A number of growth factors and cytokines can induce an Epithelial-Mesenchymal Transition (EMT) in tumor cells. These factors may be secreted by the tumor cells themselves, or by the stromal cells in the tumor microenvironment. These soluble ligands interact with their cognate receptors (e.g., tyrosine kinase receptors (RTKs), TGF-β receptors) leading to the activation of intracellular signaling pathways that induce EMT via the upregulation of selected zinc finger (e.g., SNAI1, SLUG, ZEB1, ZEB2) or basic helix-loop-helix (bHLH, e.g., TWIST1) transcription factors.
The TGF-β signaling pathway is strongly implicated in the process of EMT induction, despite paradoxically functioning as a tumor suppressor in some contexts. Canonical TGF-β/BMP signaling is initiated via the binding of ligands (e.g., TGF-β1, TGF-β2, BMPs) to their cognate Type I and Type II kinase receptors, forming a heterotetrameric ligand-receptor complex that recruits and phosphorylates receptor-regulated SMADs (R-SMADs). Phosphorylated R-SMADs (R-SMAD1/5/8 for BMP signaling; R-SMAD2/3 for TGF-β signaling) bind to a common mediator co-SMAD (SMAD4), which induces translocation of the SMAD complex to the nucleus, where it drives the expression of target genes involved in EMT (e.g., SNAIL, SLUG, etc.). Selected proteins (e.g., SARA) promote pathway activation, whereas others serve an inhibitory function. Foremost among the latter are inhibitory SMADs (SMAD6/7), which directly inhibit R-SMAD phosphorylation, thereby attenuating the induction of EMT. SMURF proteins (SMURF1/2) can promote this inhibition, by recruiting SMAD7 to the plasma membrane, enabling its competition with R-SMADS for receptor binding.
EMT may also be induced by the activation of non-canonical TGF-β signaling pathways. For example, oncogenic RTK activation of PI3K and Ras/Raf signaling pathways in conjunction with TGF-β signaling have been considered central features of cancer EMT. The Ras/Raf pathway promotes transcriptional activation of key EMT promoting genes while the PI3K pathway suppresses GSK3β-mediated phosphorylation of β-catenin, allowing β-catenin-mediated transcription of EMT target genes. The TGFR also activates p38 MAPK leading to activation of the EMT transcription factor FOXC2.
One of the hallmark features of EMT is the down-regulation of E-cadherin, a critical constituent of the adherens junctions that maintain epithelial integrity. EMT-associated transcription factors (e.g. SNAIL) repress the expression of E-Cadherin, while concomitantly inducing the expression of matrix metalloproteinases (MMPs) that degrade epithelial E-Cadherin, leading to the hallmark loss of epithelial integrity that defines EMT. Degradation of E-cadherin also releases β-catenin from the adherens junctions, which can translocate to the nucleus to activate oncogenic Wnt target genes.
Aberrant Wnt signaling is a prominent feature of several cancers particularly colorectal cancers, 90% of which exhibit overexpression of β-catenin. In the absence of Wnt signaling, unbound β-catenin in the cytoplasm is rapidly phosphorylated and ubiquitinated by the β-catenin destruction complex, marking β-catenin for proteasomal degradation. Upon Wnt binding to the Frizzled receptor, the β-catenin destruction complex is inhibited through the action of GSK3β and the β-catenin translocates to the nucleus, where it displaces the Groucho/HDAC inhibition complex to activate TCF/LEF mediated transcription of EMT effectors (e.g., SNAIL, N-Cadherin). Wnt/β-catenin mediated EMT has been demonstrated in a number of cancers (osteosarcoma, gastric, prostate). In addition to activating expression of EMT effectors, LEF1 is also involved in the expression of micro RNAs (miRNAs) implicated in the promotion of EMT. Additional factors also serve to regulate WNT/β-catenin/LEF activity, such as SRY-Box 10 (SOX 10), which inhibits EMT effector transcription by competing with β-catenin for binding to TCF/LEF.
Wnt5A and Wnt5B bind to the Frizzled2 (Fzd2) receptor to induce EMT through activation of the non-canonical STAT and MEK/ERK signaling pathways that have been shown to be elevated in metastatic liver, lung, colon, and breast cancer cell lines and high-grade tumors. Wnt5A/Frizzled signaling has also been shown to induce EMT via other non-canonical Wnt signaling pathways, which include JNK signaling in pancreatic carcinoma and PKC signaling in melanoma, and in both pathways Wnt5a expression also increased cellular motility. Consistent with the increased motility, WNT5a-mediated PKC signaling has also been shown to promote filamin A expression and processing, which in turn induces actin remodeling and stress fiber formation, processes required for motility in melanoma cell lines. In contrast to canonical Wnt signaling, Wnt5a expression is down-regulated in most colon carcinoma tumors and cell lines and negatively correlates with EMT markers while tumors with high Wnt5A expression were shown to exhibit increased intracellular calcium and non-canonical Wnt signaling, reduced EMT, cellular motility and invasiveness. Overexpression of Wnt5a in colon carcinoma cell lines with low basal levels of Wnt5A was shown to elevate calcium ion levels and inhibit EMT marker expression, cellular motility and proliferation while activating PKC and CaMKII activities, preventing the nuclear localization of β-catenin and abrogating EMT effector expression (e.g., TWIST, ZEB1).
NOTCH signaling is a juxtracine signaling pathway that involves the activation of NOTCH receptors (NOTCH1-4) by ligands (Jagged 1/2, DLL1/3/4) expressed on the surface of adjacent cells. Ligand binding leads to proteolytic cleavage of Notch by the ADAM/TACE protease followed by γ-secretase cleavage and release of the NOTCH intracellular domain (NICD) which translocates to the nucleus and regulates the transcription of downstream target genes. In the nucleus, NICD displaces the transcriptional repressor, KDM5A from a transcription factor complex, thereby inducing the expression of target genes that can promote EMT. Although Notch signaling is most commonly associated with T cell cancers (e.g. T-ALL) a Notch-driven EMT-like process has been described in selected epithelial cancers, including prostate and pancreatic carcinomas.
We would like to thank Dr. Dimiter Avtanski, Director, Endocrine Research Laboratory, Friedman Diabetes Institute at Lenox Hill Hospital, Northwell Health, for reviewing this diagram.
