Expression of antisense CD44 variant 6 inhibits colorectal tumor metastasis and tumor growth in a wound environment

Up-regulation of CD44 variant isoforms has been linked to the progression of epithelial tumors and the metastatic phenotype. Here we report a functional role for CD44 variant isoforms in colorectal cancer metastasis. An antisense mRNA approach was used to down-regulate CD44 variant isoforms containing CD44 variant 6 (v6) in the metastatic colorectal tumor cell line HT29. Cell lines stably expressing antisense CD44 exon 10 (v6) showed reduced expression of alternatively spliced CD44 variant isoforms but no significant change in expression of CD44 core protein, as judged by immunohistochemical analysis using CD44 domain-specific monoclonal antibodies. Expression of antisense exon 10 (v6) had no effect on HT29 tumor cell proliferation in vitro or the ability of the cells to bind immobilized hyaluronan, but it resulted in a reduced capacity to form liver metastases in nude mice following intrasplenic injection. Metastases were not detected in nude mice inoculated with antisense CD44 exon 10 (v6)-expressing cell lines after 4 months, against a background of a 30% metastasis rate in the control HT29 parental and vector alone transfected lines. Furthermore, whereas 82% of mice intrasplenically injected with control HT29 parental and vector alone cell lines developed tumors in incisional wound sites, none of the mice injected with antisense exon 10 expressing HT29 cells developed similar tumors. This is the first demonstration that antisense RNA can be used to selectively inhibit expression of specific domains of a molecule generated through alternative mRNA splicing while allowing expression of core domains to remain unaffected. Furthermore, these results provide direct evidence for a functional role of CD44 variant isoforms in the metastasis of human colorectal tumor cells and may suggest a critical role for CD44 variants in promoting cell growth specifically in the cytokine/growth factor-enriched environment of a wound site.     

Schwann cell tumors express characteristic patterns of CD44 splice variants

Members of the CD44 family of cell surface hyaluronate-binding proteins have been implicated in cell migration, cell-matrix interactions and tumor progression. To determine whether these proteins might play a role in the normal functions of Schwann cells and in their tumorigenesis, we examined the patterns of CD44 expression in Schwann cells from rat peripheral nerve, rat Schwann cell tumor lines, and human schwannomas. Normal rat spinal nerves and primary Schwann cell cultures expressed standard CD44 (CD44s) but not alternatively spliced variant isoforms. In contrast, rat Schwann cell tumor lines expressed both CD44s and a number of variants, including proteins containing sequences encoded by exon v6. Furthermore, we found that these cell lines bind hyaluronate, and that their cell surface hyaluronate binding correlates with CD44 expression. All of the human schwannomas also expressed CD44 variants, especially epitopes encoded by exon v5, the border between v7 and v8, and v9-10. These data indicate that Schwann cells normally express CD44s, that Schwann cell tumors express both CD44s and particular variants of CD44, and that CD44s and possibly variants of CD44 are involved in hyaluronate recognition by Schwann cell tumors.     

Expression of CD44 variant isoforms in malignant melanoma

In a variety of human tumors, expression of splice variants of the adhesion molecule CD44 (CD44v) has been described as correlating with tumor progression. Here, we report on the expression of CD44v in melanocytes, nevi, primary melanomas, and cutaneous and lymph node metastases. Thirteen nevi, 65 primary melanomas of varying thickness, 39 cutaneous and 15 lymph node metastases, and melanocytes and a panel of melanoma lines were tested for surface expression of the standard form of CD44 and the variant exons v5, v6, v7, v7-v8, and v10 by immunohistology or fluorescence-activated cell sorting. Melanocytes did not express any variant isoform of CD44. However, nevi, as well as primary melanoma and melanoma metastases, stained to a varying degree with anti-CD44v5, anti-CD44v7-v8, and anti-CD44v10. Exons v6 and v7 were not detected on any of these tissue specimens. Compared with nevi, expression of exon v10 was up-regulated in thick primary tumors and skin metastases. Lymph node metastases displayed elevated levels of exon v5. Expression of CD44v in melanoma lines (n = 20) differed, inasmuch as many lines did not express variant isoforms; in particular, exon v10. Interestingly, however, the few CD44v5-positive melanoma lines metastasized in the nu/nu mouse. Because benign as well as malignant growth of melanocytes was accompanied by expression of CD44 variant isoforms, a linkage between expression of CD44 variant isoforms and malignant transformation or tumor progression was excluded. Considering the function of distinct isoforms, one might speculate that expression of exon CD44v5, which was up-regulated in lymph node metastases compared with nevi and primary melanoma, provided a growth stimulus. Exon v10 is present at high density in epidermal cells. The de novo expression of this exon in nevi and the increased expression in thick melanoma and skin metastases would be in line with the assumption of an anchoring advantage in the surrounding epidermal tissue.     

