Constitutive K-RasG12D activation of ERK2 specifically regulates 3D invasion of human pancreatic cancer cells via MMP-1.
Constitutive K-RasG12D activation of ERK2 specifically regulates 3D invasion of human pancreatic cancer cells via MMP-1.
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DOI：
10.1158/1541-7786.mcr-11-0399
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发表时间：
2012-02
期刊：
影响因子：
--
通讯作者：
Lelkes PI
中科院分区：
文献类型：
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作者：
Botta GP;Reginato MJ;Reichert M;Rustgi AK;Lelkes PI
胰腺导管腺癌（PDAC）是一种高度侵袭性和转移性的肿瘤，通常对目前的药物治疗无反应。绝大多数情况下，PDAC在K-RasG12D中包含侵袭前存在的早期构成性致癌突变。人类PDAC活检的组织学和遗传学分析也显示ERK1/2和前侵袭性基质金属蛋白酶（MMPs）的表达增加；预后不良指标。然而，K-Ras - ERK1/2信号传导及其对原代人胰腺导管上皮细胞（PDECs） mmp定向基质侵袭的影响所需的独特分子机制尚未在3D中阐明。在基因定义的PDECs中，致癌基因K-RasG12D的表达显示浸润性和上皮到间质转化标记物的增加，但只有在含有基膜类似物的3D模型中培养时才会增加。细胞外信号相关激酶2 （ERK2）的激活，而不是ERK1，也只发生在3D培养的K-RasG12D突变的PDECs中，并且是基于药理学和shRNA抑制的侵袭所需的细胞内信号事件。K-RasG12D PDECs通过基底膜模型侵袭活性的增加与特定的微阵列基因表达特征和MMP内肽酶的诱导有关。具体来说，通过RT - q-PCR、ELISA和荧光共振能量转移（FRET）检测，K-RasG12D PDECs中扩增了MMP-1 RNA、其分泌蛋白及其蛋白水解裂解活性。重要的是，shRNA沉默MMP-1模拟ERK2抑制并破坏活跃的垂直PDEC入侵。ERK2-isoform和MMP-1靶向被证明是在3D肿瘤微环境中减弱K-RasG12D突变的人胰腺癌细胞侵袭的可行策略。
Pancreatic ductal adenocarcinomas (PDAC) are highly invasive and metastatic neoplasms commonly unresponsive to current drug therapy. Overwhelmingly, PDAC harbors early constitutive, oncogenic mutations in K-RasG12D that exist prior to invasion. Histologic and genetic analyses of human PDAC biopsies also exhibit increased expression of ERK1/2 and pro-invasive matrix metalloproteinases (MMPs); indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras – ERK1/2 signaling and its influence on MMP-directed stromal invasion in primary human pancreatic ductal epithelial cells (PDECs) has yet to be elucidated in 3D. Expression of oncogenic K-RasG12D alone in genetically-defined PDECs reveals increased invadopodia and epithelial-to-mesenchymal transition markers, but only when cultured in a 3D model incorporating a basement membrane analog. Activation of extracellular signal-related kinase 2 (ERK2), but not ERK1, also occurs only in K-RasG12D mutated PDECs cultured in 3D and is a necessary intracellular signaling event for invasion based upon pharmacologic and shRNA inhibition. Increased active invasion of K-RasG12D PDECs through the basement membrane model is associated with a specific microarray gene expression signature and induction of MMP endopeptidases. Specifically, MMP-1 RNA, its secreted protein, and its proteolytic cleavage activity are amplified in K-RasG12D PDECs when assayed by RT q-PCR, ELISA, and fluorescence resonance energy transfer (FRET). Importantly, shRNA silencing of MMP-1 mimics ERK2 inhibition and disrupts active, vertical PDEC invasion. ERK2-isoform and MMP-1 targeting are shown to be viable strategies to attenuate invasion of K-RasG12D mutated human pancreatic cancer cells in a 3D tumor microenvironment.