC in the epidermis, prevent the binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.     

An Essential Role for CD44 Variant Isoforms in Epidermal Langerhans Cell and Blood Dendritic Cell Function

Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.     

Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model

Background

Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized.

Methods

HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice.

Results

CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF) stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance.

Conclusions

Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.     

Expression of A and H blood-group and of CD44 antigens during chemical rat colonic carcinogenesis

Using an experimental model of rat colon adenocarcinoma, we have recently shown that the presence of H blood-group antigen on variants of the CD44 adhesion molecule carrying amino acids encoded by exon v6 (CD44v6), increased the cells' tumorigenicity. In the present study, colon adenocarcinomas were induced by 1,2-dimethylhydrazine treatment in rats. Using immunohistochemistry, biopsies of normal, precancerous and carcinomatous colon mucosa were evaluated for expression A and H blood group antigens and CD44s and CD44v6 antigens. Normal rat colon showed strong and homogeneous expression of blood-group antigen A, but weak expression of H antigen. Several weeks before the appearance of tumours, dysplastic glands were strongly stained with anti-H reagents, while their A antigen was lost. Expression of CD44v6 was weak and restricted to some cells at the bottom of normal crypts. No obvious change was observed before appearance of severe dysplasia. In carcinomas, a strong but irregular expression of A, H and CD44v6 antigens was observed. In moderately differentiated carcinomas, A and H antigens were present at the apical surface of cells, whereas CD44v6 was found at the basolateral side. Only carcinomatous cells with loss of polarity, found in poorly differentiated cancers or infiltrated in the muscularis mucosae, were found to coexpress blood-group H or A and CD44v6 antigens at their surface. This revised version was published online in November 2006 with corrections to the Cover Date.     

Differential regulation of CDX1 and CDX2 gene expression by deficiency in methyl group donors

The CDX2 and CDX1 homeobox genes have respectively a tumour suppressor and proliferative role in the intestinal epithelium. We analyzed DNA methylation and histones modifications associated with CDX2 and CDX1 promoters in two human colon cancer cell lines expressing differentially these genes, Caco2/TC7 [CDX2 positive-CDX1 negative] and HT29 [CDX2 negative-CDX1 negative] cells. Chromatin immunoprecipitation experiments indicated that CDX2 and CDX1 gene expression correlated with a histone modifications pattern characterizing active chromatin (H3K4 trimethylated and H3 acetylated). Bisulfite DNA sequencing and methylation-specific PCR showed that CDX2 and CDX1 promoters display no methylation in HT29 cells even though both genes are not expressed. In contrast, the CDX1 promoter is methylated in Caco2/TC7. DNA demethylation by 5aza-dC or the combination of 5aza-dC plus SAHA, an inhibitor of histone deacetylases, restored CDX1 expression in Caco2/TC7 cells but these treatments were inefficient on both CDX2 and CDX1 in HT29 cells. Thus, in colon cancer cells the changes in chromatin conformation are heterogeneous and repression of CDX2 and CDX1 in HT29 cells is not due to epigenetic mechanisms. In vivo, dietary deprivation of methyl groups in rats upregulated CDX1 mRNA and downregulated to a lesser extent CDX2 mRNA expression. Moreover, methyl group deprivation downregulated CDX2 protein by changing its phosphorylation pattern. The changes in CDX2 and CDX1 expression determined by methyl group deprivation may constitute one of the mechanisms sustaining the protective role attributed to folate in colon cancer.     

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.     

Hyaluronidase can modulate expression of CD44

CD44 is a family of transmembrane glycoproteins with multiple isoforms generated by alternative exon splicing of a single gene. CD44 and its variants are expressed on a wide variety of cells including cancer cells. The mechanisms by which splice variant exons are selected are unknown. The presence of hyaluronan in the environment of the cell appears to influence that selection process. The expression of particular splice variants of CD44 as well as the simultaneous presence of hyaluronan is important for motility, invasion, and the metastatic spread of some tumors. The influence of hyaluronidase digestion on the expression of CD44 in human cancer cell lines was examined. CD44 isoforms containing alternatively spliced exons were sensitive to hyaluronidase digestion in all lines examined, but differences between cell lines were observed. Expression of CD44s, the standard form, was resistant to digestion in two of three cell lines. A tentative model was formulated proposing that CD44 isoforms containing splice variants are unstable, requiring the continuous presence of ligand for expression. CD44s is relatively more stable, not requiring the continuous presence of hyaluronan. Additionally, a number of new CD44 variant isoforms, not previously observed, were identified.     

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.     

