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Abstract：
Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 (MMP-2) and E-cadherin (CDH1). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma.
摘要：
精原细胞瘤是最常见的睾丸癌。垂体肿瘤转化基因1（PTTG1）是一种在几种肿瘤中显示致癌活性的securin。我们先前证明了核PTTG1通过其对基质金属蛋白酶2（MMP-2）和E-cadherin（CDH1）的转录活性来促进精原细胞瘤肿瘤的侵袭。我们想知道特定的相互作用物是否会影响其亚细胞分布。为了这个目标,我们研究了精原细胞瘤细胞系中的PTTG1相互作用体，这些细胞系显示出不同的PTTG1核水平与侵袭特性相关。PTTG1免疫沉淀的蛋白质组学方法揭示了新的特异性securin相互作用物。蛋白质印迹,共聚焦显微镜，细胞质/细胞核分级分离，球体形成测定，进行和Atlas数据库的询问以验证蛋白质组学结果并调查PTTG1和新发现的伴侣之间的相互作用。我们观察到血影蛋白β链（SPTBN1）和PTTG1是辅因子，用SPTBN1将securin锚定在细胞质中。SPTBN1下调决定了PTTG1核易位，提升其侵入能力。此外，缺乏SPTBN1结合的PTTG1缺失突变体强烈定位在细胞核中。Atlas数据库显示，与非精原细胞瘤相比，含有较高核PTTG1水平的精原细胞瘤显示出明显较低的SPTBN1水平。在人类精原细胞瘤标本中，我们发现PTTG1/SPTBN1强烈的共定位在PTTG1核分布区域减少。总的来说,这些结果表明,SPTBN1与PTTG1一起是一个潜在的预后因素,可用于精原细胞瘤的临床治疗.
