In the phenomenon of epithelial-mesenchymal transition (EMT)1
, polarized epithelial cells loosen their cell-cell junctions and acquire the ability to migrate through extracellular matrices as single cells in a mesenchymal manner (1
). Although great progress has been made on identifying and understanding components and mechanisms involved in the process of EMT induction
)), the “before” versus
of this transition for signaling pathway control of cell migration has not yet been investigated from a multipathway, network-wide perspective. Cell migration results from a set of carefully orchestrated biophysical processes regulated by numerous key signaling pathways whose activities can be influenced downstream of a range of growth factor receptors. It is appreciated that these growth factor receptor-elicited signaling activities may be modulated in “before” versus
“after” manner by EMT induction (5
), whether by TGFβ or other developmental cues or inflammation-related stimuli (6
). However, a current challenge is to characterize this likely complex modulation from a multipathway network perspective and to establish an approach for predictive understanding of how the multiple pathway activities integrate to yield different migration behavior in postinduction compared with pre-induction conditions. This challenge is especially important for, among other motivations, gaining insights concerning how prospective targeted drug effects are influenced by whether tumor cells are in epithelial or mesenchymal state (8
As one currently clinically urgent application example, the epidermal growth factor receptor (EGFR) is commonly overexpressed or mutated in epithelium-derived tumors, and its activation is linked to progression and poor prognosis (9
). Therefore, EGFR has been the target of many small molecule inhibitors and monoclonal antibody antagonists, which have met with limited clinical success (10
). Recent studies exploiting EMT markers and gene expression signatures suggest that cells with low levels of epithelial markers, such as E-cadherin, and high levels of mesenchymal protein expression, such as N-cadherin and vimentin, display resistance against these inhibitors (13
). Therefore, the decreased sensitivity of mesenchymal-like tumors to EGFR antagonists argues for an ability to bypass EGFR dependence to activate the downstream signaling pathways necessary for cell migration and survival (15
). Cell activation through other receptors including the insulin-like growth factor-1 receptor (IGF-1R), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR), has been suggested to play a role in resistance to EGFR antagonists (14
). Thus, improved understanding of how EMT-mediated changes in multiple growth factor signaling networks contribute to cell invasion may necessarily shift investigational focus toward the design of novel therapeutics targeting tangential tyrosine kinase pathways or intracellular signaling nexi for use in treating EGFR inhibition-resistant carcinomas.
As a first multipathway network level study of how signaling pathway activities governing cell migration downstream of receptor tyrosine kinase stimulation differ between “before EMT” and “after EMT” conditions, we use here an established human mammary epithelial cell line (hMLE) immortalized and transformed via introduction of a minimal set of oncogenes (17
) and focus on EMT induction by Twist1 (18
), via its ectopic expression in hMLEs as previously characterized (19
). Twist expression has been demonstrated in multiple studies in vitro
, in mouse models, and in human patients, to be associated with breast tumor invasiveness, metastasis, and poor disease prognosis (e.g.
)), and thus represents a pathophysiologically and clinically important system for analysis. It also may be as simple an induction process as can be examined, because other EMT inducers such as TGFβ and TNFα act via multiple transcription factors including Twist along with others (7
), so our initial study here may indicate basic signaling network modulation insights that can be expanded upon in future analogous investigations of the more pleiotropic EMT inducers.
In this basic study, we quantitatively characterize the migration characteristics of hMLEs before and after Twist-mediated induction in both monolayer (indicative of epithelial mode) and single cell (indicative of mesenchymal mode) migration assays under stimulation by a panel of growth factors present in carcinoma environments including EGF, HRG, IGF, and HGF (16
). Across this broad landscape of extracellular treatment conditions, we measured phosphorylation states of 14 signaling pathway nodes to ascertain how Twist-mediated changes in numerous of these signals may be associated with consequent changes in the cell motility behaviors. Computational modeling with a partial least-squares regression (PLSR) framework demonstrated that quantitative combinations of multiple signals can account for the various motility behaviors across all growth factor treatments in both epithelial and mesenchymal migration modes—and, in fact, can successfully predict a priori
the motility behavior for epithelial and mesenchymal modes in a new growth factor context, PDGF stimulation. We then constructed a complementary computational model, using a correlative topology framework, to identify influences among the signaling nodes that were modulated by the Twist-mediated EMT induction.