title: Constitutive K-RasG12D activation of ERK2 specifically regulates 3D invasion of human pancreatic cancer cells via MMP-1.
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-Ras(G12D) 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-Ras(G12D) 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-Ras(G12D)-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-Ras(G12D) 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-Ras(G12D) 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-Ras(G12D)-mutated human pancreatic cancer cells in a three-dimensional tumor microenvironment.
authors.affiliation: Department of Biochemistry and Molecular BiologyDrexel University College of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Botta
authors.forename: Mauricio J
authors.initials: MJ
authors.lastname: Reginato
authors.forename: Maximilian
authors.initials: M
authors.lastname: Reichert
authors.forename: Anil K
authors.initials: AK
authors.lastname: Rustgi
authors.forename: Peter I
authors.initials: PI
authors.lastname: Lelkes
mesh_terms: D002478:Cells, Cultured; D004789:Enzyme Activation / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics* / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009361:Neoplasm Invasiveness / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010183:Pancreatic Ducts / Q000378:metabolism / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction
chemical_list: C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D020781:Matrix Metalloproteinase 1