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OBJECTIVE: Downregulation of N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor gene, has been associated with poor clinical outcomes in various cancers. However, the prognostic significance of NDRG2 in oral squamous cell carcinoma (OSCC) remains unknown. This study aimed to evaluate the prognostic value of NDRG2 downregulation in OSCC and to elucidate the mechanism by which NDRG2 is downregulated and the biological role of NDRG2 in tumor progression. METHODS: Immunohistochemical and in silico analyses of NDRG2 expression were performed, and the correlation between NDRG2 expression and clinicopathological data was analyzed. The effect of NDRG2 knockdown on the biological behavior of OSCC cells was investigated and the effect of 5-aza-2'-deoxycytidine (5-aza-dC) on NDRG2 expression was determined. RESULTS: NDRG2 expression was significantly downregulated and DNA hypermethylation of NDRG2 was frequently found in head and neck SCC, including OSCC. Low NDRG2 expression was significantly correlated with adverse clinicopathological features and worse survival in OSCC. NDRG2 knockdown could enhance the oncogenic properties of OSCC cells. NDRG2 mRNA levels in OSCC cells could be restored by 5-aza-dC. CONCLUSION: Downregulation of NDRG2 promotes tumor progression and predicts poor prognosis in OSCC. Therefore, restoration of NDRG2 expression may be a potential therapeutic strategy in OSCC.

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The protein, N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor, is significantly decreased or absent in many types of cancer. There is a significant negative correlation between the levels of NDRG2 and the development and progression of cancer tumor recurrence and tumor invasion, in different cancers. In contrast, the in vitro and in vivo overexpression of the NDRG2 protein decreases the proliferation, growth, adhesion and migration of many types of cancer cells. The in vitro overexpression of NDRG2 increases the efficacy of certain anticancer drugs in specific types of cancer cells. We hypothesize that the delivery of the mRNA of the NDRG2 protein, encapsulated by lipid nanoparticles, could represent a potential treatment of metastatic and drug-resistant cancers. This would be accomplished using a self-amplifying mRNA that encodes the NDRG2 protein and an RNA-dependent-RNA polymerase, obtained from an in vitrotranscribed (IVT) mRNA. The IVT mRNA would be encapsulated in a lipid nanoformulation. The efficacy of the nanoformulation would be determined in cultured cancer cells and if the results are positive, nude mice transplanted with either drug-resistant or metastatic drug-resistant cancer cells, would be treated with the nano- formulation and monitored for efficacy and adverse effects. If the appropriate preclinical studies indicate this formulation is efficacious and safe, it is possible it could be evaluated in clinical trials.

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Background: Cisplatin is a commonly used nephrotoxic drug and can cause acute kidney injury (AKI). In the present study, isobaric tags for relative and absolute quantification (iTRAQ) and parallel reaction monitoring (PRM)-based comparative proteomics were used to analyze differentially expressed proteins (DEPs) to determine the key molecular mechanism in mice with cisplatin-induced AKI in the presence or absence of SIS3, a specific p-smad3 inhibitor, intervention. Methods: The cisplatin-induced AKI mouse model was established and treated with SIS3. We used iTRAQ to search for DEPs, PRM to verify key DEPs and combined Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for bioinformatics analysis. We then assessed lipid deposition, malondialdehyde (MDA) and reactive oxygen species (ROS) and detected the expression of SREBF1, SCD1, CPT1A, PPARα and NDRG1 in vitro. Results: Proteomic analysis showed that the identified DEPs were mainly enriched in energy metabolism pathways, especially in lipid metabolism. When SIS3 was applied to inhibit the phosphorylation of Smad3, the expression of NDRG1 and fatty acid oxidation key proteins CPT1A and PPARα increased, the expression of lipid synthesis related proteins SREBF1 and SCD1 decreased and the production of lipid droplets, MDA and ROS decreased. Conclusion: SIS3 alleviates oxidative stress, reduces lipid accumulation and promotes fatty acid oxidation through NDRG1 in cisplatin-induced AKI. Our study provides a new candidate protein for elucidating the molecular mechanisms of fatty acid metabolism disorders in cisplatin-induced acute kidney injury.

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Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody.

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The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/ß-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased ß-catenin and downregulated glycogen synthase kinase-3ß (GSK-3ß) protein levels and its activation. However, ß-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3ß was significantly increased after NDRG1 overexpression, suggesting a GSK-3ß-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Cα (PKCα), with PKCα silencing preventing ß-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing ß-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in ß-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCα, NDRG1, and ß-catenin was identified, with the formation of a potential metabolon that promotes the latter ß-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in ß-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased ß-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCα and inhibit WNT signaling.

