Vestey S B, Sen C, Calder C J, Perks C M, Pignatelli M, Winters Z E
University of Bristol Division of Surgery, Bristol Royal Infirmary, Bristol, UK.
Breast Cancer Res. 2004;6(5):R571-85. doi: 10.1186/bcr912. Epub 2004 Jul 29.
p14ARF stabilises nuclear p53, with a variable expression of p14ARF mRNA in breast cancers. In vitro, nuclear p14ARF binds Hdm2 to block Hdm2-dependent nucleocytoplasmic shuttling of p53, which is required before cytoplasmic degradation of p53. p14ARF is negatively regulated by p53 and through p53-independent pathways. No studies have yet examined levels of p14ARF protein expression in breast cancer and their relationship to Hdm2/p53 immunoreactivity or subcellular localisation. Previously, immunohistochemical expression of cytoplasmic p14ARF, p53 and Hdm2 has been described. HER-2 (c-erbB2/neu) predicts prognosis and interacts with the p14ARF/Hdm2 pathway to inactivate p14ARF and to influence Hdm2 activity and localisation. This study examined p14ARF and p53/Hdm2 expression and subcellular localisation by using immunohistochemistry in a series of invasive ductal breast cancers (IDCs) with concomitant ductal carcinoma in situ (DCIS), to evaluate whether findings in vitro were related to clinicopathological parameters such as HER-2 and their effect on patient outcome.
The 4C6 anti-p14ARF monoclonal antibody and Dako Envision Plus system were used to evaluate p14ARF expression in 103 patients; p53/Hdm2 staining was performed.
p14ARF was evaluable in 96 patients, with nuclear p14ARF expression (modified Quick-score > or = 3) in 79% (n = 76) of IDCs and in associated DCIS in 74 patients. Cytoplasmic p14ARF was detectable in 23 breast cancers. Nuclear and cytoplasmic p14ARF showed no correlation with p53 subcellular immunoreactivity. Increasing levels of cytoplasmic p14ARF were associated with nuclear and cytoplasmic Hdm2 expression (P < 0.001). Subcellular ARF expression was not associated with clinicopathological parameters, and although not an independent prognosticator, these preliminary findings suggest that cytoplasmic p14ARF might be associated with a better overall survival (P = 0.09; log rank). The association between HER-2 positivity and nuclear p14ARF (P = 0.038), as well as nuclear Hdm2 (P = 0.019), reflects the in vitro findings of HER-2 interaction with the ARF/Hdm2 pathway. Cytoplasmic p53 and Hdm2 expression might have biological implications, through an association of cytoplasmic p53 with increased tumour proliferation (P = 0.005), and an improved overall survival (P = 0.002, log rank) in cytoplasmic Hdm2-expressing tumours, that independently predict favourable overall survival (P = 0.02) and disease-free survival (P = 0.03).
Nuclear p14ARF expression is similar in IDCs and DCIS and is associated with Hdm2 immunoreactivity. Nuclear p14ARF and Hdm2 might be regulated by HER-2. Clearly, our findings in vivo suggest a complexity of p14ARF/Hdm2 and p53 pathways in which consideration of cytoplasmic p14ARF and Hdm2 might have tumorigenic implications.
p14ARF可稳定细胞核中的p53，在乳腺癌中p14ARF mRNA表达存在差异。在体外，细胞核中的p14ARF与Hdm2结合，以阻断p53依赖Hdm2的核质穿梭，这是p53在细胞质中降解之前所必需的。p14ARF受到p53以及通过不依赖p53的途径进行负调控。尚无研究检测乳腺癌中p14ARF蛋白表达水平及其与Hdm2/p53免疫反应性或亚细胞定位的关系。此前，已有关于细胞质p14ARF、p53和Hdm2免疫组化表达的描述。HER-2（c-erbB2/neu）可预测预后，并与p14ARF/Hdm2途径相互作用，使p14ARF失活，并影响Hdm2的活性和定位。本研究采用免疫组化方法检测了一系列伴有原位导管癌（DCIS）的浸润性导管癌（IDC）中p14ARF和p53/Hdm2的表达及亚细胞定位，以评估体外研究结果是否与HER-2等临床病理参数相关及其对患者预后的影响。
使用4C6抗p14ARF单克隆抗体和Dako Envision Plus系统评估103例患者的p14ARF表达；进行p53/Hdm2染色。
96例患者的p14ARF可评估，79%（n = 76）的IDC及74例相关DCIS中有细胞核p14ARF表达（改良快速评分≥3）。23例乳腺癌中可检测到细胞质p14ARF。细胞核和细胞质p14ARF与p53亚细胞免疫反应性无相关性。细胞质p14ARF水平升高与细胞核和细胞质Hdm2表达相关（P < 0.001）。亚细胞ARF表达与临床病理参数无关，虽然不是独立的预后指标，但这些初步结果表明细胞质p14ARF可能与更好的总生存率相关（P = 0.09；对数秩检验）。HER-2阳性与细胞核p14ARF（P = 0.038）以及细胞核Hdm2（P = 0.019）之间的关联反映了HER-2与ARF/Hdm2途径相互作用的体外研究结果。细胞质p53和Hdm2表达可能具有生物学意义，细胞质p53与肿瘤增殖增加相关（P = 0.005），在表达细胞质Hdm2的肿瘤中总生存率提高（P = 0.002，对数秩检验），这独立预测了良好的总生存率（P = 0.02）和无病生存率（P = 0.03）。
IDC和DCIS中细胞核p14ARF表达相似，且与Hdm2免疫反应性相关。细胞核p14ARF和Hdm2可能受HER-2调控。显然，我们的体内研究结果表明p14ARF/Hdm2和p53途径存在复杂性，其中细胞质p14ARF和Hdm2的考量可能具有致瘤意义。
