Tactacan Carole M, Phua Yu Wei, Liu Ling, Zhang Luxi, Humphrey Emily S, Cowley Mark, Pinese Mark, Biankin Andrew V, Daly Roger J
Cancer Research Division, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia.
Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia.
Mol Cancer. 2015 Jul 29;14:139. doi: 10.1186/s12943-015-0412-3.
Characterization of molecular mechanisms underpinning development of pancreatic ductal adenocarcinoma (PDAC) may lead to the identification of novel therapeutic targets and biomarkers. SgK223, also known as Pragmin, is a pseudokinase and scaffolding protein closely related to SgK269/PEAK1. Both proteins are implicated in oncogenic tyrosine kinase signaling, but their mechanisms and function remain poorly characterized.
Expression of SgK223 in PDAC and PDAC cell lines was characterized using gene expression microarrays, mass spectrometry (MS)-based phosphoproteomics and Western blotting. SgK223 was overexpressed in human pancreatic ductal epithelial (HPDE) cells via retroviral transduction, and knocked down in PDAC cells using siRNA. Cell proliferation was determined using a colorimetric cell viability assay, and cell migration and invasion using transwells. Expression of markers of epithelial-mesenchyme transition (EMT) was assayed by quantitative PCR. SgK223 and Stat3 signaling was interrogated by immunoprecipitation, Western blot and gene reporter assays. The functional role of specific kinases and Stat3 was determined using selective small molecule inhibitors.
Elevated site-selective tyrosine phosphorylation of SgK223 was identified in subsets of PDAC cell lines, and increased expression of SgK223 detected in several PDAC cell lines compared to human pancreatic ductal epithelial (HPDE) cells and in PDACs compared to normal pancreas. Expression of SgK223 in HPDE cells at levels comparable to those in PDAC did not alter cell proliferation but led to a more elongated morphology, enhanced migration and invasion and induced gene expression changes characteristic of a partial EMT. While SgK223 overexpression did not affect activation of Erk or Akt, it led to increased Stat3 Tyr705 phosphorylation and Stat3 transcriptional activity, and SgK223 and Stat3 associated in vivo. SgK223-overexpressing cells exhibited increased JAK1 activation, and use of selective inhibitors determined that the increased Stat3 signaling driven by SgK223 was JAK-dependent. Pharmacological inhibition of Stat3 revealed that Stat3 activation was required for the enhanced motility and invasion of SgK223-overexpressing cells.
Increased expression of SgK223 occurs in PDAC, and overexpression of SgK223 in pancreatic ductal epithelial cells promotes acquisition of a migratory and invasive phenotype through enhanced JAK1/Stat3 signaling. This represents the first association of SgK223 with a particular human cancer, and links SgK223 with a major signaling pathway strongly implicated in PDAC progression.
阐明胰腺导管腺癌（PDAC）发生发展的分子机制可能有助于识别新的治疗靶点和生物标志物。SgK223，也称为Pragmin，是一种与SgK269/PEAK1密切相关的假激酶和支架蛋白。这两种蛋白都参与致癌性酪氨酸激酶信号传导，但其机制和功能仍不清楚。
使用基因表达微阵列、基于质谱（MS）的磷酸化蛋白质组学和蛋白质免疫印迹法对SgK223在PDAC及PDAC细胞系中的表达进行分析。通过逆转录病毒转导在人胰腺导管上皮（HPDE）细胞中过表达SgK223，并使用小干扰RNA（siRNA）在PDAC细胞中敲低其表达。采用比色法细胞活力测定法检测细胞增殖，使用Transwell小室检测细胞迁移和侵袭。通过定量PCR检测上皮-间质转化（EMT）标志物的表达。通过免疫沉淀、蛋白质免疫印迹和基因报告基因检测来研究SgK223和信号转导及转录激活因子3（Stat3）信号传导。使用选择性小分子抑制剂确定特定激酶和Stat3的功能作用。
在部分PDAC细胞系中发现SgK223的位点选择性酪氨酸磷酸化水平升高，与人类胰腺导管上皮（HPDE）细胞相比，在几种PDAC细胞系中检测到SgK223表达增加，与正常胰腺相比，在PDAC中SgK223表达也增加。在HPDE细胞中以与PDAC相当的水平表达SgK223不会改变细胞增殖，但会导致细胞形态更细长，迁移和侵袭增强，并诱导部分EMT特征性的基因表达变化。虽然SgK223过表达不影响细胞外信号调节激酶（Erk）或蛋白激酶B（Akt）的激活，但它会导致Stat3酪氨酸705位点磷酸化增加和Stat3转录活性增强，且SgK223和Stat3在体内相互关联。过表达SgK223的细胞表现出Janus激酶1（JAK1）激活增加，使用选择性抑制剂确定SgK223驱动的Stat3信号增加是JAK依赖性的。对Stat3进行药理学抑制表明，Stat3激活是SgK223过表达细胞运动性和侵袭性增强所必需的。
SgK223在PDAC中表达增加，胰腺导管上皮细胞中SgK223过表达通过增强JAK1/Stat3信号传导促进获得迁移和侵袭表型。这是SgK223与特定人类癌症的首次关联，并将SgK223与一条在PDAC进展中起重要作用的信号通路联系起来。
