15417786mcr110399-sup-supplementary_movie_s2a.avi (8.26 MB)
Supplementary Movie 2A from Constitutive K-RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP-1
mediaposted on 2023-04-03, 17:49 authored by Gregory P. Botta, Mauricio J. Reginato, Maximilian Reichert, Anil K. Rustgi, Peter I. Lelkes
AVI file - 8MB, Real-time imaging of E6/E7/Ras PDECs in 3D extending invadapodial protrusions and moving along the ECM over 30 m.
ARTICLE ABSTRACTPancreatic 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.
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