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 progress ion of epithelial tumors and the metastatic phenotype, Here me report a functional role for CD44 variant isoforms in colorectal cancer metas tasis, An antisense mRNA approach was used to down-regulate CD44 varia nt isoforms containing CD44 variant 6 (v6) in the metastatic colorecta l tumor cell line HT29, Cell lines stably expressing antisense CD44 ex on 10 (v6) showed reduced expression of alternatively spliced CD44 var iant isoforms but no significant change in expression of CD44 core pro tein, as judged by immunohistochemical analysis using CD44 domain-spec ific monoclonal antibodies. Expression of antisense exon 10 (v6) had n o effect on HT29 tumor cell proliferation ill 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, agai nst 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 mic e injected with antisense exon 10 expressing HT29 cells developed simi lar tumors, This is the first demonstration that antisense RNA can be used to selectively inhibit expression of specific domains of a molecu le generated through alternative mRNA splicing while allowing expressi on of core domains to remain unaffected. Furthermore, these results pr ovide direct evidence for a functional role of CD44 variant isoforms i n the metastasis of human colorectal tumor cells and may suggest a cri tical role for CD44 variants in promoting cell growth specifically in the cytokine/growth factor-enriched environment of a wound site.