Abstract
Immunohistochemical (IHC) staining in breast cancer shows both gain and loss of COX2 expression with disease risk and progression. We investigated four common COX2 antibody clones and found high specificity for purified human COX2 for three clones; however, recognition of COX2 in cell lysates was clone dependent. Biochemical characterization revealed two distinct forms of COX2, with SP21 recognizing an S-nitrosylated form, and CX229 and CX294 recognizing non-nitrosylated COX2 antigen. We found S-nitrosylated and non-nitrosylated COX2 occupy different subcellular locations in normal and breast cancer tissue, implicating distinct synthetic/trafficking pathways and function. Dual stains of ~2000 breast cancer cases show early-onset breast cancer had increased expression of both forms of COX2 compared to postmenopausal cases. Our results highlight the strengths of using multiple, highly characterized antibody clones for COX2 IHC studies and raise the prospect that S-nitrosylation of COX2 may play a role in breast cancer biology.
Highlights
The cyclooxygenase enzyme COX2, a key mediator of tissue inflammation via prostaglandin production, has been investigated extensively as a cancer biomarker and therapeutic target[1,2,3,4,5]
We found that SP21 detected HCA-7 COX2 only after incubation with SNOG (Fig. 2B, lane 4), which is consistent with SP21 recognizing an S-nitrosylated form of COX2
Since COX2 biology is thought to be important in many cancer types and diseases, our discovery of S-nitrosylation state-specific COX2 antibody clones is likely to have broad impact
Summary
The cyclooxygenase enzyme COX2, a key mediator of tissue inflammation via prostaglandin production, has been investigated extensively as a cancer biomarker and therapeutic target[1,2,3,4,5]. While some breast cancer studies corroborate these results[35,36,37,38,39], others show loss of COX2 in invasive disease compared to adjacent normal tissue, even when the same antibody clones are used[31,40,41,42]. One explanation for these divergent results may be methodological, as no standardized approach to COX2 IHC detection has been adopted. One concern regarding αCOX2 antibodies is cross-reactivity, as COX1 is closely related to COX2, with 65%
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