Understanding Spatially Regulated Tumor-Inhibitory Function of Profilin-1 and its Deregulation in Breast Cancer

项目摘要 改变的蛋白质核质运输越来越多地被认为是癌症的真正驱动因素 进展许多肿瘤抑制蛋白（如p53，Rb）在细胞核中起作用，但在细胞核中表达增加。 在许多类型的癌症中，核输出是由于其核输出蛋白XPO 1/CRM 1的过表达而导致的。 Profilin-1（Pfn1）是一种肌动蛋白结合蛋白，在肿瘤的发生发展中具有抗肿瘤和抗转移活性。 各种癌症。然而，它作为肿瘤抑制因子的作用仍然存在争议，因为它很少被 由于其促进肌动蛋白聚合，它发生了突变，并且对细胞生长和生存至关重要。 虽然通常认为Pfn1是一种细胞质蛋白，但它存在于许多哺乳动物细胞的细胞核中 功能不太清楚。我们先前已经发现Pfn1在乳腺癌中的体外抗肿瘤作用， 癌细胞需要有进入细胞核的能力。这表明Pfn1的肿瘤抑制活性可能是由于 从它在细胞核中的"兼职"功能来看，它在空间上与其基本的细胞质肌动蛋白分离， 监管职能。pfn1与肌动蛋白复合，由核输出者主动进行核输出 exportin-6（XPO 6）。与XPO 1/CRM 1不同，XPO 6具有高度选择性，Pfn1/actin是其唯一已知的货物。 我们在TCGA数据集中发现，XPO 6的mRNA水平在65 - 100%的肿瘤中显著上调， 12种不同类型的样本（包括乳房）。我们还发现，XPO 6蛋白水平是 在一组管腔和基底样乳腺癌细胞系中， 未转化的乳腺上皮细胞XPO 6敲低选择性抑制了几种细胞的生长和存活。 乳腺癌细胞系，而对未转化的对照细胞几乎没有影响。这些数据表明， 癌症中Pfn1失调的主要模式可能是其增加的核输出， 其核输出蛋白XPO 6的过度表达。此外，我们还发现了一种新的相互作用， 核Pfn1和多蛋白超延伸复合物（SEC）通过co-IP和质谱。SEC 正调控许多促肿瘤和促EMT/转移基因的转录延长， 磷酸化（通过其组分CDK9/细胞周期蛋白T1）和不暂停启动子近端RNA聚合酶 二是引发生产延伸。我们证明，Pfn1敲低增加磷酸化RNA Pol II水平， 乳腺癌细胞并使其对CDK9抑制敏感。总之，我们的数据支持这样的假设， Pfn1通过抑制SEC介导的基因转录在细胞核中发挥肿瘤抑制作用 并且其亚细胞定位在癌症中经常通过其 核出口国XPO 6。在这项资助中，我们将使用各种方法来测试这一假设，包括在体外 以及体内肿瘤模型、转录组和表观基因组分析和原发性肿瘤样品分析。

PROJECT SUMMARY Altered protein nucleocytoplasmic trafficking is increasingly recognized as a bona fide driver of cancer progression. Many tumor suppressor proteins (e.g. p53, Rb) function in the nucleus, but undergo increased nuclear export in many types of cancer resulting from overexpression of their nuclear exporter XPO1/CRM1. Profilin-1 (Pfn1) is an actin-binding protein with well-documented anti-tumor and anti-metastatic activities in various types of cancer. However, its role as a tumor suppressor remains controversial because it is rarely mutated and paradoxically essential for cell growth and survival owing to its facilitation of actin polymerization. Though generally considered a cytoplasmic protein, Pfn1 is present in the nucleus of many mammalian cells with poorly understood function. We have previously found that the in vitro anti-tumor effect of Pfn1 in breast cancer cells requires its ability to enter nucleus. This suggests that Pfn1's tumor suppressor activity may stem from its “moonlighting” function in the nucleus that is spatially separated from its essential cytoplasmic actin- regulatory functions. Pfn1, in a complex with actin, undergoes active nuclear export by the nuclear exporter exportin-6 (XPO6). Unlike XPO1/CRM1, XPO6 is highly selective with Pfn1/actin being its only known cargo. We found in the TCGA datasets that the mRNA level of XPO6 is significantly upregulated in 65-100% of tumor samples across 12 different types (including breast). We also discovered that XPO6 protein level is significantly increased in a panel of luminal and basal-like breast cancer cell lines as compared to untransformed breast epithelial cells. XPO6 knockdown selectively inhibited the growth and survival of several breast cancer cell lines while having little on the untransformed control cells. Together, these data suggest that the primary mode of Pfn1 deregulation in cancer may be its increased nuclear export resulting from overexpression of its nuclear exporter XPO6. In addition, we have uncovered a novel interaction between nuclear Pfn1 and the multi-protein super elongation complex (SEC) by co-IP and mass spectrometry. SEC positively regulates the transcription elongation of many pro-tumor and pro-EMT/metastasis genes by phosphorylating (through its components CDK9/cyclin T1) and un-pausing promoter proximal RNA polymerase II to trigger productive elongation. We demonstrate that Pfn1 knockdown increases phospho-RNA Pol II level in breast cancer cells and sensitizes them to CDK9 inhibition. Together, our data support the hypothesis that Pfn1 functions in the nucleus as a tumor suppressor by inhibiting SEC-mediated gene transcription and its subcellular localization is frequently deregulated in cancer through overexpression of its nuclear exporter XPO6. In this grant, we will test this hypothesis using various approaches including in vitro and in vivo tumor models, transcriptome and epigenome analysis, and primary tumor sample analysis.