The splice-pattern of CD44 is altered in chronic pancreatitis exhibiting dysplastic changes

Recent studies have shown that several splice variants of CD44, might be involved in tumor progression. Since chronic pancreatitis is suggested to be a risk factor for pancreatic cancer we investigated the splice pattern of CD44 in chronic pancreatitis to elucidate the role of CD44 in pancreas tumorigenesis. The expression of CD44-isoforms was examined in 40 specimens of chronic pancreatitis and 12 specimens of normal pancreas by immunohistochemistry, Westernblotting and exon specific RT-PCR. Pancreatic cancer tissue from two patients who developed pancreatic cancer 2 and 3 years following surgery for chronic pancreatitis were analyzed. Strong expression of CD44s was found in all cells, whereas the expression of CD44v6 was restricted to ductal cells. Westernblotting revealed an overexpression of CD44v6 in chronic pancreatitis as compared to normal pancreas. Exon specific analysis revealed an altered splice pattern of CD44, similar to that in pancreatic cancer, in 12.5% of the chronic pancreatitis specimens. Both patients who developed pancreatic cancer after chronic pancreatitis exhibited this altered splice pattern in both, chronic pancreatitis and pancreatic cancer. These results suggest that variant forms of CD44-mRNA might be expressed in early dysplastic alterations in chronic pancreatitis.     

CD44H expression by human neuroblastoma cells: relation to MYCN amplification and lineage differentiation

The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment, and tumor metastasis. Due to the alternative splicing of the single gene, a large family of different variants or isoforms is generated. Several reports have indicated an up-regulation of CD44 variant (v) isoforms in malignant process, conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumor characterized by an aggressive and metastatic behavior in advanced stages with amplification of the MYCN protooncogene. In this report we show that the CD44 standard molecule is highly expressed in 100% of stage I-III, IVs neuroblastomas and ganglioneuromas but only in a subset of stage IV tumors. In contrast, no expression of CD44 was detected on MYCN amplified stage IV tumors, thus demonstrating a highly significant negative relationship between MYCN amplification and CD44 expression in neuroblastoma. The expression of CD44 on neuroblastoma cultured cell lines was not shown to be related to MYCN amplification but rather linked to the S-type, schwann/glial differentiation lineage. Immunochemical analysis of tumor samples with anti-CD44v3 and -v6 antibodies and Northern blot analysis of mRNA from cell lines with probes spanning exons 4-10 did not reveal any expression of splice variants on neuroblastomas of all stages and cell lines, thus ruling out a major role of these isoforms in neuroblastoma progression and metastasis.     

Differential expression of CD44v6 in adenocarcinoma of the pancreas: an immunohistochemical study

