Constitutive K-Ras^[G12D] activation of ERK2 specifically regulates oncogenesis of pancreatic cancer cells in 3D
Year: 2012
Type: dissertation
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 ERK1/2, pro-invasive matrix metalloproteinases (MMPs), as well as their inhibitors tissue inhibitors of MMPs (TIMPs) - all indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras - ERK1/2 signaling and its influence on stromal invasion and proliferation 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, epithelial-to-mesenchymal transition markers, and hyperplasia but only when cultured in a 3D model incorporating a basement membrane. 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 is associated with a specific microarray gene expression signature and induction of MMP endopeptidases. Specifically, MMP-1 and TIMP-1 RNA, their secreted proteins, and MMP-1's 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. TIMP-1 silencing is dispensable but overexpression induces proliferation in only K-RasG12D mutated PDECs. 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 while TIMP-1 use to attenuate growth is cautioned.
DOI: 10.17918/00000488
Authors: Gregory P. Botta, Peter I. Lelkes
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4207039907