Constitutive K-RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP-1

摘要

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 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-Ras^G12D 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-Ras^G12D 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-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 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-Ras^G12D mutated human pancreatic cancer cells in a 3D tumor microenvironment.