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BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

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Cerebral ischemic preconditioning (CIP) has been shown to improve brain ischemic tolerance against subsequent lethal ischemia. Reactive astrocytes play important roles in cerebral ischemia-reperfusion. Recent studies have shown that reactive astrocytes can be polarized into neurotoxic A1 phenotype (C3d) and neuroprotective A2 phenotype (S100A10). However, their role in CIP remains unclear. Here, we focused on the role of N-myc downstream-regulated gene 2 (NDRG2) in regulating the transformation of A1/A2 astrocytes and promoting to brain ischemic tolerance induced by CIP. A Sprague Dawley rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used. Rats were divided into the following six groups: (1) sham group; (2) CIP group: left middle cerebral artery was blocked for 10 min; (3) MCAO/R group: left middle cerebral artery was blocked for 90 min; (4) CIP + MCAO/R group: CIP was performed 72 h before MCAO/R; (5) AAV-NDRG2 + CIP + MCAO/R group: adeno-associated virus (AAV) carrying NDRG2 was administered 14 days before CIP + MCAO/R; (6) AAV-Ctrl + CIP + MCAO/R group: empty control group. The rats were subjected to neurological evaluation 24 h after the above treatments, and then were sacrificed for 2, 3, 5-triphenyltetraolium chloride staining, thionin staining, immunofluorescence and western blot analysis. In CIP + MCAO/R group, the neurological deficit scores decreased, infarct volume reduced, and neuronal density increased compared with MCAO/R group. Notably, CIP significantly increased S100A10 expression and the number of S100A10+/GFAP+ cells, and also increased NDRG2 expression. MCAO/R significantly decreased S100A10 expression and the number of S100A10+/GFAP+ cells yet increased C3d expression and the number of C3d+/GFAP+ cells and NDRG2 expression, and these trends were reversed by CIP + MCAO/R. Furthermore, over-expression of NDRG2 before CIP + MCAO/R, the C3d expression and the number of C3d+/GFAP+ cells increased, while S100A10 expression and the number of S100A10+/GFAP+ cells decreased. Meanwhile, over-expression of NDRG2 blocked the CIP-induced brain ischemic tolerance. Taken together, these results suggest that CIP exerts neuroprotective effects against ischemic injury by suppressing A1 astrocyte polarization and promoting A2 astrocyte polarization via inhibiting NDRG2 expression.

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AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is resistant to immune checkpoint blockade (ICB) therapies. Emerging evidence suggests that NDRG1 may be an important target for the development of new therapies for PDAC. Herein, we investigated the novel roles of NDRG1 and Combretastatin A-4 (CA-4) in the treatment of PDAC ICB resistance. METHODS: Enrichment of MHC class I was detected by RNA sequence and verified by RT-qPCR and immunoblotting in NDRG1-knockdown human pancreatic cancer cell lines. The protein degradation mode was found by stimulation with various inhibitors, and the autophagy degradation pathway was found by immunoprecipitation and immunolocalization. The roles of NDRG1 and MHC-I in immunotherapy were investigated by orthotopic solid tumors, histology, immunohistochemistry, multiplex immunofluorescence staining and flow cytometry. RESULTS: Here, we identified a previously undescribed role of NDRG1 in activating major histocompatibility complex class 1 (MHC-1) expression in pancreatic ductal adenocarcinoma (PDAC) cells through lysosomal-autophagy-dependent degradation. In mouse models of PDAC, either tumor cell overexpression or pharmacologic activation of NDRG1 leads to MHC-1 upregulation in tumor cells, which in turn promotes the infiltration and activity of CD8 + T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. Moreover, combination therapy of CA-4 and ICB overcomes the drug resistance of pancreatic cancer to ICB therapy. In PDAC patients, NDRG1 expression correlates with high MHC-1 expression and better survival. CONCLUSION: Our results reveal NDRG1 in PDAC cancer cells as a tumor suppressor and suggest that pharmaceutically targeting NDRG1 is a promising way to overcome pancreatic cancer resistance to immunotherapy and provides a potential therapeutic strategy for the treatment of pancreatic cancer patients.

