Abstract
Rac1 is a ubiquitously expressed Rho GTPase and an important regulator of the actin cytoskeleton. Its splice variant Rac1b exhibits a 19-amino acid (aa) in-frame insertion and is predominantly active. Both proteins were described in tumorigenesis or metastasis. We investigated the contribution of Rac1 and Rac1b to tumor progression of human non-small-cell lung adenocarcinoma (NSCLA). Rac1 protein was present in 8/8 NSCLA cell lines analyzed, whereas Rac1b was expressed in only 6/8. In wound-healing assays, enhanced green fluorescence protein (EGFP)-Rac1 slightly decreased cell migration, whereas proliferation was increased in both, Rac1- and Rac1b-expressing cells. In the in vivo chorioallantoic invasion model, EGFP-Rac1-expressing cells formed more invasive tumors compared to EGFP-Rac1b. This increased invasiveness correlated with enhanced phosphorylation of p38α, AKT and glycogen synthase kinase 3β (GSK3β), and activation of serum response- and Smad-dependent gene promoters by Rac1. In contrast, Rac1b solely activated the mitogen-activated protein kinase (MAPK) JNK2, together with TCF/LEF1- and nuclear factor kappa B (NFκB)-responsive gene reporters. Rac1b, as Rac1, phosphorylated p38α, AKT and GSK3β. Knockdown of the splicing factor epithelial splicing regulatory protein 1 (ESRP1), which mediates out-splicing of exon 3b from Rac1 pre-messenger RNA, resulted in increased Rac1b messenger RNA (mRNA) and suppression of the epithelial-mesenchymal transition (EMT)-associated transcription factor ZEB1. Our data demonstrate different signaling and functional activities of Rac1 and Rac1b and an important role for Rac1 in lung cancer metastasis.
Highlights
The small GTPases of the Rho family, namely Rho, Rac, Cdc42 and its isoforms are well-known regulators of the actin cytoskeleton dynamics and cellular migration, as well as transducers of multiple signals in a variety of oncogenic pathways (Wolpert et al, 2000; Hall, 2005)
Most assays were repeated with enhanced green fluorescence protein (EGFP)-Rac1/1b-transfected HCC44 lung adenocarcinoma cells (Supplementary Figures S1, S2) with results comparable to those obtained with H23 cells
We provide evidence that enhanced amounts of the GTPase Rac1, in contrast to its closely related isoform Rac1b, contribute to progression of lung adenocarcinoma by increasing tumor invasion, whereas both Rac isoforms led to increased proliferation of H23 and HCC44 lung adenocarcinoma cells
Summary
The small GTPases of the Rho family, namely Rho, Rac, Cdc and its isoforms are well-known regulators of the actin cytoskeleton dynamics and cellular migration, as well as transducers of multiple signals in a variety of oncogenic pathways (Wolpert et al, 2000; Hall, 2005). The splice isoform Rac1b, which was first described in 1999 (Jordan et al, 1999; Schnelzer et al, 2000), includes an additional exon 3b immediately behind the switch II region comprising a 19-amino acid (aa) in-frame insertion. This insertion resulted in a strong increase in its nucleotide exchange rate and a delayed GTP hydrolysis (Fiegen et al, 2004). Unlike Rac, Rac1b does not associate with GDP dissociation inhibitors (GDIs), further
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