Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-ß signalling.

In advanced cancers, the TGF-ß pathway acts as an oncogenic factor and is considered to be a therapeutic target. Here using a genome-wide cDNA screen, we identify nuclear receptor NR4A1 as a strong activator of TGF-ß signalling. NR4A1 promotes TGF-ß/SMAD signalling by facilitating AXIN2-RNF12/ARKADIA-induced SMAD7 degradation. NR4A1 interacts with SMAD7 and AXIN2, and potently and directly induces AXIN2 expression. Whereas loss of NR4A1 inhibits TGF-ß-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-ß-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-ß-mediated breast cancer cell migration, invasion and metastasis in vitro and in vivo. Notably, NR4A1 expression is elevated in breast cancer patients with high immune infiltration and its expression weakly correlates with phosphorylated SMAD2 levels, and is an indicator of poor prognosis. Our results uncover inflammation-induced NR4A1 as an important determinant for hyperactivation of pro-oncogenic TGF-ß signalling in breast cancer.

An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy.

The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings.

Treatment with anti-gremlin 1 antibody ameliorates chronic hypoxia/SU5416-induced pulmonary arterial hypertension in mice.

The expression of the bone morphogenetic protein antagonist, Gremlin 1, was recently shown to be increased in the lungs of pulmonary arterial hypertension patients, and in response to hypoxia. Gremlin 1 released from the vascular endothelium may inhibit endogenous bone morphogenetic protein signaling and contribute to the development of pulmonary arterial hypertension. Here, we investigate the impact of Gremlin 1 inhibition in disease after exposure to chronic hypoxia/SU5416 in mice. We investigated the effects of an anti-Gremlin 1 monoclonal antibody in the chronic hypoxia/SU5416 murine model of pulmonary arterial hypertension. Chronic hypoxic/SU5416 exposure of mice induced upregulation of Gremlin 1 mRNA in lung and right ventricle tissue compared with normoxic controls. Prophylactic treatment with an anti-Gremlin 1 neutralizing mAb reduced the hypoxic/SU5416-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy. Importantly, therapeutic treatment with an anti-Gremlin 1 antibody also reduced pulmonary vascular remodeling and right ventricular hypertrophy indicating a role for Gremlin 1 in the progression of the disease. We conclude that Gremlin 1 plays a role in the development and progression of pulmonary arterial hypertension in the murine hypoxia/SU5416 model, and that Gremlin 1 is a potential therapeutic target for pulmonary arterial hypertension.

TRAF4 promotes TGF-ß receptor signaling and drives breast cancer metastasis.

TGF-ß signaling is a therapeutic target in advanced cancers. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a key component mediating pro-oncogenic TGF-ß-induced SMAD and non-SMAD signaling. Upon TGF-ß stimulation, TRAF4 is recruited to the active TGF-ß receptor complex, where it antagonizes E3 ligase SMURF2 and facilitates the recruitment of deubiquitinase USP15 to the TGF-ß type I receptor (TßRI). Both processes contribute to TßRI stabilization on the plasma membrane and thereby enhance TGF-ß signaling. In addition, the TGF-ß receptor-TRAF4 interaction triggers Lys 63-linked TRAF4 polyubiquitylation and subsequent activation of the TGF-ß-activated kinase (TAK)1. TRAF4 is required for efficient TGF-ß-induced migration, epithelial-to-mesenchymal transition, and breast cancer metastasis. Elevated TRAF4 expression correlated with increased levels of phosphorylated SMAD2 and phosphorylated TAK1 as well as poor prognosis among breast cancer patients. Our results demonstrate that TRAF4 can regulate the TGF-ß pathway and is a key determinant in breast cancer pathogenesis.

USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-ß type I receptor.

The stability and membrane localization of the transforming growth factor-ß (TGF-ß) type I receptor (TßRI) determines the levels of TGF-ß signalling. TßRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-ß signalling. USP4 was found to directly interact with TßRI and act as a deubiquitylating enzyme, thereby controlling TßRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-ß-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TßRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-ß and AKT signalling pathways.

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation.

TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.

PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta.

Engagement of the immunoinhibitory receptor, programmed death-1 (PD-1) attenuates T-cell receptor (TCR)-mediated activation of IL-2 production and T-cell proliferation. Here, we demonstrate that PD-1 modulation of T-cell function involves inhibition of TCR-mediated phosphorylation of ZAP70 and association with CD3zeta. In addition, PD-1 signaling attenuates PKCtheta activation loop phosphorylation in a cognate TCR signal. PKCtheta has been shown to be required for T-cell IL-2 production. A phosphorylated PD-1 peptide, corresponding to the C-terminal immunoreceptor tyrosine-switch motif (ITSM), acts as a docking site in vitro for both SHP-2 and SHP-1, while the phosphorylated peptide containing the N-terminal PD-1 immunoreceptor tyrosine based inhibitory motif (ITIM) associates only with SHP-2.