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Using N-Myc61-89 as a starting template we showcase the systematic use of truncation and maleimide constraining to develop peptidomimetic inhibitors of the N-Myc/Aurora-A protein-protein interaction (PPI); a potential anticancer drug discovery target. The most promising of these - N-Myc73-94-N85C/G89C-mal - is shown to favour a more Aurora-A compliant binding ensemble in comparison to the linear wild-type sequence as observed through fluorescence anisotropy competition assays, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Further in silico investigation of this peptide in its Aurora-A bound state, by molecular dynamics (MD) simulations, imply (i) the bound conformation is more stable as a consequence of the constraint, which likely suppresses dissociation and (ii) the constraint may make further stabilizing interactions with the Aurora-A surface. Taken together this work unveils the first orthosteric N-Myc/Aurora-A inhibitor and provides useful insights on the biophysical properties and thus design of constrained peptides, an attractive therapeutic modality.

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Photoreceptor apoptosis is the main pathological feature of retinal degenerative diseases; however, the underlying molecular mechanism has not been elucidated. Recent studies have shown that N-myc downstream regulated gene 2 (NDRG2) exerts a neuroprotective effect on the brain and spinal cord. In addition, our previous studies have confirmed that NDRG2 is expressed in mouse retinal photoreceptors and counteracts N-methyl-N-nitrosourea (MNU)-induced apoptosis. However, the underlying molecular mechanism remains unclear. In this study, we observed that the expression of NDRG2 was not only significantly inhibited in photoreceptors after MNU treatment but also after hydrogen peroxide treatment, and photoreceptor apoptosis was alleviated or aggravated after overexpression or knockdown of NDRG2 in the 661W photoreceptor cell line, respectively. The apoptosis inhibitor Z-VAD-FMK rescued photoreceptor apoptosis induced by MNU after NDRG2 knockdown. Next, we screened and identified tissue inhibitor of metalloproteinases 3 (TIMP3) as the downstream molecule of NDRG2 in 661W cells by using quantitative real-time polymerase chain reaction. TIMP3 exerts a neuroprotective effect by inhibiting the expression of matrix metalloproteinases (MMPs). Subsequently, we found that signal transducer and activator of transcription 3 (STAT3) mediated the NDRG2-associated regulation of TIMP3. Finally, we overexpressed NDRG2 in mouse retinal tissues by intravitreally injecting an adeno-associated virus with mouse NDRG2 in vivo. Results showed that NDRG2 upregulated the expression of phospho-STAT3 (p-STAT3) and TIMP3, while suppressing MNU-induced photoreceptor apoptosis and MMP expression. Our findings revealed how NDRG2 regulates the STAT3/TIMP3/MMP pathway and uncovered the molecular mechanism underlying its neuroprotective effect on mouse retinal photoreceptors.

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Objective To observe the changes in serum a proliferation inducing ligand(a proliferation inducing ligand,APRIL)and N-myc downstream regulated gene 1(N-myc downstream regulated gene 1,NDRG1)levels,and analyze their diagnostic value for ovarian endometrioma(OEM).Methods From July 2021 to July 2022,132 patients with OEM who visited Zigong First People's Hospital were regarded as the observation group,and regular follow-up was conducted.According to the prognosis of these patients,they were grouped into the recurrence group(n=50)and the non recurrence group(n=82).Meanwhile,78 healthy individuals who had their medical checkups at the hospital during the same period were the control group.Enzyme linked immunosorbent assay(ELISA)was applied to detect serum APRIL and NDRG1 levels,and the general data of the recurrent and non recurrent groups were compared.Logistic regression analysis was applied to analyze the relevant factors affecting the prognosis of OEM.Pearson analysis was applied to explore the correlation between serum APRIL and NDRG1 levels in patients with OEM.Receiver operating characteristic(ROC)curve was applied to evaluate the diagnostic value of serum APRIL,NDRG1 levels and their combination for OEM.Results Compared with the control group,APRIL level(35.28±6.81ng/ml vs 26.37±3.19ng/ml)and NDRG1 level(124.39±15.67μg/L vs 9.67±10.82μg/L)in observation group were increased,and the differences were significant(t=10.864,17.278,all P<0.05).Compared with the non recurrence group,the serum levels of APRIL(40.38±7.88ng/ml vs 32.16±6.18ng/ml)and NDRG1(132.04±19.83μg/L vs 119.73±13.16μg/L)in the recurrence group were increased,and the differences were significant(t=6.668,4.287,all P<0.05).Logistic regression analysis showed that serum APRIL and NDRG1 levels were risk factors for the prognosis of patients with OEM(Waldχ2=11.839,28.437,all P＜0.001).Pearson method analysis results showed a positive correlation between serum APRIL level and NDRG1 level in patients with OEM(r=0.439,P<0.001).The area under the curve(AUC)of combined diagnosis of serum APRIL and NDRG1 levels in patients with OEM was 0.849,with a sensitivity and specificity of 73.95%and 85.37%,respectively,which was better than the single prediction of APRIL and NDRG1(Z =2.644,2.094,P=0.008,0.036).Conclusion The serum levels of APRIL and NDRG1 were increased in patients with OEM.The combination of the two has high clinical value in the diagnosis of OEM,which may be closely related to the prognosis of patients with OEM.

