EMT, Signaling Pathway & Stemness FAQs

Find scientific technical answers on frequently asked questions (FAQs):

1. What is EMT and how is it significant in biosciences?
2. How is EMT different from de-differentiation of the cultured cells?
3. What are the key signaling pathways involved in EMT regulation?
4. How do transcription factors regulate EMT?
5. What are the most important EMT markers?
6. Is it possible to perform a quantitative analysis of EMT?
7. Can you suggest any preliminary in vitro assays for detecting EMT before I study the molecular markers?
8. How can I analyze EMT in cancer xenograft tissues from control and drug-treated mice?
9. I am running IHC on frozen tissue sections to determine alpha-SMA expression. Do I need to perform antigen retrieval?
10. How can I increase the expression of E-cadherin and ZEB1/ZEB2 in my Western blots?
11. What is an ideal housekeeping gene/protein for use as a control in EMT studies?
12. Do you offer any EMT-inducing cell culture medium?
13. Does Bio-Techne offer any small molecules for induction of stem cell differentiation?
14. Why do cancer cells undergo EMT and acquire increased stemness?
15. What are cancer-associated fibroblasts (CAFs) and how do they affect EMT?
16. How does hypoxia impact EMT in cancers?
17. How do exosomes facilitate EMT in cancers?
18. Is there any link between autophagy and EMT or stemness?

What is EMT and how is it significant in biosciences?

Epithelial-Mesenchymal Transition (EMT) refers to the trans-differentiation of stationary epithelial cells into motile mesenchymal cells. Major changes which occur during EMT are the down-regulation of epithelial markers (such as E-Cadherin, TJP1/ZO-1 and Occludin), rearrangement of the cytoskeleton, loss of cell-cell adhesion and apical-basal polarity, and the acquisition of mesenchymal phenotype markers (such as Vimentin, N-Cadherin and Fibronectin). These mesenchymal proteins confer increased cell protrusions and motility/migratory potential of the differentiated cells. EMT was initially described by Elizabeth Hay in the 1980s as ‘epithelial to mesenchymal transformation’. However, because of its transient or transitional nature, this process was renamed later on as “epithelial-mesenchymal transition”. On the other hand, Mesenchymal-Epithelial Transition (MET) is the reverse process (of EMT) which results in the differentiation of motile mesenchymal cells to stationary epithelial cell type. A coordinated interplay between EMT and MET signals is fundamental to a wide range of biological processes including primary/secondary epithelia generation during embryogenesis/organogenesis, wound healing, and also to the pathophysiology of fibrosis and malignancies. In cancer, EMT drives invasion, therapeutic resistance, stemness and cancer spread to distant sites in the body (Stone et al. 2016; Nieto et al. 2016).

How do transcription factors regulate EMT?

At the transcriptional control level, EMT is driven by the factors: SNAI1, ZEB and basic helix–loop–helix (bHLH) proteins. These factors repress epithelial marker genes and activate genes associated with the mesenchymal phenotype. The activities of these transcription factors are regulated through their post-translational modifications, subcellular localization, and stability. Specifically, GSK-3 beta and/or PKD1 (PKC mu) phosphorylates SNAI1 which leads to its nuclear export followed by degradation via the ubiquitin (Ub)-conjugation system. SNAI1’s nuclear retention and signaling activity are promoted through its phosphorylation by PAK1 or LATS2, or its de-phosphorylation by small C-terminal domain phosphatase1 (SCP1). SNAI2/Slug, a transcriptional repressor of E-Cadherin/CDH1, on the other hand gets degraded upon p53-mediated recruitment to the MDM2 complex. Transcriptional regulator TWIST’s nuclear translocation/activity is controlled through its phosphorylation by MAPK p38, JNK and ERK. ZEB2, another transcriptional inhibitor of CDH, gets sumoylated by PRC2 and this modification leads to its translocation into the cytoplasm which reduces its activity (Lamouille et al. 2014). The genes/proteins which get repressed (down-arrow) or up-regulated (up-arrow) through transcriptional regulators of EMT are shown below.

Is it possible to perform a quantitative analysis of EMT?

Yes, one of the most commonly employed approaches is to quantify changes in the expression of molecular markers responsible for epithelial and mesenchymal phenotypes. WB, Flow cytometry, ICC/IF, and IHC are some of the assays which are useful for immuno-analysis of EMT markers. It is advisable to include 2-4 targets for both the epithelial markers (E-cadherin, pan-cytokeratin, TJP1/ZO-1, laminin-1, COL4A1, MUC1, etc.) and mesenchymal markers (N-cadherin, Vimentin, Tenascin C, alpha-SMA, F-actin, SPARC, etc). For mechanistic studies, regulators of EMT, such as SIP1, SNAI1/Snail, SNAI2/Slug, Twist1, FOXC2, and ZEB1/2 may also be analyzed.

