Osteopontin silencing by small interfering RNA suppresses in vitro and in vivo CT26 murine colon adenocarcinoma metastasis

• Published in: Carcinogenesis (New York) Vol. 26; no. 4; pp. 741 - 751
• Main Authors: Wai, Philip Y., Mi, Zhiyong, Guo, Hongtao, Sarraf-Yazdi, Shiva, Gao, Chengjiang, Wei, Junping, Marroquin, Carlos E., Clary, Bryan, Kuo, Paul C.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01-04-2005; Oxford Publishing Limited (England)
• Subjects: Adenocarcinoma - genetics; Adenocarcinoma - secondary; Adenocarcinoma - therapy; Animals; Biological and medical sciences; Blotting, Western; Carcinogenesis, carcinogens and anticarcinogens; Cell Movement; Colonic Neoplasms - genetics; Colonic Neoplasms - pathology; Colonic Neoplasms - therapy; CT26 cells transiently transfected with OPN siRNA to target 1; CT26 cells transiently transfected with OPN siRNA to target 2; Down-Regulation; EGFR; epidermal growth factor receptor; Female; GAPDH; Gene Expression Regulation, Neoplastic - physiology; Gene Silencing - drug effects; Genetic Therapy; glyceraldehyde-3-phosphate dehydrogenase; In Vitro Techniques; Liver Neoplasms - genetics; Liver Neoplasms - secondary; Liver Neoplasms - therapy; Matrix Metalloproteinase 2 - metabolism; Medical sciences; Mice; Mice, Inbred BALB C; OPN; Organ Size; Osteopontin; PCNA; plasmid vector expressing mismatch siRNA; plasmid vector expressing siRNA against OPN; proliferating cell nuclear antigen; pS-MM; pS-OPN; RNA interference; RNA, Small Interfering - physiology; RNAi; Sialoglycoproteins - genetics; Sialoglycoproteins - metabolism; siRNA; small interfering RNA; sR1; sR2; Tumors; uPA; urokinase plasminogen activator; vascular endothelial growth factor; VGEF; Small; Colon adenocarcinoma; RNA; Rodentia; Osteopontin; Malignant tumor; Metastasis; In vitro; In vivo; Gene silencing; Vertebrata; Mammalia; Mouse; Animal
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/bgi027

Hepatic metastasis is a primary cause for failure of locoregional therapy in colorectal cancer. Increased expression of osteopontin (OPN), a ligand for αvβ3 integrin and CD44 receptors, is associated with metastasis in several types of cancer. However, the mechanism by which OPN mediates metastasis in colorectal cancer remains unknown. We hypothesized that OPN mediates invasion of colon cancer cells through basement membrane and migration through extracellular matrix (ECM). In this study, we used CT26 murine colon adenocarcinoma cells syngeneic to BALB/c mice to generate cell lines (pS-OPN) in which OPN expression was suppressed through small interfering RNA (siRNA) plasmids. CT26 wild-type cells (WT) and CT26 cells stably expressing murine-mismatch siRNA (pS-MM) served as controls. Western blotting quantified OPN protein levels and our most downregulated clone, pS-OPN-A4, demonstrated a mean 3.0-fold decrease in OPN protein expression versus WT. In vitro cell motility and invasiveness were decreased in pS-OPN-A4 by 3.6-fold (P = 0.004 versus WT) and 4.1-fold (P = 0.01 versus WT), but proliferation was similar amongst cell lines. We demonstrated that OPN suppression significantly correlates with MMP-2 downregulation. In vivo hepatic metastasis was assessed by quantifying liver weights and surface tumor nodules in 33 BALB/c mice (11/group) subjected to intrasplenic injection of tumor cells. pS-OPN-A4 resulted in a 50.4% decrease in mean liver weight compared with WT (3.79 ± 1.49 g versus 1.88 ± 1.34 g, P = 0.009). Only 18% of pS-OPN-A4 livers had >20 metastatic surface nodules compared with 89% for WT and 75% for pS-MM-V6. This study demonstrates that RNA interference stably reduces CT26 tumor expression of OPN and significantly attenuates CT26 colon cancer metastasis by diminishing tumor cell motility and invasiveness.