De Petris Luigi, Orre Lukas M, Kanter Lena, Pernemalm Maria, Koyi Hirsh, Lewensohn Rolf, Lehtiö Janne
Karolinska Biomics Center, Karolinska Intitutet, Stockholm, Sweden.
Lung Cancer. 2009 Mar;63(3):410-7. doi: 10.1016/j.lungcan.2008.06.003. Epub 2008 Jul 11.
In a previously published in vitro study based on top-down proteomics we found that the calcium-binding proteins S100A6 and S100A4 were affected by exposure to ionizing radiation in a p53-dependent fashion. Both proteins showed post-translational modification changes, and S100A6 also showed increased expression and translocation in response to irradiation. Aim of the present study was to evaluate the expression of S100A6 and S100A4 in non-small-cell lung cancer (NSCLC).
S100A6 expression on archival tumor cell lysates from 39 patients with radically resected NSCLC was assessed with SELDI-TOF-MS. S100A6 identity was confirmed using a SELDI-based antibody-capture method on lysates from the A549 lung cancer cell line, cell lysates from two freshly prepared NSCLC samples, four plasma samples and one pleural effusion sample. Immunostainings for S100A6, S100A4 and p53 were performed on tissue microarrays containing 103 stage I surgically resected NSCLC cases and 14 normal lung parenchyma specimens.
The presence of post-translationally modified S100A6 forms was confirmed with SELDI-MS on enriched tumor cell lysates, as well as in plasma and pleural effusion samples. In addition, high S100A6 peak intensity was associated with longer median survival (35 months vs. 18 months for high and low peak intensity, respectively; p=n.s.). The immunohistochemical analysis showed that 25% of tumors were S100A6 positive. S100A6 expression correlated directly with non-squamous histology (p<0.0001) and S100A4 expression (p=0.005), and inversely with p53 expression (p=0.01). S100A6-positive cases showed a trend of longer survival compared with S100A6-negative cases (p=0.07). This difference became significant when the analysis was restricted to p53-negative cases (n=72). In this subgroup of patients, whose tumors likely exhibit a functional p53, S100A6 was an independent prognostic factor of improved survival at multivariate analysis (HR 0.49, 95% CI 0.27-0.81, p=0.017).
In this study we have validated on clinical material our previous findings on cell lines in terms of S100A6 expression and post-translational modifications pattern in NSCLC. Moreover, the survival results obtained in p53-negative stage I NSCLC cases support the proposed pro-apoptotic function of S100A6 and suggest the hypothesis of a cross regulation between these two proteins.
在先前发表的一项基于自上而下蛋白质组学的体外研究中，我们发现钙结合蛋白S100A6和S100A4以p53依赖的方式受到电离辐射的影响。这两种蛋白均显示出翻译后修饰的变化，并且S100A6在辐射后还显示出表达增加和易位。本研究的目的是评估S100A6和S100A4在非小细胞肺癌（NSCLC）中的表达。
使用表面增强激光解吸电离飞行时间质谱（SELDI-TOF-MS）评估39例接受根治性切除的NSCLC患者存档肿瘤细胞裂解物中S100A6的表达。使用基于SELDI的抗体捕获方法在A549肺癌细胞系的裂解物、两个新制备的NSCLC样品的细胞裂解物、四个血浆样品和一个胸腔积液样品中确认S100A6的身份。对包含103例I期手术切除的NSCLC病例和14例正常肺实质标本的组织微阵列进行S100A6、S100A4和p53的免疫染色。
通过SELDI-MS在富集的肿瘤细胞裂解物以及血浆和胸腔积液样品中证实了翻译后修饰的S100A6形式的存在。此外，S100A6峰强度高与中位生存期较长相关（高、低峰强度组的中位生存期分别为35个月和18个月；p=无显著性差异）。免疫组织化学分析显示，25% 的肿瘤为S100A6阳性。S100A6表达与非鳞状组织学直接相关（p<0.0001）和S100A4表达相关（p=0.005），与p53表达呈负相关（p=0.01）。与S100A6阴性病例相比，S100A6阳性病例显示出生存期较长的趋势（p=0.07）。当分析仅限于p53阴性病例（n=72）时，这种差异变得显著。在这一亚组患者中，其肿瘤可能具有功能性p53，在多变量分析中，S100A6是生存改善的独立预后因素（风险比0.49，95% 置信区间0.27-0.81，p=0.017）。
在本研究中，我们在临床材料上验证了我们先前在细胞系上关于NSCLC中S100A6表达和翻译后修饰模式的发现。此外，在p53阴性的I期NSCLC病例中获得的生存结果支持了所提出的S100A6的促凋亡功能，并提出了这两种蛋白之间交叉调节的假设。
