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.     

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.     

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.     

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 6 and lymphatic invasion: importance to lymph node metastasis in gastric cancer

Lymph node metastasis is a critical prognostic factor for gastric cancer. In the present investigation we examined clinicopathologic factors influencing the metastatic processes to the lymph mode and their prognostic importance. A randomly selected group of 98 patients with adenocarcinomas of the stomach who underwent gastrectomy plus systematic lymph node dissection at Osaka Police Hospital from 1991 to 1996 were analyzed. Altogether 37 (38%) cancers were positive for CD44 variant 6 (v6) staining, 31 (32%) were intermediately stained, and 30 (30%) were negative. CD44-v6 expression correlated well with lymph node metastasis. Expression of CD44-v6 and lymphatic invasion were independent risk factors for metastatic lymph nodes. Among the patients with CD44-v6-positive and lymphatic invasion-positive cancers, 88% had lymph node metastasis, whereas only 13% of patients negative for both factors had lymph node metastasis. Although CD44-v6 expression and lymphatic invasion have been reported to be risk factors for recurrence and a poor prognosis, in this investigation these factors were found not to be significant for hematogenous and lymphatic recurrences or overall survival rates. Thus expression of CD44-v6 and lymphatic invasion may regulate lymph node metastases from gastric cancer.     

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.     

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.     

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.     

Changes in body image, depression, and anxiety levels among women with human papillomavirus infection

In the present study, we examined the expression of CD44 variant exons in human oral squamous cell carcinoma (OSCC). Of ten cell lines from OSCCs, two (KB and H357), as well as the HeLa cell line, failed to express CD44 variant exons. In surgical specimens, all normal mucosa expressed CD44v9 (both mRNA and protein). Of 40 primary OSCCs, 19 (47.5%) showed downregulation of CD44v9, which correlated with tumor cell differentiation, primary metastasis to lymph nodes and secondary metastasis to lymph nodes. The results suggest that the downregulation of CD44v9 may play a role in lymphatic metastasis of OSCC and changes in its expression may be a useful diagnostic tool to determine the metastatic potential of OSCC to lymph nodes. Moreover, three cell lines that failed to express CD44 variant exons might become a useful experimental model to study the role of variant exons in the progression of OSCC.     

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.     

Complex CD44 splicing combinations in synovial fibroblasts from arthritic joints

CD44 is a broadly expressed cell surface glycoprotein which is the major cell surface receptor for the glycosaminoglycan, hyaluronan. In humans, alternative splicing of up to 9 variant exons (v2-v10) into CD44 mRNA, together with post-translational modification via glycosylation and chondroitin sulfate attachment has the potential of generating a large number of CD44 isoforms. Insertion of these various exons has the potential to change the functional capacities of the molecule and has implications in disease. We have analyzed CD44 splice variant expression in cultured VCAM-1-positive synovial fibroblasts isolated from patients with osteo- or rheumatoid arthritis and from normal synovium. Rheumatoid and osteoarthritic tissue express CD44 splice variants at the cell surface level. At the mRNA level exons v3, v6, v7, v8, v9 and v10 were detected in different splicing combinations. Rheumatoid tissue showed high expression, osteoarthritic tissues showed great variation. In contrast, non-inflamed tissue showed no splicing events. Our results indicate that the nature of CD44 splice variant expression may be linked to the inflammatory state of the synovial joint.     

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 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.