Nir Ben-Chetrit

A reciprocal tensin-3-cten switch mediates EGF-driven mammary cell migration

Cell migration driven by the epidermal growth factor receptor (EGFR) propels morphogenesis and involves reorganization of the actin cytoskeleton. Although de novo transcription precedes migration, transcript identity remains largely unknown. Through their actin-binding domains, tensins link the cytoskeleton to integrin-based adhesion sites. Here we report that EGF downregulates tensin-3 expression, and concomitantly upregulates cten, a tensin family member that lacks the actin-binding domain. Knockdown of cten or tensin-3, respectively, impairs or enhances mammary cell migration. Furthermore, cten displaces tensin-3 from the cytoplasmic tail of integrin β1, thereby instigating actin fibre disassembly. In invasive breast cancer, cten expression correlates not only with high EGFR and HER2, but also with metastasis to lymph nodes. Moreover, treatment of inflammatory breast cancer patients with an EGFR/HER2 dual-specificity kinase inhibitor significantly downregulated cten expression. In conclusion, a transcriptional tensin-3–cten switch may contribute to the metastasis of mammary cancer.

Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC’s cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

EGR1 and the ERK-ERF axis drive mammary cell migration in response to EGF

The signaling pathways that commit cells to migration are incompletely understood. We employed human mammary cells and two stimuli: epidermal growth factor (EGF), which induced cellular migration, and serum factors, which stimulated cell growth. In addition to strong activation of ERK by EGF, and AKT by serum, early transcription remarkably differed: while EGF induced early growth response‐1 (EGR1), and this was required for migration, serum induced c‐Fos and FosB to enhance proliferation. We demonstrate that induction of EGR1 involves ERK‐mediated down‐regulation of microRNA‐191 and phosphorylation of the ETS2 repressor factor (ERF) repressor, which subsequently leaves the nucleus. Unexpectedly, knockdown of ERF inhibited migration, which implies migratory roles for exported ERF molecules. On the other hand, chromatin immunoprecipitation identified a subset of direct EGR1 targets, including EGR1 autostimulation and SERPINB2, whose transcription is essential for EGF‐induced cell migration. In summary, EGR1 and the EGF‐ERK‐ERF axis emerge from our study as major drivers of growth factor‐induced mammary cell migration.—Tarcic, G., Avraham, R., Pines, G., Amit, I., Shay, T., Lu, Y., Zwang, Y., Katz, M., Ben‐Chetrit, N., Jacob‐Hirsch, J., Virgilio, L., Rechavi, G., Mavrothalassitis, G., Mills, G. B., Domany, E., Yarden, Y. EGR1 and the ERK‐ERF axis drive mammary cell migration in response to EGF. FASEB J. 26, 1582‐1592 (2012). www.fasebj.org

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.

EGF induces microRNAs that target suppressors of cell migration: miR-15b targets MTSS1 in breast cancer

Growth factor–induced metastasis involves microRNA-mediated repression of a tumor suppressor. Micromanaging growth factor–induced metastasis Epidermal growth factor (EGF) stimulates cell proliferation and tumor growth in part by triggering kinase-dependent changes in gene expression. Noncoding RNAs, such as microRNAs (miRNAs), reduce gene expression by binding to protein-encoding transcripts. Kedmi et al. found that EGF stimulated migration in mammary epithelial cells and also increased the abundance of a set of miRNAs. Of these, miR-15b promoted EGF-induced migration and reduced the abundance of metastasis suppressor protein 1 (MTSS1). The expression of miR-15b was higher in aggressive tumors than in adjacent normal tissue and inversely correlated with that of MTSS1. Knockdown of MTSS1 promoted the migratory behavior and the formation of migration-associated structures in cultured cells. Low abundance of MTSS1 correlated with shorter survival in patients, and low expression of MTSS1 correlated with high expression of miR-15b in aggressive basal breast cancer tissue, suggesting that this pathway is important in breast cancer and could be targeted to reduce metastatic disease in patients. Growth factors promote tumor growth and metastasis. We found that epidermal growth factor (EGF) induced a set of 22 microRNAs (miRNAs) before promoting the migration of mammary cells. These miRNAs were more abundant in human breast tumors relative to the surrounding tissue, and their abundance varied among breast cancer subtypes. One of these miRNAs, miR-15b, targeted the 3′ untranslated region of MTSS1 (metastasis suppressor protein 1). Although xenografts in which MTSS1 was knocked down grew more slowly in mice initially, longer-term growth was unaffected. Knocking down MTSS1 increased migration and Matrigel invasion of nontransformed mammary epithelial cells. Overexpressing MTSS1 in an invasive cell line decreased cell migration and invasiveness, decreased the formation of invadopodia and actin stress fibers, and increased the formation of cellular junctions. In tissues from breast cancer patients with the aggressive basal subtype, an inverse correlation occurred with the high expression of miRNA-15b and the low expression of MTSS1. Furthermore, low abundance of MTSS1 correlated with poor patient prognosis. Thus, growth factor–inducible miRNAs mediate mechanisms underlying the progression of cancer.

Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR’s positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR’s feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

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 tumors microenvironment.

Tumor cell invasion can be blocked by modulators of collagen fibril alignment that control assembly of the extracellular matrix

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.

Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer.

Small-molecule inhibitors of the lipid phosphatase synaptojanin 2 may prevent breast cancer metastasis. Blocking Receptor Recycling to Prevent Metastasis Blocking cancer cell metastasis can prolong patient survival. Ben-Chetrit et al. found that many patients with aggressive breast cancer have tumors with increased expression of SYNJ2, which encodes the lipid phosphatase synaptojanin 2. In cultured breast cancer cells, epidermal growth factor (EGF) triggered the localization of SYNJ2 to lamellipodia and invadopodia, which are cellular protrusions associated with invasive behavior. Knocking down SYNJ2 inhibited recycling of the EGF receptor to the cell surface and decreased the invasive behavior of cultured breast cancer cells. Expressing a phosphatase-deficient mutant of SYNJ2 in xenografted breast cancer cells suppressed tumor growth and lung metastasis in mice. A chemical screen identified SYNJ2 inhibitors that reduced cell invasion through a 3D matrix, suggesting that targeting SYNJ2 may prevent metastasis in breast cancer patients. Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5′-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

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.