Different signaling and functionality of Rac1 and Rac1b in the progression of lung adenocarcinoma.

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 3ß (GSK3ß), 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 GSK3ß. 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.

CRISPR-Cas9-based target validation for p53-reactivating model compounds.

Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9-based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.