Can you suggest any preliminary in vitro assays for detecting EMT before I study the molecular markers?

There are some assays that you can use to study the migratory and invasive potential of cells which is linked to the mesenchymal character. The most commonly used assays to assess EMT are: wound healing assays, migration chamber assays, matrigel invasion assays, gelatin zymography, and aggregation and adhesion assays. Spindle Index analysis is a relatively new assay which involves the analysis of the ratio of maximum length to maximum width of cells.

I am running IHC on frozen tissue sections to determine alpha-SMA expression. Do I need to perform antigen retrieval?

It is a common misconception that antigen retrieval is optional when performing IHC on frozen sections. Actually, the requirement for an antigen retrieval step in immunostaining protocols is primarily dependent upon the nature of the fixative used and the fixation time. In situations wherein the tissues are fixed in formalin/para-formaldehyde solution for more than a few hours, an antigen retrieval step is mandatory before probing the sections with primary antibody. The underlying reason is that formalin cross-links the antigenic proteins in tissues or cells hindering the antigen-antibody reaction. Without antigen retrieval, protein expression may appear low or negligible in formalin fixed tissues. For frozen sections from tissues fixed for >4 hours in formalin, we generally recommend antigen retrieval with 10 mM sodium citrate buffer (pH 6.0) for 5-10 minutes at 90 - 100 °C.

What is an ideal housekeeping gene/protein for use as a control in EMT studies?

Because EMT is a process which involves critical changes in the molecular expression, as well as the structural organization of various proteins in the cells, some researchers find it concerning to use structural proteins such as beta-Actin or CD44 as controls in immuno-blotting or immuno-staining analyses. GAPDH is a protein that is commonly used as a control in EMT studies, however, the expression of this protein may change with hypoxia which makes it unsuitable as a control when working on hypoxic samples. Tubulin, PARP, or other proteins, such as total-EGFR and pan-AKT, are some other options which are useful for loading control purpose. When working on Western blots, staining of the blots with Ponceau S or Amido black stain may also be used to normalize the protein.

Does Bio-Techne offer any small molecules for induction of stem cell differentiation?

We provide a large range of small molecules which are useful for differentiating stem cells into various cell types.
 

Why do cancer cells undergo EMT and acquire increased stemness?

A growing tumor creates a hypoxic microenvironment to promote its own growth. It does this through the secretion of stress-related nutrients and growth factors, which prevent immune recognition and destruction. In response to the tumor microenvironment, cancer cells undergo EMT and acquire a mesenchymal phenotype and stem cell-like features. A mesenchymal or stemness-rich phenotype (cancer stem cells/CSCs) promotes tumor cell evasion and invasion/metastasis of secondary sites which are rich in nutrients, growth factors, and oxygen.

What are cancer-associated fibroblasts (CAFs) and how do they affect EMT?

In solid tumors, CAFs form a major component of the tumor stroma. Cancer cells activate the normal stromal fibroblasts (NSFs) into CAFs via the stimulation of paracrine growth factors. CAFs play a critical role in tumor growth and angiogenesis, and they are known to stimulate cancer cell invasiveness via secretion of various cytokines/factors, such as TGF-β1, PDGF, TNFα and IL-6. In comparison to NSFs, CAFs are more effective in promoting cancer cell survival, growth and progression. Importantly, some of the growth factors released from CAFs, especially TGF-β1 and CXCL12/SDF-1, are well-known EMT inducers in cancer cells (Zhuang et al. 2015).

How does hypoxia impact EMT in cancers?

Inadequate or low levels of oxygen, also known as hypoxia, is a hallmark of tumor generation. Hypoxia in the tumor microenvironment develops in growing tumors as a pathophysiologic outcome of disrupted microcirculation. Hypoxia induces the expression of hypoxia-inducing factors (HIFs), and through their target genes, HIFs regulate several signaling proteins and pathways, which in turn regulate tumor growth, metabolism, angiogenesis, EMT and stemness, and metastasis. HIF-1 alpha, the master regulator of hypoxia, directly or indirectly controls the expression of several transcriptional regulators of EMT (Twist1, Snail, Slug, ZEB1/2, and E12/E47) and of pro-EMT/stemness proteins, including TGF-beta, beta-catenin, Wnt and Notch (Kao et al. 2016).

How do exosomes facilitate EMT in cancers?

Exosomes are small membrane-enclosed vesicles which are generated during the maturation of endosomes as intra-luminal vesicles of multi-vesicular bodies. Exosomes are released into the extracellular environment via exocytosis and are now known to act as extracellular signalosomes, facilitating intercellular communication. In cancers, tumor-derived exosomes (TDEs) carry pro-EMT molecules such as TGF-β, caveolin-1, HIF-1α, and β-catenin. Upon reaching the recipient cells, these molecules trigger the generation of mesenchymal phenotype, stromal remodeling, and the formation of the pre-metastatic niche (Syn et al. 2016).