Alternative splicing gives rise to numerous CD44 isoforms, some of which seem to have a role in tumour metastasis. Specifically, a variant form of CD44 with sequences encoded by exon v6 (CD44v6) confers metastatic potential when transfected into a nonmetastasizing cell line of rat pancreatic adenocarcinoma. This study has investigated standard CD44 (CD44s) and CD44v6 expression immunohistochemically in 6 samples of normal pancreatic tissue, 4 of tissue affected by chronic pancreatitis, and 24 of tissue from metastasizing and nonmetastasizing pancreatic adenocarcinomas. In addition, 18 samples from lymph node or visceral metastases were included in the study. CD44s was expressed in nonneoplastic tissue and in tissue from pancreatic adenocarcinomas. In contrast, CD44v6 was not detected in any of the normal tissue or chronic pancreatitis specimens, whereas 54% of pancreatic adenocarcinomas and 55% of metastases expressed this variant exon. Although it is not clear whether CD44 isoforms containing exon v6 play a part in malignant progression in the human exocrine pancreas, it seems plausible that the expression of multiple isoforms containing this and other variant exon confers a selective advantage on pancreatic adenocarcinoma.     

ATP-induced, P2U purinoceptor-mediated constriction of isolated, perfused mesenteric beds of the rat

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.     

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.     

In vivo chiro-inositol metabolism in the rat: a defect in chiro-inositol synthesis from myo-inositol and an increased incorporation of chiro-[3H]inositol into phospholipid in the Goto-Kakizaki (G.K) rat

BACKGROUND: CD44 is a cell adhesion molecule often expressed in the form of various splice variants. The role of standard CD44 isoform (CD44s) and its variants in colorectal carcinogenesis is partly conflicting. Therefore, we compared the expression of CD44s (hermes-3) and its splice variants (v3 and v6) with traditional prognostic factors in 194 colorectal cancer patients treated at Kuopio University Hospital and followed up for a mean of 14 years. METHODS: Formalin-fixed, paraffin-embedded tissue sections from 194 patients with colorectal carcinoma were examined immunohistochemically to detect the expression of different forms of CD44. The hypothesis that CD44s, CD44v3, and CD44v6 expression intensities and distribution in cancer cells correlated with survival was tested with the log-rank test, hazard ratios, and their confidence intervals. RESULTS: In high-grade tumours CD44s and CD44v6 expression intensities and CD44s percentages were stronger than in low-grade tumours. CD44v6, CD44v3, and CD44s expression intensities in tumour epithelium were also stronger in Dukes C and D tumours than in A and B tumours. In the univariate survival analysis patients with strong CD44s, CD44v3, and CD44v6 expression intensities in tumour epithelium had lower cancer-related survival than the patients who had weak CD44s, CD44v3, and CD44v6 expression intensities. Recurrence-free survival was also shorter in patients with intense signals for CD44v3 and CD44v6 in tumour epithelium. In the multivariate analysis the CD44v6 expression intensity in tumour epithelium predicted independently both cancer-related and recurrence-free survival in T1-4N0-3M0 and T1-3N0M0 cases. In addition, the CD44v3 expression intensity in tumour epithelium was a significant predictor of RFS in T1-3N0M0 cases. CONCLUSIONS: These results strongly suggest that the CD44 splice variants v6 and v3 have prognostic significance in colorectal cancer.     

Expression of CD44 containing variant exon 9 (CD44v9) in gastric adenomas and adenocarcinomas: relation to the proliferation and progression

The expression of CD44 splice variant containing exon 14 (variant exon 9: CD44v9) was examined immunohistochemically in non-neoplastic mucosa, adenoma and adenocarcinoma of the stomach and analyzed the relation with the expression of Ki-67 antigen and p53 protein. In non-neoplastic gastric mucosa, basolateral membrane of the epithelial cells in the pyloric glands showed the expression of CD44v9. The epithelial cells in the intestinal metaplastic mucosa of the stomach sometimes expressed CD44v9. In the neoplastic lesions, the expression of CD44v9 was detected in 20% (34/170) of the adenomas and 28% (132/478) of the adenocarcinomas, respectively. The incidence of CD44v9 expression did not differ among histological type of gastric carcinoma. Twelve per cent of the adenocarcinomas showed strong expression of CD44v9, whereas non of the adenomas did. The incidence of CD44v9 expression was significantly higher in carcinomas invading into muscularis propria or the cases of stages 3 and 4 in comparison with that in carcinomas limited to submucosa or the stages 1 and 2 cases (p<0.05). The incidence of positive cases was higher in carcinomas with lymph node metastasis than those without metastasis (p<0.05). The expression of CD44v9 was significantly correlated with the expression of Ki-67 (p<0.05). It was also correlated with the expression of p53 protein in the tumor cells (p<0.01). These findings overall suggest that the expression of CD44v9 may be associated with the development as well as progression of the gastric carcinomas.     

