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Cyclooxygenase-2 Is a Target of KRAS^D12, Which Facilitates the Outgrowth of Murine C26 Colorectal Liver Metastases

Departments of ¹ Surgery and ² Pathology, University Medical Center Utrecht, Utrecht, the Netherlands

Requests for reprints: Onno Kranenburg, Department of Surgery, University Medical Center Utrecht, G04-228, P.O. Box 85500, 3508 GA Utrecht, the Netherlands. Phone: 31-30-2506681; Fax: 31-30-2541944; E-mail: o.kranenburg{at}azu.nl.

Purpose: Mutational activation of the KRAS oncogene and overexpression of cyclooxygenase-2 (COX-2) contribute to colorectal carcinoma (CRC) development, but the relationship between these two events is unclear. This study was designed to clarify that relationship and to assess the contribution of KRAS-dependent COX-2 to the seeding of CRC cells in the liver and to their outgrowth as liver metastases in an experimental mouse model.

Experimental Design: The effect of RNA interference–mediated KRAS knockdown on COX-2 expression and activity was tested in murine C26 CRC cells. The contribution of KRAS-dependent COX-2 to early metastatic tumor cell seeding (by intravital microscopy) and outgrowth of metastases in the liver (by bioluminescence imaging) was studied by using parecoxib, a novel and highly selective liver-activated COX-2 inhibitor. Intratumoral cell proliferation, apoptosis, and tumor-associated angiogenesis were assessed by immunohistochemistry on liver tissue sections.

Results: Stable knockdown of mutant KRAS^D12 in murine C26 CRC cells by RNA interference lead to a dramatic reduction of COX-2 synthesis and prostaglandin E₂ production. Inhibition of host or tumor cell COX-2 activity had no effect on early metastatic cell seeding in the liver but greatly reduced intrahepatic tumor cell proliferation and the rate of liver metastasis outgrowth. COX-2 inhibition had no effect on early tumor vascularization or on tumor cell apoptosis.

Conclusions: The high levels of COX-2 enzyme and prostaglandin production in C26 CRC cells are primarily caused by the presence of endogenous mutant KRAS^D12. Furthermore, COX-2 inhibition affects the tumoral rather than the vascular compartment during the early stages of C26 liver metastasis outgrowth.

Key Words: parecoxib • bioluminescence • hepatic replacement area • seeding • angiogenesis

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