The cysteine protease cathepsin B (CTSB) is frequently overexpressed in human breast cancer and correlated with a poor prognosis. Genetic deficiency or pharmacological inhibition of CTSB attenuates tumor growth, invasion and metastasis in mouse models of human cancers. CTSB is expressed in both cancer cells and cells of the tumor stroma, in particular in tumor-associated macrophages (TAM). In order to evaluate the impact of tumor- or stromal cell-derived CTSB on Polyoma Middle T (PyMT)-induced breast cancer progression, we used in vivo and in vitro approaches to induce human CTSB overexpression in PyMT cancer cells or stromal cells alone or in combination. Orthotopic transplantation experiments revealed that CTSB overexpression in cancer cells rather than in the stroma affects PyMT tumor progression. In 3D cultures, primary PyMT tumor cells showed higher extracellular matrix proteolysis and enhanced collective cell invasion when CTSB was overexpressed and proteolytically active. Coculture of PyMT cells with bone marrow-derived macrophages induced a TAM-like macrophage phenotype in vitro, and the presence of such M2-polarized macrophages in 3D cultures enhanced sprouting of tumor spheroids. We employed a doxycycline (DOX)-inducible CTSB expression system to selectively overexpress human CTSB either in cancer cells or in macrophages in 3D cocultures. Tumor spheroid invasiveness was only enhanced when CTSB was overexpressed in cancer cells, whereas CTSB expression in macrophages alone did not further promote invasiveness of tumor spheroids. We conclude that CTSB overexpression in the PyMT mouse model promotes tumor progression not by a stromal effect, but by a direct, cancer cell-inherent mode of action: CTSB overexpression renders the PyMT cancers more invasive by increasing proteolytic extracellular matrix protein degradation fostering collective cell invasion into adjacent tissue.

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.

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.