Abstract 1505: The role of cathepsin B in colorectal tumorigenesis

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