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  • Cellular signal transduction and cancer metastasis

Metastatic disease is largely incurable because of its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents. To colonize distant organs, circulating tumor cells must overcome many obstacles, including surviving in circulation, infiltrating distant tissues, evading immune defenses, adapting to supportive niches, surviving as latent tumor-initiating seeds and eventually breaking out to replace the host tissue. Metastasis is a highly inefficient process and the mechanisms are poorly understood. Mutiple cellular signaling are involved in all these metastatic processes. We are searching for mechanisms of metastasis and the therapeutic methods of preventing it from occurence. 

  • Exosome in human diseases 

Exosome are cell-derived vesicles that are present in many and perhaps all eukaryotic fluids, including blood, urine, and cultured medium of cell cultures. The reported diameter of exosomes is between 50 and 150 nm. Exosomes are either released from the cell whenmutivesicular bodies fuse with the plasma membrane or released directly from the plasma membrane. Evidence is accumulating that exosomes have specialized functions and play a key role in processes such as coagulation, intercellular signaling, and waste management. Cancer exosome secreated from tumor initiating cell, circulating tuomr cells, mediate cancer progression. Thus exosomes can potentially be used for prognosis, for therapy, and as biomarkers for health and disease. We are working on exosome and looking for new technology, new understanding and new application of exosome.  

2019 highlight: On going!!!


2018 highlight: Breast cancer metastasis suppressor OTUD1 deubiquitinates SMAD7 (Nature Communications) 

Metastasis is the main cause of death in cancer patients. TGF-β is pro-metastatic for malignant cancer cells. Here we report a loss-of-function screen in mice with metastasis as readout and identify OTUD1 as a metastasis-repressing factor. OTUD1 silenced cancer cells show mesenchymal and stem-cell-like characteristics. Further investigation reveal that OTUD1 directly deubiquitinates the TGF-β pathway inhibitor SMAD7 and prevents its degradation. Moreover, OTUD1 cleaves Lysine 33-linked poly-ubiquitin chains of SMAD7 Lysine 220, which exposes its PY motif to allow for SMURF2 binding and their capacity of promoting cell surface TβRI turnover. Importantly, OTUD1 is lost in multiple types of human cancers and loss of OTUD1 increases metastasis in intracardial xenograft and orthotopic transplantation models, and correlates with poor prognosis among breast cancer patients. High level of OTUD1 inhibits cancer stemness and shuts off metastasis. Thus, OTUD1 represses breast cancer metastasis by mitigating TGF-β-induced pro-oncogenic responses via deubiquitinating SMAD7.


2017 highlight: (1) YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKe-mediated phosphorylation (Nature Immunology

The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKɛ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.  

(2) USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling (EMBO Journal
SMAD4 is a common intracellular effector for TGF-β family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-β family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.


2016 highlight: Loss of FAF1 accumulates TβRII for metastasis  (Nature Communications)

We found FAF1 destabilizes TβRII on the cell surface by recruiting VCP/E3 ligase complex thereby limiting excessive TGF-β response. The activated AKT directly phosphorylates FAF1 at Ser 582 then disrupts the FAF1-VCP complex and reduces FAF1 at the plasma membrane. The latter results in an increase in TβRII at the cell surface that promotes both TGF-β-induced SMAD and non-SMAD signaling. We uncover metastasis suppressing role for FAF1 through analyses of FAF1-knockout animals, various in vitro and in vivo models of epithelial to mesenchymal transition and metastasis, an MMTV-PyMT transgenic model of mammary tumor progression and clinical breast cancer samples. These findings uncover a previously uncharacterized mechanism by which TβRII is tightly controlled. Importantly, we reveal how SMAD and AKT pathways interact to confer pro-oncogenic responses to TGF-β.


2015 highlight: c-Myb drives Wnt dependent metastasis (Cancer Research)
The molecular underpinnings of aggressive breast cancers remain mainly obscure. Here we demonstrate that activation of the transcription factor c-Myb is required for the pro-metastatic character of basal breast cancers. An analysis of breast cancer patients led us to identify c-Myb as an activator of Wnt/β-catenin signaling. c-Myb interacted with the intracellular Wnt effector β-catenin and co-activated the Wnt/β-catenin target genes Cyclin D1 and Axin2. Moreover, c-Myb controlled metastasis in an Axin2 dependent manner. Expression microarray analyses revealed a positive association between Axin2 and c-Myb, a target of the pro-inflammatory cytokine IL-1β that was found to be required for IL-1β-induced breast cancer cell invasion. Overall, our results identified c-Myb as a promoter of breast cancer invasion and metastasis through its ability to activate Wnt/β-catenin/Axin2 signaling. 


2014 highlight: Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-β signalling (Nature Communications)
In advanced cancers, the TGF-β 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-β signalling. NR4A1 promotes TGF-β/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-β-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-β-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-β-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-β signalling in breast cancer.

2013 highlight: TRAF4 Promotes TGF-β Receptor Signaling and Drives Breast Cancer Metastasis (Molecular Cell)

TGF-β 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-β-induced SMAD and non-SMAD signaling. Upon TGF-β stimulation, TRAF4 is recruited to the active TGF-β receptor complex, where it antagonizes E3 ligase SMURF2 and facilitates the recruitment of deubiquitinase USP15 to the TGF-β type I receptor (TβRI). Both processes contribute to TβRI stabilization on the plasma membrane and thereby enhance TGF-β signaling. In addition, the TGF-β receptor-TRAF4 interaction triggers Lys 63-linked TRAF4 polyubiquitylation and subsequent activation of the TGF-β-activated kinase (TAK)1. TRAF4 is required for efficient TGF-β-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-β pathway and is a key determinant in breast cancer pathogenesis.


2012 highlight: USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-β type I receptor (Nature Cell Biology)

The stability and membrane localization of the transforming growth factor-β (TGF-β) type I receptor (TβRI) determines the levels of TGF-β signalling. TβRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. We performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-β signalling. USP4 was found to directly interact with TβRI and act as a deubiquitylating enzyme, thereby controlling TβRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-β-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 TβRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-β and AKT signalling pathways. 


2011 highlightRNF12 controls embryonic stem cell fate by targeting SMAD7 for degradation (Molecular Cell) 
Smad7 is a potent antagonist of TGF-β 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-β signaling. Depletion of RNF12 dramatically reduced TGF-β/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-β family signaling.


 
Cell signaling pathways
We also explored widely the other cellular signaling that are involved in human cancer metastasis, such as Wnt, BMP, NFκB, PI3K/AKT, Hippo, p53 and innate immune pathways.
 
Focusing on human deubiquitinating enzymes (DUBs)
Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, are functionally important but less well understood. We systematically study DUBs in tumor oncogenesis with focus on their function, specificity, and the regulation of their activity.
                      
 (human DUB family members. Cell. 2005 Dec)