CD44 expression is aberrant in benign Schwann cell tumors possessing mutations in the neurofibromatosis type 2, but not type 1, gene

Atypical expression of CD44 splice variants has been implicated in the progression of numerous tumors. This abnormal CD44 expression is presumed to result from gene alterations that cause tumorigenic transformation. Two tumor types that have been linked to specific gene alterations are schwannomas, which have mutations in the neurofibromatosis (NF) type 2 (NF2) gene, and neurofibromas, which characteristically possess NF type 1 (NF1) gene mutations. We examined CD44 expression in normal sciatic nerves, in schwannomas with confirmed NF2 mutations, and in neurofibromas and malignant peripheral nerve sheath tumor tissue and cell lines from NF1 patients. Compared to normal nerves, schwannomas express higher total levels of CD44 and additional splice variants, whereas CD44 expression in neurofibromas is unaltered. Malignant peripheral nerve sheath tumor tissue and cell lines express the CD44v6 epitope, which is not expressed by normal Schwann cells or by other Schwann cell tumors. These data indicate that altered CD44 expression correlates strictly with mutations in the NF2 but not NF1 gene and suggest that CD44v6 might be a marker for the malignant transformation of Schwann cells.     

Expression of CD44 and variant isoforms in cervical intraepithelial neoplasia

The cell adhesion molecule CD44 and its variant isoforms have been found to be related to invasive and metastatic character of cancer cells. Their expression in gynecologic precancerous lesions has not yet been reported. Mouse monoclonal antibodies directed against a common epitope (CD44s) and exons 4v, 6v, and 9v were used to study the expression of CD44 and variant isoforms by immunohistochemistry in cervical intraepithelial neoplasia (CIN). Twenty tissue samples with normal cervical epithelium and 57 samples with CIN of different histological grades and different HPV status were included in this study. The standard CD44, CD44-4v, CD44-6v, and CD44-9v were expressed in normal cervical epithelium and in precancerous lesions. In distinct contrast to the normal epithelium, however, the standard CD44, CD44-4v, and 6v showed a reduced expression in precancerous lesions, whereas CD44-9v was significantly overexpressed. Expression of CD44 standard and CD44-4v was correlated with the histological grade but not with the HPV status. Compared with mild and moderate dysplasia, severe dysplasia and carcinoma in situ are associated with low expression of CD44s (P = 0.007) and of CD44-4v (P = 0.03). These observations reveal dynamic changes in CD44 expression during neoplastic cell transformation in cervical intraepithelial neoplasia.     

Expression of CD44 variant isoforms in peripheral blood leukocytes in malignant lymphoma and leukemia: inverse correlation between expression and tumor progression

In a variety of human tumors, including high grade Non-Hodgkin's lymphoma (hgNHL), a linkage between expression of CD44 variant isoforms (CD44v) and tumor progression has been described. In search of an easily accessible diagnostic parameter, expression of CD44 standard (CD44s) and CD44 variant isoforms (exons v5, v6, v7 and v10) in peripheral blood lymphocytes (PBLs) of patients with hematological malignancies was evaluated by fluorescence activated cell scanning. The analysis of 30 blood samples of healthy donors and patients with non-malignant diseases and of 183 blood samples of patients with malignant hematological disorders revealed that only in patients with malignant disorders did a measurable proportion of PBLs express CD44 variant isoforms, mostly exons v5, v6, v7 and, less frequently, exon v10. Elevated levels of CD44v expression were noted in PBLs of patients with acute and chronic myeloid leukemia (AML: 16%, CML: 25%), Hodgkin's disease (HD: 17%), multiple myeloma (MM: 22%), polycythemia vera (PV: 33%), acute lymphoid leukemia (ALL: 23%) and, most frequently, in PBLs of patients with non-Hodgkin's lymphoma (NHL:54%). CD44v expression was not restricted to the malignant phenotype, but instead was also noted in T cells, B cells and monocytes, preferentially in a subpopulation of large cells. Furthermore, expression of CD44v in PBLs was not linked to the histological grading or clinical staging. There was, however, an inverse correlation with tumor progression, whereas response to therapy was frequently accompanied by upregulation of CD44v. Thus, expression of CD44v in the PBLs of patients with NHL mainly reflected immune responsiveness. Since NHL manifests itself primarily in lymphoid organs, its progression is difficult to follow. Monitoring of CD44v in PBLs could be used as an additional and convenient parameter for surveying the course of disease.     

