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Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis
Published on: May 16, 2020
Expression of the FGFR2c mesenchymal splicing variant in human keratinocytes inhibits differentiation and promotes
Danilo Ranieri1, Benedetta Rosato1, Monica Nanni1
1Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Clinical and Molecular Medicine, Sapienza University of Rome, Roma, Lazio, Italy.
Abstract:
The altered isoform switching of the fibroblast growth factor receptor 2 (FGFR2) and aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells is involved in cancer progression. We have recently described that the ectopic expression of FGFR2c in normal human keratinocytes induces epithelial-mesenchymal transition and leads to invasiveness and anchorage-independent growth. Here, we extended our analysis to the effects of this FGFR2c forced expression on human keratinocyte differentiation and stratification. Our findings demonstrated that, differently from cells overexpressing the epithelial splicing variant FGFR2b, keratinocytes ectopically expressing FGFR2c are not able to form a monolayer and display decreased expression of early differentiation markers. This impaired ability to enter the differentiation program is related to the up-modulation of the transcription factor ΔNp63. In addition, FGFR2c-expressing keratinocytes undergo defective stratification and invasion of the collagen matrix in 3D organotypic cultures, further suggesting their tumorigenic potential. Taken together, our results support the hypothesis that the receptor switching and the consequent appearance of the mesenchymal FGFR2c variant in the epithelial context would drive early steps of carcinogenesis, unbalancing the p63/FGFR interplay, and altering the paracrine response to the microenvironment.
Insights
Forced expression of fibroblast growth factor receptor 2c (FGFR2c) in keratinocytes impairs differentiation and stratification, promoting early carcinogenesis by disrupting the p63/FGFR balance.
Area of Science:
- Cell Biology
- Cancer Research
- Molecular Biology
Background:
- Altered fibroblast growth factor receptor 2 (FGFR2) isoform switching, particularly FGFR2c in epithelial cells, is linked to cancer progression.
- Ectopic FGFR2c expression in keratinocytes induces epithelial-mesenchymal transition, invasiveness, and anchorage-independent growth.
Purpose of the Study:
- To investigate the effects of forced FGFR2c expression on human keratinocyte differentiation and stratification.
- To elucidate the role of FGFR2c in early carcinogenesis and its interplay with transcription factors like ΔNp63.
Main Methods:
- Forced expression of FGFR2c in human keratinocytes.
- Analysis of keratinocyte monolayer formation and differentiation markers.
- 3D organotypic cultures to assess stratification and invasion.
- Western blot analysis for transcription factor expression (ΔNp63).
Main Results:
- Keratinocytes expressing FGFR2c failed to form a monolayer and showed reduced early differentiation markers compared to FGFR2b-expressing cells.
- FGFR2c expression led to increased ΔNp63 levels, correlating with impaired differentiation.
- FGFR2c-expressing keratinocytes exhibited defective stratification and matrix invasion in 3D cultures, indicating tumorigenic potential.
Conclusions:
- The aberrant expression of FGFR2c in epithelial cells disrupts normal keratinocyte differentiation and stratification, contributing to early carcinogenesis.
- The FGFR2c-driven carcinogenesis involves an imbalance in the p63/FGFR signaling pathway and altered microenvironment responses.
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