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Predicting the prognosis of hepatocellular carcinoma (HCC) is a major medical challenge and of guiding significance for treatment. This study explored the actual relevance of RNA expression in predicting HCC prognosis. Cox's multiple regression was used to establish a risk score staging classification and to predict the HCC patients' prognosis on the basis of data in the Cancer Genome Atlas (TCGA). We screened seven gene biomarkers related to the prognosis of HCC from the perspective of oxidative stress, including Alpha-Enolase 1(ENO1), N-myc downstream-regulated gene 1 (NDRG1), nucleophosmin (NPM1), metallothionein-3, H2A histone family member X, Thioredoxin reductase 1 (TXNRD1) and interleukin 33 (IL-33). Among them we measured the expression of ENO1, NGDP1, NPM1, TXNRD1 and IL-33 to investigate the reliability of the multi-index prediction. The first four markers' expressions increased successively in the paracellular tissues, the hepatocellular carcinoma samples (from patients with better prognosis) and the hepatocellular carcinoma samples (from patients with poor prognosis), while IL-33 showed the opposite trend. The seven genes increased the sensitivity and specificity of the predictive model, resulting in a significant increase in overall confidence. Compared with the patients with higher-risk scores, the survival rates with lower-risk scores are significantly increased. Risk score is more accurate in predicting the prognosis HCC patients than other clinical factors. In conclusion, we use the Cox regression model to identify seven oxidative stress-related genes, investigate the reliability of the multi-index prediction, and develop a risk staging model for predicting the prognosis of HCC patients and guiding precise treatment strategy.

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The high heterogeneity and low percentage of neuroendocrine cells in prostate cancer limit the utility of traditional bulk RNA sequencing and even single-cell RNA sequencing to find better biomarkers for early diagnosis and stratification. Re-clustering of specific cell-type holds great promise for identification of intra-cell-type heterogeneity. However, this has not yet been used in studying neuroendocrine prostate cancer heterogeneity. Neuroendocrine cluster(s) were individually identified in each castration-resistant prostate cancer specimen and combined for trajectory analysis. Three neuroendocrine states were identified. Neuroendocrine state 2 with the highest AR score was considered the initial starting state of neuroendocrine transdifferentiation. State 1 and state 3 with distinct high neuroendocrine scores and marker genes enriched in N-Myc and REST target genes, respectively, were considered as two different types of neuroendocrine differentiated cancer cells. These two states contained distinct groups of prostate cancer biomarkers and a strong distinguishing ability of normal versus cancerous prostate across different pathological grading was found in the N-Myc-associated state. Our data highlight the central role of N-Myc and REST in mediating lineage plasticity and classifying neuroendocrine phenotypes.

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Background: Pulmonary hypertension (PH) represents a threatening pathophysiologic state that can be induced by chronic hypoxia and is characterized by extensive vascular remodeling. However, the mechanism underlying hypoxia-induced vascular remodeling is not fully elucidated. Methods and Results: By using quantitative polymerase chain reactions, western blotting, and immunohistochemistry, we demonstrate that the expression of N-myc downstream regulated gene-1 (NDRG1) is markedly increased in hypoxia-stimulated endothelial cells in a time-dependent manner as well as in human and rat endothelium lesions. To determine the role of NDRG1 in endothelial dysfunction, we performed loss-of-function studies using NDRG1 short hairpin RNAs and NDRG1 over-expression plasmids. In vitro, silencing NDRG1 attenuated proliferation, migration, and tube formation of human pulmonary artery endothelial cells (HPAECs) under hypoxia, while NDRG1 over-expression promoted these behaviors of HPAECs. Mechanistically, NDRG1 can directly interact with TATA-box binding protein associated factor 15 (TAF15) and promote its nuclear localization. Knockdown of TAF15 abrogated the effect of NDRG1 on the proliferation, migration and tube formation capacity of HPAECs. Bioinformatics studies found that TAF15 was involved in regulating PI3K-Akt, p53, and hypoxia-inducible factor 1 (HIF-1) signaling pathways, which have been proved to be PH-related pathways. In addition, vascular remodeling and right ventricular hypertrophy induced by hypoxia were markedly alleviated in NDRG1 knock-down rats compared with their wild-type littermates. Conclusions: Taken together, our results indicate that hypoxia-induced upregulation of NDRG1 contributes to endothelial dysfunction through targeting TAF15, which ultimately contributes to the development of hypoxia-induced PH.

