Abstract 1505: The role of cathepsin B in colorectal tumorigenesis

Abstract

Abstract Many cancers express elevated protease levels which contribute to certain aspects of tumor behavior such as growth and metastatic spread. Specifically, elevation of the cysteine protease cathepsin B has been shown to correlate with malignant progression of tumors. The majority of reports on cathepsin B expression in tumors have focused on measurements of activity or protein staining. However, multiple transcripts species have been detected resulting from alternative splicing in the 5’- or 3-untranslated regions, and possibly the use of alternative promoter regions. The specific role of these variants remains to be established. Methods: Expression of cathepsin B was analyzed in normal human intestinal epithelial cells (HIEC) and colorectal cancer cell lines (Caco-2/15, HCT116, DLD1, HT29, SW480, T84, Colo205) by qPCR and Western blot. The role of cathepsin B was examined in HT29 and DLD1 cell lines through recombinant lentiviruses encoding anti-cathepsin B short hairpin RNA (shRNA). Results: RT-PCR analyses demonstrated that the levels of the full transcript for cathepsin B containing exons 1-12 (CB) were similar between normal and cancerous cell lines. By contrast, the splice variant lacking exons 2 and 3 (CB-2,3) was expressed only in cancer cells and in biopsies from human colorectal tumors when compared to the adjacent healthy tissues. Immunofluorescence studies showed that this novel tumor form of cathepsin B (CB-2,3) was associated with nuclei and mitrochondria while the full-length cathepsin B protein (CB) was found in the lysosomes. Western blot analyses on culture media showed a strong secretion of various cathepsin B isoforms by cancer cell lines. The lentiviral infection of a shRNA which specifically knocked-down cathepsin B expression, while decreasing enzyme activity in cell lysates, did not influence colorectal cancer cell proliferation under anchorage-dependent conditions. By contrast, decrease of cathepsin B expression in HT29 and DLD1 colon cancer cell lines resulted in a marked reduction of their capacity to grow in soft agar. Levels of β-catenin protein and phosphorylated ERK1/2 were not affected following cathepsin B silencing in contrast to phosphorylated Akt which was significantly reduced. Further, migration and invasion through Matrigel was reduced by 60% in cells expressing the shRNA against cathepsin B compared to cells expressing a control shRNA. Conclusion: These results suggest that the alternative splicing of cathepsin B could contribute to the aberrant intracellular trafficking and function of cathepsin B in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2011-1505