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Induction and Analysis of Epithelial to Mesenchymal Transition
Published on: August 27, 2013
Kinetics of v-src-induced epithelial-mesenchymal transition in developing glandular stomach
Y Shimizu1, N Yamamichi, K Saitoh
1Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Japan.
Abstract:
The oncogene function in primary epithelial cells is largely unclear. Recombination organ cultures in combination with the stable and transient gene transfer techniques by retrovirus and electroporation, respectively, enable us to transfer oncogenes specifically into primary epithelial cells of the developing avian glandular stomach (proventriculus). In this system, the epithelium and mesenchyme are mutually dependent on each other for their growth and differentiation. We report here that either stable or transient expression of v-src in the epithelium causes budding and migration of epithelial cells into mesenchyme. In response to the transient expression of v-Src or a constitutive active mutant of MEK, we observed immediate downregulation of the Sonic hedgehog gene and subsequent elimination of E-cadherine expression in migrating cells, suggesting the involvement of MAP kinase signaling pathway in these processes. v-src-expressing cells that were retained in the epithelium underwent apoptosis (anoikis) and detached from the culture. Continuous expression of v-src by, for example, Rous sarcoma virus (RSV) was required for the epithelial cells to acquire the ability to express type I collagen and fibronectin genes (mesenchymal markers), and finally to establish the epithelial-mesenchymal transition. These observations would partly explain why RSV does not apparently cause carcinoma formation, but induces sarcomas exclusively.
Insights
Oncogene v-src expression in primary epithelial cells triggers migration and mesenchymal marker expression. This process involves MAP kinase signaling and explains why Rous sarcoma virus induces sarcomas, not carcinomas.
Area of Science:
- Developmental Biology
- Cell Biology
- Oncogenesis
Background:
- The role of oncogenes in primary epithelial cells remains poorly understood.
- Investigating oncogene function requires specific gene transfer methods into distinct cell types.
Purpose of the Study:
- To elucidate the function of oncogenes in primary epithelial cells using a novel organ culture system.
- To investigate the molecular mechanisms underlying oncogene-induced epithelial-mesenchymal transition (EMT).
Main Methods:
- Utilized recombination organ cultures of developing avian glandular stomach (proventriculus).
- Employed retroviral and electroporation techniques for stable and transient gene transfer of oncogenes.
- Analyzed gene expression changes (Sonic hedgehog, E-cadherin, collagen I, fibronectin) and cellular behaviors (migration, apoptosis).
Main Results:
- Expression of v-src in epithelial cells induced budding and migration into the mesenchyme.
- Transient v-src or MEK activation led to Sonic hedgehog downregulation and E-cadherin loss, implicating MAP kinase signaling.
- Continuous v-src expression promoted acquisition of mesenchymal markers and established epithelial-mesenchymal transition (EMT).
Conclusions:
- Oncogene expression can reprogram epithelial cells, driving migration and EMT.
- The MAP kinase pathway plays a critical role in mediating oncogene-induced cellular changes.
- These findings provide insights into the distinct oncogenic outcomes of Rous sarcoma virus (RSV), explaining sarcoma formation.
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