ADA3: a novel target for HPV16 E6 oncoprotein

描述（由申请人提供）：人类癌起源于正常上皮细胞，通过肿瘤抑制检查点的渐进式放松。HPV E6癌基因可以显著地使乳腺上皮细胞永生，否则乳腺上皮细胞会表现出有限的寿命，这为鉴定上皮细胞转化过程中失活的肿瘤抑制途径提供了一种实用的方法。该模型揭示了p53功能失活的关键作用；然而，选择性地消除p53功能并不能导致有效的永生化，这表明了其他途径的作用。我们已经确定了酵母转录辅激活子yADA3的人类同源物，作为一个新的E6靶点。yADA3是连接转录激活因子与组蛋白乙酰化和基础转录机制的辅激活因子复合物的关键成分。重组哺乳动物核受体介导的酵母转录，揭示了对yADA3及其相关蛋白yADA2和yGCN5的需求。基于这些研究，以及hADA3选择性结合高危HPV E6蛋白和永生化E6突变体，我们假设hADA3参与维持上皮细胞正常状态的基因的转录调控。由于hADA3与HPV E6的相互作用或通过其他方式，hADA3功能的丧失或失调可能是HPV相关癌症和其他癌症的重要致癌机制。为了验证我们的假设，我们将生成针对人类ADA3和ADA2的抗体，并检测它们的表达，并确定含有hada3的组蛋白乙酰化酶复合物的存在及其参与上皮细胞中类视黄醇受体介导的基因转录。然后，我们将定义ADA3的结构域，介导其与活化的类视黄醛受体和组蛋白乙酰化酶复合物的物理和功能相互作用。我们将研究E6是否通过取代结合伙伴或ADA3降解来灭活ADA3功能。最后，我们将确定ADA3在介导细胞分化和生长抑制功能中的潜在作用，并评估ADA3显性阴性突变体诱导永生化的潜力。这些研究的成功结果应该确定一种新的肿瘤抑制途径，其成分可能为癌症的早期诊断提供新的标志物，并为开发合理的治疗方法提供靶点。

DESCRIPTION (provided by applicant): Human carcinomas arise from normal epithelial cells through progressive relaxation of tumor suppressor checkpoints. HPV E6 oncogene can dominantly immortalize mammary epithelial cells, which otherwise exhibit a finite life span, providing a practical approach to identify tumor suppressor pathways inactivated during transformation of epithelial cells. This model revealed a crucial role for inactivation of p53 functions; however, selective elimination of p53 function did not lead to efficient immortalization, indicating the role of additional pathways. We have identified the human homologue of yeast transcriptional coactivator yADA3, as a novel E6 target. yADA3 is a critical component of coactivator complexes that link transcriptional activators to histone acetylation and basal transcriptional machinery. Reconstitution of mammalian nuclear receptor-mediated transcription in yeast revealed a requirement for yADA3, and associated proteins, yADA2 and yGCN5. Based on these studies, and selective binding of hADA3 to high-risk HPV E6 proteins and immortalizing E6 mutants, we hypothesize that hADA3 participates in transcriptional regulation of genes that maintain the normal state of epithelial cells. Loss or deregulation of hADA3 function, due to its interaction with HPV E6 or by other means, is likely to represent an important oncogenic mechanism in HPV-associated and other cancers. To address our hypotheses, we will generate antibodies to human ADA3 and ADA2, and examine their expression, and establish the presence of hADA3-containing histone acetylase complexes and their involvement in retinoid receptor-mediated gene transcription in epithelial cells. We will then define the domains of ADA3 that mediate its physical and functional interactions with activated retinoid receptors and histone acetylase complexes. We will examine whether E6 inactivates ADA3 function by displacement of binding partners or ADA3 degradation. Finally, we will determine the potential role of ADA3 in mediating cell differentiation and growth inhibitory functions, and assess the potential of dominant-negative ADA3 mutants to induce immortalization. Successful outcome of these studies should identify a novel tumor suppressor pathway whose components may provide new markers for early diagnosis of carcinomas, and targets for development of rational therapeutics.