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Data Supplement from Targeted Deletion and Lipidomic Analysis Identify Epithelial Cell COX-2 as a Major Driver of Chemically Induced Skin Cancer

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posted on 2023-04-03, 16:25 authored by Jing Jiao, Tomo-O Ishikawa, Darren S. Dumlao, Paul C. Norris, Clara E. Magyar, Carol Mikulec, Art Catapang, Edward A. Dennis, Susan M. Fischer, Harvey R. Herschman

Keratinocytes from Cox-2deltaE Cox-2flox/flox;K14Cre+) mice undergo effective targeted Cox-2 gene deletion. Keratinocyte were isolated from Cox-2deltaE and Cox-2flox/flox (Cox-2fl/fl) mice. Briefly, skin specimens isolated from 7-9 week old mice were treated with 3 mg/ml Dispase (Invitrogen, Carlsbad, CA) for 16 hrs at 4oC, and the epidermis was separated from the dermis. The isolated keratinocytes were plated on dishes containing keratinocyte serum-free medium. After seven days of culture, DNAs from the keratinocytes were extracted and the presence of the Cox-2fl, Cox-2del and Cre alleles were examined by PCR. PCR analysis demonstrates both the presence of the Cre+ and the Cox-2del alleles in Cox-2deltaE keratinocytes and the absence of the Cre+ allele and the presence of the Cox-2fl allele in Cox-2fl/fl keratinocytes.

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ARTICLE ABSTRACT

Pharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX-2) plays a critical role in DMBA/TPA–induced skin tumor induction. Although many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell type–specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell–specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell–specific Cox-2 gene deletion, compared with littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2–expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA–treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2–dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell type–specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biologic responses.Implications:Cox-2 gene deletion demonstrates that intrinsic COX-2 expression in initiated keratinocytes is a principal driver of skin carcinogenesis; lipidomic analysis identifies likely prostanoid effectors. Mol Cancer Res; 12(11); 1677–88. ©2014 AACR.