The phenotypic switching called epithelial-to-mesenchymal transition is frequently associated with epithelial tumor cell progression from a comparatively benign to an aggressive, invasive malignancy. Coincident with the emergence of such cellular plasticity is an altered response to transforming growth factor-B (TGF-B) as well as epidermal growth factor (EGF) receptor amplification. TGF-B in the tumor microenvironment promotes invasive traits largely through reprogramming gene expression, which paradoxically supports matrix-disruptive as well as stabilizing processes. ras-transformed HaCaT II-4 keratinocytes undergo phenotypic changes typical of epithelial-to-mesenchymal transition, acquire a collagenolytic phenotype, and effectively invade collagen type 1 gels as a consequence of TGF-B1 + EGF stimulation in a three-dimensional physiologically relevant model system that monitors collagen remodeling. Enhanced collagen degradation was coupled to a significant increase in matrix metalloproteinase (MMP)-10 expression and involved a proteolytic axis composed of plasmin, MMP-10, and MMP-1. Neutralization of any one component in this cascade inhibited collagen gel lysis. Similarly, addition of plasminogen activator inhibitor type 1 (SERPINE1) blocked collagen degradation as well as the conversion of both proMMP-10 and proMMP-1 to their catalytically active forms. This study therefore identifies an important mechanism in TGF-B1 + EGF-initiated collagen remodeling by transformed human keratinocytes and proposes a crucial upstream role for plasminogen activator inhibitor type 1-dependent regulation in this event. [Cancer Res 2009;69(9):4081-91]
Serum-stimulation of quiescent (G 0 ) keratinocytes initiates a temporally regulated program of transcriptional activity required for G 0 /G 1 transit and subsequent entry into the proliferative cycle (Qi and Higgins, 2003). Expression profiling of such "activated" keratinocytes identified physiologically relevant subsets of cell cycle/growth state-regulated genes (Gromov et al., 2002; Gazel et al., 2003). Indeed, non-cycling human (HaCaT) keratinocytes express a differentiated (i.e., super-basal) genetic signature, whereas the serum-stimulated transcriptome approximates that of transient amplifying cells (Pivarcsi et al., 2001; Lemaitre et al., 2004). Clearly, the associated transcriptional responses dictate epidermal cell lineage commitments by impacting the expression of pathway-relevant genes (Banno et al., 2004; Lemaitre et al., 2004).This report provides early evidence regarding the comprehensive inventory of genes expressed by human HaCaT-II4 keratinocytes during the initial stage of cell-cycle re-entry. Reintroduction of serum to quiescent HaCaT-II4 cells stimulates G 0 exit and residence in a shortlived "activated G 0 substate" (i.e., the kinetically defined G 0 →G 1 transition state) (Qi et al., 2006). Microarray analysis of quiescent and 2 hours fetal bovine serum (FBS)-" activated" HaCaT-II4 cells defined the transcriptional signature of this early G 0 →G 1 window. A total of 54,675 expressed sequence-tagged genes were analyzed with 41,083 directly compared for groups A (quiescent) and B (2 hours FBS-stimulated) and a total of 35,991 reproducibly assessed for all three experimental conditions (i.e., 66% of the total sequences available; this includes group C [FBS for 2 hours in the presence of puromycin included as a first approximation of the immediate-early response cluster]). Genes exhibiting statistically significant (analysis of variance) changes (two-fold increase or decrease) distributed as follows: 1151 for A versus B, 1241 for A versus C, and 1319 for B versus C. Among the most prominently upregulated mRNA transcripts were those encoding proteins involved in the initial growth response (EGR1-4), extracellular matrix remodeling and tissue invasion (uPA,
Supplementary Video 2C from TGF-β1 + EGF-Initiated Invasive Potential in Transformed Human Keratinocytes Is Coupled to a Plasmin/MMP-10/MMP-1–Dependent Collagen Remodeling Axis: Role for PAI-1
<div>Abstract<p>The phenotypic switching called epithelial-to-mesenchymal transition is frequently associated with epithelial tumor cell progression from a comparatively benign to an aggressive, invasive malignancy. Coincident with the emergence of such cellular plasticity is an altered response to transforming growth factor-β (TGF-β) as well as epidermal growth factor (EGF) receptor amplification. TGF-β in the tumor microenvironment promotes invasive traits largely through reprogramming gene expression, which paradoxically supports matrix-disruptive as well as stabilizing processes. <i>ras</i>-transformed HaCaT II-4 keratinocytes undergo phenotypic changes typical of epithelial-to-mesenchymal transition, acquire a collagenolytic phenotype, and effectively invade collagen type 1 gels as a consequence of TGF-β1 + EGF stimulation in a three-dimensional physiologically relevant model system that monitors collagen remodeling. Enhanced collagen degradation was coupled to a significant increase in matrix metalloproteinase (MMP)-10 expression and involved a proteolytic axis composed of plasmin, MMP-10, and MMP-1. Neutralization of any one component in this cascade inhibited collagen gel lysis. Similarly, addition of plasminogen activator inhibitor type 1 (SERPINE1) blocked collagen degradation as well as the conversion of both proMMP-10 and proMMP-1 to their catalytically active forms. This study therefore identifies an important mechanism in TGF-β1 + EGF-initiated collagen remodeling by transformed human keratinocytes and proposes a crucial upstream role for plasminogen activator inhibitor type 1–dependent regulation in this event. [Cancer Res 2009;69(9):4081–91]</p></div>
Supplementary Figure 2 from TGF-β1 + EGF-Initiated Invasive Potential in Transformed Human Keratinocytes Is Coupled to a Plasmin/MMP-10/MMP-1–Dependent Collagen Remodeling Axis: Role for PAI-1
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