Abstract
BackgroundThe 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.MethodsCommercially 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.ResultsGene 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.ConclusionsOur 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.
Highlights
The small Guanosine triphosphate (GTP)-binding protein Rab31 plays an important role in the modulation of tumor biologicalrelevant processes, including cell proliferation, adhesion, and invasion
Our results demonstrate that Rab31 is a potent modulator of the expression of Transforming growth factor-ß (TGF-ß) and other components of the TGF-ß signaling pathway in breast cancer cells
Rab31 overexpression in CAMA‐1 breast cancer cells leads to increased proliferation of microtumor‐like spheroids The effects of Rab31 overexpression were analyzed in a 3D spheroid model, which more closely mimics the in vivo tumor microenvironment compared to classical 2D cell monolayer cultures
Summary
The small GTP-binding protein Rab plays an important role in the modulation of tumor biologicalrelevant processes, including cell proliferation, adhesion, and invasion. Rab ( known as rab22B) belongs to the Rab-II supergroup, which encompasses 9 human members It is ubiquitously expressed in normal human tissue (Bao et al 2002; Kloepper et al 2012) and is mainly localized in the trans-Golgi network and endosomes (Bao et al 2002; Ng et al 2007; Rodriguez-Gabin et al 2001). In these cellular compartments, it regulates vesicle transport from the Golgi apparatus to early and late endosomes, e.g. the transport of mannose-phosphate receptors (RodriguezGabin et al 2009), or in retrograde transport from the plasma membrane into late endosomes, e.g. in case of the EGF receptor (EGFR) (Chua and Tang 2015). Contrariwise, Rab overexpression enhances EGFR trafficking to late endosomes resulting in an enhanced degradation of the receptor (Chua and Tang 2015)
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