Hyaluronan, CD44 and its variant exons in human trophoblast invasion and placental angiogenesis

Both hyaluronan and one of its receptors, CD44, can be demonstrated in the early human conceptus and in placental stroma. The variants of CD44 resulting from variable exon splicing are found in metastasizing human malignancies and are also involved in hyaluronan uptake and degradation. The resulting hyaluronan fragments are known to be highly angiogenic. We postulated that the self-limited process of trophoblast invasion of the uterine decidua results in part from the strategy of alternative splicing of CD44, similar to that used by invasive cancer cells in the course of metastatic spread and possibly angiogenesis. Monoclonal antibodies specific for CD44s and for an exon expressed during metastatic tumour progression, CD44v7, were used to examine this hypothesis. In this study we found human trophoblasts, for the first time, to express CD44. Intermediate trophoblasts of first and second trimester exhibited the standard form of CD44 while extravillous trophoblasts, which are responsible for the invading characteristics of the placenta, were positive for the alternatively spliced form, the CD44v7-8. Moreover, in the case of placenta accreta there was a prominent membrane staining of the trophoblasts that were embedded in the fibrin layer over the myometrium. The highly metastatic choriocarcinoma cells also expressed CD44v7-8. We propose, therefore, that the invading trophoblasts utilize the alternatively splicing machinery. These cells retain their invasive capabilities through the permissive ECM by carrying the CD44v7-8 isoform, which binds weakly to hyaluronan and thus prevents it from being degraded by intracellular hyaluronidase.     

A directly spliced exon 10-containing CD44 variant promotes the metastasis and homotypic aggregation of aggressive non-Hodgkin's lymphoma

Variants of the CD44 cell-surface adhesion molecule include additional sequences encoded by combinations of exons from the membrane proximal domain (exons 6-14). Preliminary studies suggest that these additional variable membrane proximal sequences may alter the ligand specificity, glycosylation, and biologic function of CD44. In earlier studies, we found that primary extranodal and widely disseminated aggressive non-Hodgkin's lymphomas (NHLs) and normal activated B cells expressed a directly spliced exon 10-containing variant (CD44ex10), whereas normal resting B cells expressed larger exon 10-containing variants (CD44ex10-14 and CD44ex7-14). To obtain additional information regarding the function of exon 10-containing CD44 variants in aggressive NHL, we generated aggressive NHL transfectants that expressed CD44ex10, CD44ex10-14, CD44ex7-14, the standard CD44 isoform (CD44H), or vector alone, and evaluated the local tumorogenicity, aggregation, and metastatic potential of these transfectants. CD44ex10 aggressive NHL transfectants were more likely to cause local tumor formation in nude mice than transfectants expressing the larger exon 10-containing variants, CD44H, or vector alone. In addition, cell suspensions derived from CD44ex10 local tumors exhibited far greater homotypic aggregation than those obtained from other CD44 or vector-only local tumors. In nude mice that received CD44ex10 transfectants, distant metastases were also significantly more likely to develop than in animals that were given either the CD44ex10-14, CD44ex7-14, CD44H, or vector-only transfectants. These data provide the first evidence that the directly spliced exon 10-containing CD44 variant (CD44ex10) has a unique biologic function in aggressive NHL.