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Updated: Oct 10, 2025
Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells
Published on: October 27, 2020
Rab31-dependent regulation of transforming growth factor ß expression in breast cancer cells
Susanne Soelch1, Nathalie Beaufort2, Daniela Loessner3,4
1Clinical Research Unit, Department of Obstetrics and Gynecology, Technische Universität München, Ismaninger Str. 22, 81576, Munich, Germany.
Background:
The small GTP-binding protein Rab31 plays an important role in the modulation of tumor biological-relevant processes, including cell proliferation, adhesion, and invasion. As an underlying mechanism, Rab31 is presumed to act as a molecular switch between a more proliferative and an invasive phenotype. This prompted us to analyze whether Rab31 overexpression in breast cancer cells affects expression of genes involved in epithelial-to-mesenchymal transition (EMT)-like processes when compared to Rab31 low-expressing cells.
Methods:
Commercially available profiler PCR arrays were applied to search for differentially expressed genes in Rab31 high- and low-expressing CAMA-1 breast cancer cells. Differential expression of selected candidate genes in response to Rab31 overexpression in CAMA-1 cells was validated by independent qPCR and protein assays.
Results:
Gene expression profiling of key genes involved in EMT, or its reciprocal process MET, identified 9 genes being significantly up- or down-regulated in Rab31 overexpressing CAMA-1 cells, with the strongest effects seen for TGFB1, encoding TGF-ß1 (> 25-fold down-regulation in Rab31 overexpressing cells). Subsequent validation analyses by qPCR revealed a strong down-regulation of TGFB1 mRNA levels in response to increased Rab31 expression not only in CAMA-1 cells, but also in another breast cancer cell line, MDA-MB-231. Using ELISA and Western blot analysis, a considerable reduction of both intracellular and secreted TGF-ß1 antigen levels was determined in Rab31 overexpressing cells compared to vector control cells. Furthermore, reduced TGF-ß activity was observed upon Rab31 overexpression in CAMA-1 cells using a sensitive TGF-ß bioassay. Finally, the relationship between Rab31 expression and the TGF-ß axis was analyzed by another profiler PCR array focusing on genes involved in TGF-ß signaling. We found 12 out of 84 mRNAs significantly reduced and 7 mRNAs significantly increased upon Rab31 overexpression.
Conclusions:
Our results demonstrate that Rab31 is a potent modulator of the expression of TGF-ß and other components of the TGF-ß signaling pathway in breast cancer cells.
Insights
Rab31 overexpression in breast cancer cells significantly down-regulates TGF-ß1 expression and impacts TGF-ß signaling. This suggests Rab31 influences tumor invasion by modulating the TGF-ß pathway.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Rab31, a small GTP-binding protein, is implicated in tumor progression, including cell proliferation, adhesion, and invasion.
- Rab31 is hypothesized to function as a molecular switch regulating the transition between proliferative and invasive cellular phenotypes.
Purpose of the Study:
- To investigate the effect of Rab31 overexpression on gene expression related to epithelial-to-mesenchymal transition (EMT) in breast cancer cells.
- To determine if Rab31 influences the expression of genes within the transforming growth factor beta (TGF-ß) signaling pathway.
Main Methods:
- Differential gene expression analysis using PCR arrays in Rab31 high- and low-expressing breast cancer cell lines (CAMA-1, MDA-MB-231).
- Validation of gene and protein expression using quantitative PCR (qPCR), ELISA, and Western blot.
- Assessment of TGF-ß activity using a TGF-ß bioassay.
Main Results:
- Rab31 overexpression led to significant down-regulation of TGFB1 (encoding TGF-ß1) by over 25-fold in CAMA-1 cells, also observed in MDA-MB-231 cells.
- Reduced intracellular and secreted TGF-ß1 protein levels and decreased TGF-ß activity were detected in cells overexpressing Rab31.
- Analysis of TGF-ß signaling pathway genes revealed significant down-regulation of 12 mRNAs and up-regulation of 7 mRNAs upon Rab31 overexpression.
Conclusions:
- Rab31 acts as a significant modulator of TGF-ß1 expression in breast cancer.
- Rab31 influences the TGF-ß signaling pathway, suggesting a role in regulating tumor cell invasion and metastasis.
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