Supplementary Movie 2A from Constitutive K-RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP-1
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posted on 2023-04-03, 17:49 authored by Gregory P. Botta, Mauricio J. Reginato, Maximilian Reichert, Anil K. Rustgi, Peter I. LelkesAVI file - 8MB, Real-time imaging of E6/E7/Ras PDECs in 3D extending invadapodial protrusions and moving along the ECM over 30 m.
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ARTICLE ABSTRACT
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 extracellular signal-regulated kinase (ERK) 1/2 and proinvasive matrix metalloproteinases (MMP), 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 (PDEC) have yet to be elucidated in three-dimensions. 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 three-dimensional model incorporating a basement membrane analog. Activation of ERK2, but not ERK1, also occurs only in K-RasG12D–mutated PDECs cultured in three-dimensions and is a necessary intracellular signaling event for invasion based upon pharmacologic and short hairpin RNA (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 real-time quantitative 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 three-dimensional tumor microenvironment. Mol Cancer Res; 10(2); 183–96. ©2011 AACR.Usage metrics
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Drug TargetsProtein kinase & phosphatase drug targetsGastrointestinal CancersPancreatic cancerGene RegulationPhosphorylation and gene expressionGenome BiologySilencing and reactivation of gene expressionOncogenes & Tumor SuppressorsPreclinical ModelsProgression, Invasion & MetastasisCell adhesion and extracellular matrixMetastasisTumor MicroenvironmentTumor microcirculationTumor-stromal cell interactions
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