Reassessment of Id1 Protein Expression in Human Mammary, Prostate, and Bladder Cancers Using a Monospecific Rabbit Monoclonal Anti-Id1 Antibody JonathanPerk, 1 Ignacio Gil-Bazo, 1 YvetteChin, 1 PaoladeCandia, 1 JohnJ.S.Chen, 3 YuntaoZhao, 3 Shirley Chao, 3 WaiCheong, 3 YaohuangKe, 4 Hikmat Al-Ahmadie, 2 WilliamL.Gerald, 2 Edi Brogi, 2 andRobertBenezra 1 1 Cancer Biology and Genetics Program and 2 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York; 3 BioCheck, Inc., Foster City, California; and 4 Epitomics, Inc., Burlingame, California Abstract Id proteins are a class of dominant-negative antagonists of helix-loop-helix transcription factors and have been shown to control differentiation of a variety of cell types in diverse organisms. Although the importance of Id1 in tumor endothelial cells is well established, the expression and role of the Id1 protein in human cancer cells is controversial. To explore this issue, we developed and characterized a highly specific rabbit monoclonal antibody against Id1 to assess its expression in human breast, prostate, and bladder malignan- cies. Our results show that in usual types of human mammary carcinomas, the Id1 protein is expressed exclusively in the endothelium. Interestingly, we detected nuclear expression of the Id1 protein in the tumor cells in 10 of 45 cases of poorly differentiated and highly aggressive carcinoma with meta- plastic morphology. Similarly, only 1 of 30 prostate cancer samples showed Id1-positive tumor cells, whereas in almost all, endothelial cells showed high Id1 expression. Intriguingly, whereas normal prostate glands do not show any Id1 protein expression, basal layer cells of benign prostate glands in proximity to tumors expressed high levels of the Id1 protein. In contrast to the lack of Id1 expression in the usual types of mammary and prostate cancers, the majority of transitional cell bladder tumors showed Id1 protein expression in both tumor and endothelial cells. These results suggest that further refinement of Id1 expression patterns in a variety of tumor types will be necessary to identify and study the functional roles played by Id1 in human neoplastic processes. (Cancer Res 2006; 66(22): 10870-7) Introduction The Id proteins are a class of dominant-negative antagonists of helix-loop-helix transcription factors and have been shown to control differentiation of a variety of cell types in diverse organisms (for reviews, see refs. 1, 2). The expression and role of the Id1 protein in epithelial cancer cells, however, is controversial. For example, some investigators have reported that the neoplastic cells of breast, prostate, and bladder carcinomas express the Id1 protein, and that this finding correlates in some cases with poor prognosis (3–6). Others, however, have reported Id1 protein expression in mammary tumors only in the endothelial cells of blood vessels associated with these lesions (7). Interestingly, mRNA expression is observed in a broad spectrum of tumors, suggesting posttranscrip- tional control mechanisms perhaps at the level of protein destabilization (8). Genetic elimination of the Id1 locus in mice predisposed to develop breast or prostate neoplasias shows a profound effect on vascularization and tumor integrity but little effect on tumor initiation: results are consistent with an endothelial cell–specific localization of the protein product (7, 9). Mobilization of endothelial cell progenitors from the bone marrow is also severely perturbed in Id knockout mice consistent with this hypothesis (10, 11). On the other hand, a role for Id1 in modulating tumor epithelial cell behavior is suggested by the fact that overexpression of Id1 in cell lines results in increased invasiveness and metastatic potential of these cells, whereas reduction of high levels of Id1 present endogenously in these lines leads to an inhibition of these properties (12–17). It is likely that the discrepancy in the expression data is due to variability in the specificity of antibodies used in these studies that are obtained from commercial sources. To unequivocally deter- mine the expression pattern of the Id1 protein in human breast, prostate, and bladder cancers, we developed a rabbit monoclonal antibody against purified murine Id1, which cross-reacts with the human orthologue. The rationale for isolating a monoclonal antibody with this cross-reactivity is that the demonstration of the specificity required in immunohistochemistry experiments is most easily achieved using mouse knockout strains as negative controls. Thus, to insure antibody specificity, we have imposed the following criteria: the antibody preparation must be monospecific in Western blots of tissue extracts that contain the Id1 protein but negative over a wide molecular weight range in extracts from which the gene product is known to be absent. In addition, we demanded that the signals detected by immunohistochemistry in a murine tissue known to express Id1 are absent in Id1 knockout controls. By these criteria, we have identified a rabbit monoclonal antibody (195-14), which is suitable for human immunohistochem- istry, and have used it to measure Id1 protein expression in human breast, prostate, and bladder tumor samples. Our results show that the Id1 protein is present in some breast tumor cells derived from a restricted subset of human mammary tumors known as metaplas- tic carcinomas, usually associated with a poor prognosis. Despite the purported role of Id1 in breast cancer metastasis, lymph node metastatic lesions of the common types of breast cancer also did not show Id1 staining outside of the endothelium. In human Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Perk and I. Gil-Bazo contributed equally to this work. Requests for reprints: Robert Benezra, Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-2389; E-mail: r-benezra@ski.mskcc.org. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-2643 Cancer Res 2006; 66: (22). 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