PCLAF (PCNA clamp-associated factor), also known as PAF15/ KIAA0101, is overexpressed in most human cancers and is a predominant regulator of tumor progression. However, its biological function in neuroblastoma remains unclear. PCLAF is extremely overexpressed in neuroblastoma and is associated with poor prognosis. Through the analysis of various data sets, we found that the high expression of PCLAF is positively correlated with increased stage and high risk of neuroblastoma. Most importantly, knocking down PCLAF could restrict the proliferation of neuroblastoma cells in vitro and in vitro. By analyzing RNA-seq data, we found that the enrichment of cell cycle-related pathway genes was most significant among the differentially expressed downregulated genes after reducing the expression of PCLAF. In addition, PCLAF accelerated the G1/S transition of the neuroblastoma cell cycle by activating the E2F1/PTTG1 signaling pathway. In this study, we reveal the mechanism by which PCLAF facilitates cell cycle progression and recommend that the PCLAF/E2F1/PTTG1 axis is a therapeutic target in neuroblastoma.

Lymphangioma is a common type of congenital vascular disease in children with a broad spectrum of clinical manifestations. The current classification of lymphangioma by International Society for the Study of Vascular Anomalies is largely based on the clinical manifestations and complications and is not sufficient for selection of therapeutic strategies and prognosis prediction. The clinical management and outcome of lymphangioma largely depend on the clinical classification and the location of the disease, ranging from spontaneous regression with no treatment to severe sequelae even with comprehensive treatment. Recently, rapid progression has been made toward elucidating the molecular pathology of lymphangioma and the development of treatments. Several signaling pathways have been revealed to be involved in the progression and development of lymphangioma, and specific inhibitors targeting these pathways have been investigated for clinical applications and clinical trials. Some drugs already currently in clinical use for other diseases were found to be effective for lymphangioma, although the mechanisms underlying the anti-tumor effects remain unclear. Molecular classification based on molecular pathology and investigation of the molecular mechanisms of current clinical drugs is the next step toward developing more effective individualized treatment of children with lymphangioma with reduced side effects.

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: Minichromosome maintenance complex component 6 (MCM6), as an important replication permission factor, is involved in the pathogenesis of various tumors. Here we studied the expression of MCM6 in neuroblastoma and its influence on tumor characteristics and prognosis. Methods: Publicly available datasets were used to explore the influence of the differential expression of MCM6 on neuroblastoma tumor stage, risk and prognosis. In cell experiments, human neuroblastoma cell lines SK-N-SH and SK-N-BE [ (2)] were utilized to verify the ability of MCM6 to promote cell proliferation, migration and invasion. We further explored the possible molecular mechanism of MCM6 affecting the phenotype of neuroblastoma cells by mutual verification of RNA-seq and western blotting, and flow cytometry to inquire about its potential specific roles in the cell cycle. Results: Through multiple datasets mining, we found that high expression of MCM6 was positively correlated with elevated tumor stage, high risk and poor prognosis in neuroblastoma. At the cellular level, neuroblastoma cell proliferation, migration and invasion were significantly inhibited after MCM6 was interfered by siRNA. Mutual verification of RNA-seq and western blotting suggested that the downstream cell cycle-related genes were differentially expressed after MCM6 interference. Flow cytometric analysis revealed that neuroblastoma cells were blocked in G1/S phase after MCM6 interference. Conclusion: MCM6 is considered to be the driving force of G1/S cell cycle progression, and it is also a prognostic marker and a potential novel therapeutic target in neuroblastoma.

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.

The prognosis of high‑risk neuroblastoma remains poor. Clinical first‑line drugs for treating neuroblastoma have been developed over the previous half‑century; however, progress in the identification of new drugs with high efficiency is required. Bufalin, one of the major components of extracts obtained from the venom of the Chinese toad Bufo gargarizans, which is used to treat heart failure in Asian Pacific countries, has been reported to be a potential drug against multiple types of tumor; however, the detailed mechanisms underlying its antitumor activities remain unclear, largely due to lack of knowledge regarding its targets. In the present study, bufalin was revealed to exhibit potent antitumor effects against neuroblastoma, both in vitro and in vivo, using cell proliferation, colony formation, Transwell migration and flow cytometry assays, as well as a nude mouse subcutaneous xenograft model. Moreover, a chemically modified bufalin probe was designed to identify the potential targets of bufalin in neuroblastoma via chemical proteomics. With this strategy, it was revealed that the electron transport chain (ETC) on the inner membrane of mitochondria may contain potential targets for bufalin, and that bufalin‑induced mitochondrial‑dependent apoptosis may be caused by disruption of the ETC. Collectively, the present study suggests that bufalin may a promising drug for chemotherapy against neuroblastoma, and provides a foundation for further studies into the antitumor mechanisms of bufalin.