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NDRG1 is a nickel- and calcium-inducible gene that plays important roles in the primary growth of malignant tumors, as well as in invasion and metastasis. This study investigated the associations of NDRG1 expression with cell adhesion and other clinicopathological factors in ovarian cancer. The clinical records of 123 women who underwent surgery for ovarian cancer in our institute were reviewed retrospectively. The expression of NDRG1, E-cadherin, and beta-catenin in surgical specimens were evaluated immunohistochemically. The NDRG1 expression level was significantly associated with beta-catenin expression, peritoneal metastasis outside the pelvic cavity, lymph node metastasis, and FIGO stages. The Kaplan-Meier analysis showed a significant association between the NDRG1 expression level and progression-free survival: high NDRG1 expression was related to poor survival. Our results suggest that the increased expression of NDRG1 is associated with cell adhesion and may be a poor prognostic indicator in women with ovarian cancer.

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Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

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This review presents current knowledge on the molecular biology of retinoblastoma (RB). Retinoblastoma is an intraocular tumor with hereditary and sporadic forms. 8,000 new cases of this ocular malignancy of the developing retina are diagnosed each year worldwide. The major gene responsible for retinoblastoma is RB1, and it harbors a large spectrum of pathogenic variants. Tumorigenesis begins with mutations that cause RB1 biallelic inactivation preventing the production of functional pRB proteins. Depending on the type of mutation the penetrance of RB is different. However, in small percent of tumors additional genes may be required, such as MYCN, BCOR and CREBBP. Additionally, epigenetic changes contribute to the progression of retinoblastoma as well. Besides its role in the cell cycle, pRB plays many additional roles, it regulates the nucleosome structure, participates in apoptosis, DNA replication, cellular senescence, differentiation, DNA repair and angiogenesis. Notably, pRB has an important role as a modulator of chromatin remodeling. In recent years high-throughput techniques are becoming essential for credible biomarker identification and patient management improvement. In spite of remarkable advances in retinoblastoma therapy, primarily in high-income countries, our understanding of retinoblastoma and its specific genetics still needs further clarification in order to predict the course of this disease and improve therapy. One such approach is the tumor free DNA that can be obtained from the anterior segment of the eye and be useful in diagnostics and prognostics.

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Mutations in the FLT3 gene not only lead to abnormalities in its structure and function, but also affect the expression of other genes involved in leukemogenesis. This study evaluated the expression of genes that are more characteristic of neuroblastoma but less studied in leukemia. N-MYC oncogene expression was found to be more than 3-fold higher in primary AML patients carrying the FLT3-ITD mutation compared to carriers of other mutations as well as patients with normal karyotype (p = 0.03946). In contrast to the expression of several genes (C-MYC, SPT16, AURKA, AURKB) directly correlated to the allelic load of FLT3-ITD, the expression of the N-MYC oncogene is extremely weakly related or independent of it (p = 0.0405). Monitoring of N-MYC expression in some patients with high FLT3-ITD allelic load receiving therapy showed that a decrease in FLT3-ITD allelic load is not always accompanied by a decrease in N-MYC expression. On the contrary, N-MYC expression may remain elevated during the first three months after therapy, which is additional evidence of the emergence of resistance to therapy and progression of AML.

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The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

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Imatinib resistance in chronic myelogenous leukemia (CML) is a clinical problem. The present study examined the role of N­Myc downstream regulatory gene 3 (NDRG3) in imatinib resistance in CML. Quantitative PCR demonstrated that NDRG3 was highly expressed in patients with CML. Cell Counting Kit (CCK)­8 experiments proved that NDRG3 promoted the proliferation of K562 CML cells and enhanced imatinib resistance. Dual­luciferase assay showed that microRNA (miR)­204­5p inhibited expression of NDRG3 and immunofluorescence experiments showed that NDRG3 promoted accumulation of ß­catenin in the nucleus, thereby increasing the expression of downstream drug resistance­ and cell cycle­associated factors (c­Myc and MDR1). At the same time, cell proliferation experiments showed that ß­catenin played a role in cell proliferation and drug resistance. Co­transfection with small interfering (si)­ß­catenin partially reversed the effect of NDRG3. This finding indicated that NDRG3 plays an important role in imatinib resistance and miR­204­5p and ß­catenin are involved in the biological behavior of NDRG3. The present results provide theoretical support for overcoming drug resistance in CML.

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