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.     

Preparing physicians for indicatory physical examination of the nervous system

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 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.     

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.     

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.     

Expression of CD44 variant exons by primary and metastatic oral squamous carcinomas

Abnormal CD44 expression in many neoplasms correlates with behaviour, but reports on its role in oral squamous carcinoma are contradictory. CD44 expression was characterised in a closely matched series of oral carcinomas with and without metastases in both frozen and formalin-fixed tissue and correlated with behaviour and histological grading parameters. Eleven primary oral squamous carcinomas without metastases and nine primary carcinomas with 19 matched metastases were stained immunocytochemically for CD44H and products of variant exons v3, v4/5, v6 and v9. Patterns of staining in frozen and formalin-fixed tissue were correlated with invasive front grading and behaviour using exact inferential statistics. Most primary carcinomas stained for all exons tested but some showed loss of expression of v4/5. Loss of expression was more marked in metastases, but there was no correlation between expression and behaviour or grade. Stromal surfaces of epithelial cells often expressed variant exon products reflecting loss of polarity. This, together with selective loss of v4 and v5 in primary carcinomas and their more frequent loss in metastases, suggests that CD44 may play a role in metastasis of some oral squamous carcinomas.     

Expression of CD44 standard and CD44 variant 6 in human lung cancer

We immunohistochemically examined the expression of CD44 standard (CD44 st) and CD44 variant 6 (CD44 v6) in 112 cases of primary lung cancer, and their relationship to the clinical milieu, including the clinical stage. In 46 cases of squamous cell carcinoma, expression of CD44 st was observed in 45.7% of the cases, and expression of CD44 v6 was observed in 60.9%. In 43 cases of adenocarcinoma, positive staining of CD44 st and CD44 v6 was seen in 2.3% and 4.7% of the cases, respectively. None of 21 small cell carcinomas was positive for CD44 st or CD44 v6. In squamous cell carcinomas, the expression of CD44 st and CD44 v6 was observed at a rate significantly higher than in other histologic type. Most specimens positive for CD44 st stained positively for CD44 v6. Therefore, it seems likely that the CD44 expression observed in squamous cell carcinoma of the lung was a variant CD44 containing the domain encoded by variant exon 6. The expression of CD44 v6 was not related to the clinical stage. Significant association between CD44 v6 and differentiation of squamous cell carcinoma was seen; 2/7 (28.6%) for poorly differentiated, 19/31 (61.3%) for moderately differentiated, and 7/8 (87.5%) for well differentiated squamous cell carcinomas (p = 0.02 by trend test). It was previously reported that CD44 st and CD44 v6 were expressed in both normal bronchial epithelium and squamous cell metaplasia. These results suggest that the expression of CD44 v6 in squamous cell carcinoma of the lung may reflect the immunohistochemical characteristics of the tissue from which such carcinoma emerge.     

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.     

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.     

Augmented expression of CD44 splice variants in lymphoproliferative disorder of the lacrimal gland in Sj?gren's syndrome

We explored the involvement of CD44 isoforms in lymphoproliferative disorder (LPD) of the lacrimal gland of a Sj?gren's syndrome patient with unilateral LPD. The CD44 variant with the v6 exon was selectively detected from infiltrating lymphocytes in the gland with LPD, but not from infiltrating lymphocytes in the normal lacrimal gland, suggesting that the CD44 v6 variant exon may be closely associated with the development of LPD in Sj?gren's syndrome.     

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.     

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.     

Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells

CD44 is a family of molecules involved in cell-cell and cell-matrix interactions. Various isoforms of CD44 arise by insertion of one or more of the variant exons into the common backbone shared by all forms of CD44. In this work, we studied the expression of CD44 and exon v6-containing CD44 isoforms (CD44v6) in several nonmalignant and malignant conditions and the possibilities for regulating the expression of CD44v6. In primary squamocellular carcinomas of the head and neck, CD44 and CD44v6 were down-regulated in poorly differentiated tumors, whereas these molecules were uniformly expressed in the normal squamocellular epithelium, in proliferating skin diseases, and in nonmalignant tumors. When CD44v6 expression of original tumors and that of squamocellular carcinoma cell lines derived from them were compared, no CD44v6 up-regulation could be observed on in vitro growing cells. Moreover, several regulators were unable to up-regulate CD44v6 expression on cultured cell lines in vitro. When the same cell lines formed tumors after s.c. injection into severe combined immunodeficient mice, some of them up-regulated their CD44v6 expression. These data suggest that cell lines at certain differentiation stages can be induced to express CD44v6. Our results further indicate that CD44v6 positivity cannot be used as a universal indicator of tumor metastasis. Instead, the down-regulation of CD44v6 in squamocellular tumors is a sign of malignant transformation of the epithelium.     

Structural variability of CD44v molecules and reliability of immunodetection of CD44 isoforms using mAbs specific for CD44 variant exon products

CD44 can be considered structurally and functionally one of the most variable surface molecules. Alternative splicing of variant exons as well as posttranslational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 isoforms in vivo, using mAbs specific for CD44 variant exon products, is largely used to identify those CD44 molecules involved in tumor growth and progression and to interfere with CD44-mediated processes. In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon-specific epitopes can be impaired by the structural variability of the molecule. Our findings demonstrate that (1) specific exon assortment and/or posttranslational modifications of CD44v molecules can mask CD44 exon-specific epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isoforms of high molecular weight, may play a critical role in determining the exact conformation of the molecule, which is necessary for the detection of CD44 variant epitopes by specific mAbs; and (3) in a panel of stable transfectants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine substitution is sufficient per se to impair the immunoreactivity of several mAbs to pan-CD44. Thus, conformational changes due to the alternative splicing of CD44 variant exons and/or posttranslational modifications of the molecule (different degree of glycosylation), which are cell type-specific, are likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD44 expression in vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.     

The prognostic factors of pancreatic cancer--adhesion molecule CD44

The prognosis for pancreatic cancer is extremely poor, because there are usually invasion of surrounding tissues and metastases to lymph nodes, liver or peritoneum at the time of diagnosis. Many studies from the molecular-biological stand point have demonstrated this poor prognosis. However, the mechanisms underlying these invasive and metastatic capabilities have not yet been clarified. We reviewed several reports for prognostic factors of pancreatic cancer in molecular-biological examinations, and introduced our recent study on the adhesion molecule CD44. The CD44 variant 6 (v6) molecule has been noted as a marker for tumor metastasis and prognosis in several tumors. We examined whether or not v6 is a useful marker for evaluating the prognosis of pancreatic cancer patients. In addition, we attempted to assess the clinicopathological implications for pancreatic cancer of the variant 2 (v2) isoform using a recently developed monoclonal antibody against a v2 epitope. The expression of CD44 v6 and v2 was observed only in tumor cells, if at all. The expression of CD44 v6 and v2 was correlated with decreased overall survival. A significant correlation was obtained between CD44 v2 and vessel invasion. These results suggest that CD44 v2 and CD44 v6 may be useful markers of a poor prognosis.