E6-associated protein, E6-AP is involved in the carcinogenesis of human breast and prostate Free

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Aim: The E6-associated protein, E6-AP, is a dual function protein. It acts as an E3 ubiquitin-protein ligase as well as a steroid hormone receptor coactivator. Considering the importance of steroid hormone receptors and their coactivators in the normal development and tumorigenesis of reproductive organs of both genders, the roles of E6-AP in the tumorigenesis of female breast and male prostate tissues need to be investigated. Methods: 1. Immunohistochemistry. a. Formalin-fixed paraffin-embedded tumor samples from 13 human invasive breast carcinomas, 20 in-situ carcinomas, 7 human prostate carcinomas, and the corresponding normal glands were evaluated immunohistochemically for the expression of E6-AP, estrogen (ERa) and androgen (AR) receptors. The intensity of expression and the proportion of positive cells were measured using Automated Cell Imaging System (ACIS, ChromaVision). H-score of expression was obtained by multiplying the intensity with the proportion of positive cells (H=I x P). b. Breast tissues from E6-AP knockout mice were fixed, embedded, sectioned and immunohistochemically analyzed similarly as we did for human samples. 2. Protein degradation and ubiquitination assay. Human ERa protein was synthesized in vitro and radiolabeled by 35S using a transcription and translation kit. Then the ER protein was subjected to protein degradation either in the presence or absence of purified E6-AP. Results: 1. Normal mammary and prostate luminal epithelium strongly expressed E6-AP. A 25% and 27% decrease in expression intensity were observed for invasive breast carcinomas and prostate carcinoma, respectively, in comparison to their paired normal glands (p<0.001). Analysis of E6-AP expression in in-situ breast carcinomas indicated no difference when compared with their adjacent normal tissue. 2. In contrast to E6-AP, ERa expression is higher in invasive breast carcinomas than in in-situ breast carcinomas, indicating an inverse correlation between the expression of E6-AP and that of ERa. 3. The levels of ERa are higher in E6-AP knockout mammary glands compared with that of normal wild-type mammary glands. 4. E6-AP is required for the degradation of ERa through the ubiquitin-proteasome pathway. Conclusion: The expression of E6-AP is down regulated in advanced stage human breast and prostate cancers. There is an inversely correlation between the expression of E6-AP and ERa. E6-AP modulates the expression levels of ERa by promoting its degradation via the ubiquitin-proteasome pathway.