It is known that proinflammatory cytokines empower multipotent mesenchymal stromal cells (MSCs) the immunosuppressive capacity to treat various inflammatory diseases. Nevertheless, how the proinflammatory cytokines modulate the immunosuppressive capacity of MSCs is poorly understood. In the present study, we identified that the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) was upregulated in MSCs upon stimulation of proinflammatory cytokines IFN-γ plus TNF-α. Interestingly, through intervening UCHL1 by shRNA knockdown or its inhibitor LDN57444 or overexpression, we found that UCHL1 played a critical role in suppressing cytokines-induced inducible nitric oxide synthase expression in murine MSCs and indoleamine 2,3-dioxygenase expression in human MSCs, thereby restrained their immunosuppressive capacity. This effect of UCHL1 was attributed to the negative role in regulating NF-κB and STAT1 signaling, as exhibited by promoting NF-κB and STAT1 activation upon inhibition of UCHL1. Besides, inhibition of UCHL1 suppressed cytokines-induced MSC apoptosis via upregulation of Bcl-2. As a consequence, UCHL1-inhibited MSCs effectively alleviated concanavalin A-induced inflammatory liver injury. Therefore, our study demonstrates a novel role of UCHL1 in regulating the immunosuppressive capacity and survival of MSCs, which further affects their immunotherapy for inflammatory diseases.

Neuroblastoma (NB) is the most common pediatric solid tumor that originates from neural crest-derived sympathoadrenal precursor cells that are committed to development of sympathetic nervous system. The well differentiated histological phenotype of NB tumor cells has been reportedly associated with favorable patient outcome. Retinoic acid (RA) can effectively induce NB cell differentiation, thereby being used in the clinic as a treatment agent for inducing the differentiation of high-risk NB. However, the underlying molecular mechanisms of regulating differentiation remain elusive. The correlation between clinical characteristics, survival and the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) expression were assessed using a neuroblastic tumor tissue microarray, and then validated in three independent patient datasets. The different expression of UCHL1 in ganglioneuroblastoma, ganglioneuroma and NB was detected by immunohistochemistry, mass spectra and immunoblotting analysis, and the correlation between UCHL1 expression and the differentiated histology was analyzed, which was also validated in three independent patient datasets. Furthermore, the roles of UCHL1 in NB cell differentiation and proliferation and the underlying mechanisms were studied by using short hairpin RNA and its inhibitor LDN57444 in vitro. Based on our neuroblastic tumor tissue microarrays and three independent validation datasets (Oberthuer, Versteeg and Seeger), we identified that UCHL1 served as a prognostic marker for better clinical outcome in NB. We further demonstrated that high UCHL1 expression was associated with NB differentiation, indicated by higher UCHL1 expression in ganglioneuroblastomas/ganglioneuromas and well-differentiated NB than poorly differentiated NB, and the positive correlation between UCHL1 and differentiation markers. As expected, inhibiting UCHL1 by knockdown or LDN57444 could significantly inhibit RA-induced neural differentiation of NB tumor cells, characterized by decreased neurite outgrowth and neural differentiation markers. This effect of UCHL1 was associated with positively regulating RA-induced AKT and ERK1/2 signaling activation. What’s more, knockdown of UCHL1 conferred resistance to RA-induced growth arrest. Our findings identify a pivotal role of UCHL1 in NB cell differentiation and as a prognostic marker for survival in patients with NB, potentially providing a novel therapeutic target for NB.

Background: High-risk neuroblastoma patients have a 5-year survival rate of less than 50%. It’s an urgent need to identify new therapeutic targets and the appropriate drugs. Exportin-1 (XPO1), also known as chromosomal region maintenance 1, plays important roles in the progression of tumorigenesis. However, the prognostic and therapeutic values of XPO1 in neuroblastoma have not been reported. Methods: Correlations between XPO1 expression level and clinical characteristics were analyzed using the Neuroblastoma Research Consortium (NRC) dataset and tissue microarray analysis. Cell proliferation assays, colony formation assays, apoptosis assays, cell cycle analysis were performed to analyze the anti-tumor effects of verdinexor (KPT-335) in vitro. Western blot and mRNA sequencing were performed to explore underlying mechanism. In vivo anti-tumor effects of verdinexor were studied in a neuroblastoma xenograft model. Results: Higher XPO1 levels were associated with advanced stage and poor prognosis in neuroblastoma patients. The specific inhibitor of XPO1 verdinexor suppressed the neuroblastoma cell growth both in vitro and in vivo. Specifically, inhibition of XPO1 suppressed the neuroblastoma cell proliferation and induced cell apoptosis by nuclear accumulation of FOXO1 and RB1 in the neuroblastoma due to the inhibition of the PI3K/AKT pathway, and induced G0/G1 phase cell cycle arrest by activation of P53 function. Conclusions: XPO1 is a promising prognostic indicator for neuroblastoma and a novel target for antitumor treatment with selective inhibitor verdinexor.

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC-MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.

Infantile hemangioma (IH) is a common and benign vascular neoplasms, which has a high incidence in children. Although IH is benign, some patients experience complications such as pain, functional impairment, and permanent disfigurement. Treatment options for IH include corticosteroids, surgery, vincristine, interferon or cyclophosphamide. However, none of these modalities are ideal due to restrictions or potential serious side effects. There is thus a great need to explore novel treatments for IH with less side effects. Angiogenesis, vasculogenesis and tumorigenesis are the main features of IH. Tanshen is mostly used in Chinese traditional medicine to treat haematological abnormalities. Therefore, the aim of our study was to evaluate anti-proliferation and anti-angiogenesis effects on hemangiomas cells by extracted Tanshen compounds compared with propranolol, the first-line treatment for IH currently, both in vitro and in vivo. Cell viability, apoptosis, protein expression and anti-angiogenesis were analyzed by CCK8, Annexin V staining, Western blot and tube formation, respectively. The anti-tumor activity in vivo was evaluated using a mouse xenograft model. Fourteen major compounds extracting from Tanshen were screened for their ability to inhibit hemangiomas cells. Of the 14 compounds investigated, 15,16-Dihydrotanshinone I (DHTS) was the most potent modulator of EOMA cell biology. DHTS could significantly decrease EOMA cells proliferation by inducing cell apoptosis, which is much more efficient than propranolol in vitro. DHTS increased the expression of several apoptosis-related proteins, including caspase9, caspase3, PARP, AIF, BAX, cytochrome c, caspase8 and FADD and significantly inhibited angiogenesis, as indicated by reduced tube formation and diminished expression of vascular endothelial cell growth factor receptor 2 and matrix metalloproteinase 9. In nude mice xenograft experiment, DHTS (10mg/kg) could significantly inhibit the tumor growth of EOMA cells as well as propranolol (40mg/kg). Our study showed that DHTS was much more effective than propranolol in inhibiting hemangiomas proliferation and angiogenesis in vitro and in vivo, which could have potential therapeutic applications for treatment of IH.