Deubiquitination of EGFR by Cezanne-1 contributes to cancer progression.

Once stimulated, the epidermal growth factor receptor (EGFR) undergoes self-phosphorylation, which, on the one hand, instigates signaling cascades, and on the other hand, recruits CBL ubiquitin ligases, which mark EGFRs for degradation. Using RNA interference screens, we identified a deubiquitinating enzyme, Cezanne-1, that opposes receptor degradation and enhances EGFR signaling. These functions require the catalytic- and ubiquitin-binding domains of Cezanne-1, and they involve physical interactions and transphosphorylation of Cezanne-1 by EGFR. In line with the ability of Cezanne-1 to augment EGF-induced growth and migration signals, the enzyme is overexpressed in breast cancer. Congruently, the corresponding gene is amplified in approximately one third of mammary tumors, and high transcript levels predict an aggressive disease course. In conclusion, deubiquitination by Cezanne-1 curtails degradation of growth factor receptors, thereby promotes oncogenic growth signals.

A recombinant decoy comprising EGFR and ErbB-4 inhibits tumor growth and metastasis.

Epidermal growth factor (EGF)-like growth factors control tumor progression as well as evasion from the toxic effects of chemotherapy. Accordingly, antibodies targeting the cognate receptors, such as EGFR/ErbB-1 and the co-receptor HER2/ErbB-2, are widely used to treat cancer patients, but agents that target the EGF-like growth factors are not available. To circumvent the existence of 11 distinct ErbB ligands, we constructed a soluble fusion protein (hereinafter: TRAP-Fc) comprising truncated extracellular domains of EGFR/ErbB-1 and ErbB-4. The recombinant TRAP-Fc retained high-affinity ligand binding to EGF-like growth factors and partially inhibited growth of a variety of cultured tumor cells. Consistently, TRAP-Fc displayed an inhibitory effect in xenograft models of human cancer, as well as synergy with chemotherapy. Additionally, TRAP-Fc inhibited invasive growth of mammary tumor cells and reduced their metastatic seeding in the lungs of animals. Taken together, the activities displayed by TRAP-Fc reinforce critical roles of EGF-like growth factors in tumor progression, and they warrant further tests of TRAP-Fc in preclinical models.

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions.

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients' lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment.

Combining epitope-distinct antibodies to HER2: cooperative inhibitory effects on invasive growth.

Monoclonal antibodies (mAbs) to HER2 are currently used to treat breast cancer, but low clinical efficacy, along with primary and acquired resistance to therapy, commonly limit clinical applications. We previously reported that combinations of antibodies directed at non-overlapping epitopes of HER2 are endowed with enhanced antitumor effects, probably due to accelerated receptor degradation. Here, we extend these observations to three-dimensional mammary cell models, and compare the effects of single mAbs with the effects of antibody combinations. Collectively, our in vitro assays and computational image analyses indicate that combining mAbs against different epitopes of HER2 better inhibits invasive growth. Importantly, while growth factors are able to reduce intraluminal apoptosis and induce an invasive phenotype, combinations of mAbs better than single mAbs can reverse the growth factor-induced phenotypes of HER2-overexpressing spheroids. In conclusion, our studies propose that mAb combinations negate the biological effects of growth factors on invasive growth of HER2-overexpressing cells. Hence, combining mAbs offers a therapeutic strategy, potentially able to enhance clinical efficacy of existing antireceptor immunotherapeutics.