Stromal Hyaluronan Interaction with Epithelial CD44 Variants Promotes Prostate Cancer Invasiveness by Augmenting Expression and Function of Hepatocyte Growth Factor and Androgen Receptor

The main aim of our study is to determine the significance of the stromal microenvironment in the malignant behavior of prostate cancer. The stroma-derived growth factors/cytokines and hyaluronan act in autocrine/paracrine ways with their receptors, including receptor-tyrosine kinases and CD44 variants (CD44v), to potentiate and support tumor epithelial cell survival. Overexpression of hyaluronan, CD44v9 variants, and stroma-derived growth factors/cytokines are specific features in many cancers, including prostate cancer. Androgen/androgen receptor interaction has a critical role in regulating prostate cancer growth. Our previous study showed that 1) that increased synthesis of hyaluronan in normal epithelial cells promotes expression of CD44 variants; 2) hyaluronan interaction with CD44v6-v9 promotes activation of receptor-tyrosine kinase, which stimulates phosphatidylinositol 3-kinase-induced cell survival pathways; and 3) CD44v6/short hairpin RNA reduces colon tumor growth in vivo (Misra, S., Hascall, V. C., De Giovanni, C., Markwald, R. R., and Ghatak, S. (2009) J. Biol. Chem. 284, 12432–12446). Our results now show that hepatocyte growth factor synthesized by myofibroblasts associated with prostate cancer cells induces activation of HGF-receptor/cMet and stimulates hyaluronan/CD44v9 signaling. This, in turn, stabilizes the androgen receptor functions in prostate cancer cells. The stroma-derived HGF induces a lipid raft-associated signaling complex that contains CD44v9, cMet/phosphatidylinositol 3-kinase, HSP90 and androgen receptor. CD44v9/short hairpin RNA reverses the assembly of these components in the complex and inhibits androgen receptor function. Our results provide new insight into the hyaluronan/CD44v9-regulated androgen receptor function and the consequent malignant activities in prostate cancer cells. The present study describes a physiologically relevant in vitro model for studying the molecular mechanisms by which stroma-derived HGF and hyaluronan influence androgen receptor and CD44 functions in the secretory epithelia during prostate carcinogenesis.     

CD44 is not an adhesive receptor for osteopontin

Osteopontin is a secreted glycoprotein with adhesive and migratory functions. Cellular interactions with osteopontin are mediated through integrin receptors which recognize the RGD domain. Recently, CD44, a non-integrin, multifunctional adhesion molecule was identified as an osteopontin receptor. CD44 is a ubiquitous surface molecule that exists as a number of different isoforms, generated by alternative splicing. To analyze which forms of CD44 mediate binding to osteopontin, we used the standard form of CD44 as CD44-human immunoglobulin fusion proteins and several splice variants in enzyme-linked immunosorbant assays. Multiple preparations of osteopontin were used including native osteopontin derived from smooth muscle cells, human urinary osteopontin, full-length recombinant osteopontin, and two recombinant osteopontin fragments expected to be formed following thrombin cleavage. Our data show that although the CD44-hlg fusion proteins could interact with hyaluronic acid as expected, there was no interaction between CD44H, CD44E, CD44v3,v8-v10, or CD44v3 with osteopontin. These studies suggest that CD44-osteopontin interactions may not be common in vivo and may be limited to a specific CD44 isoform(s), and/or a particular modified form of osteopontin.     

Trans-acting factors regulate the expression of CD44 splice variants.

Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell-type specific trans-acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant-exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans-acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3' to 5' direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis-acting recognition sites.     

Novel di-tertiary-butyl phenylhydrazones as dual cyclooxygenase-2/5-lipoxygenase inhibitors: Synthesis,COX/LOX inhibition,molecular modeling,and insights into their cytotoxicities

Although dual inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) enzymes is highly effective than targeting COX or LOX alone, there are only a few reports of examining such compounds in case of colorectal cancers (CRC). In the present work we report that the novel di-tert-butyl phenol-based dual inhibitors DTPSAL, DTPBHZ, DTPINH, and DTPNHZ exhibit significant cytotoxicity against human CRC cell lines. Molecular docking studies revealed a good fit of these compounds in the COX-2 and 5-LOX protein cavities. The inhibitors show significant inhibition of COX-2 and 5-LOX activities and are effective against a panel of human colon cancer cell lines including HCA-7, HT-29, SW480 and intestinal Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressing colon cancer cells, through inhibition of the Hyaluronan/CD44v6 cell survival pathway. Western blot analysis and qRT-PCR analyses indicated that the di-tert-butyl phenol-based dual inhibitors reduce the expression of COX-2, 5-LOX, and CD44v6 in human colon cancer HCA-7 cells, while the combination of CD44v6shRNA and DTPSAL has an additional inhibitory effect on CD44v6 mRNA expression. The synergistic inhibitory effect of Celecoxib and Licofelone on CD44v6 mRNA expression suggests that the present dual inhibitors down-regulate cyclooxygenase and lipoxygenase enzymes through CD44v6. The compounds also exhibited enhanced antiproliferative potency compared to standard dual COX/LOX inhibitor, viz. Licofelone. Importantly, the HA/CD44v6 antagonist CD44v6shRNA in combination with synthetic compounds had a sensitizing effect on the cancer cells which enhanced their antiproliferative potency, a finding which is crucial for the anti-proliferative potency of the novel synthetic di-tert-butyl phenol based dual COX–LOX inhibitors in colon cancer cells.     

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.