Therapeutic targeting of TNIK in papillary thyroid carcinoma: a novel approach for tumor growth suppression.

Papillary thyroid carcinoma (PTC) is a common endocrine malignancy. The pathology of PTC is far from clear. As a kinase that can be targeted, the role of TNIK in PTC has not been investigated. This study was focused on the effects and molecular mechanisms of TNIK in PTC. Both public datasets and clinical specimens were used to verify TNIK expression. The effects of TNIK were investigated in both cell lines and mice models. Transcriptome analysis was used to explore the underlying mechanism of TNIK. Immunofluorescence, wound healing, and qRT-PCR assays were used to validate the mechanism of TNIK in PTC. The therapeutic effects of TNIK inhibitor NCB-0846 were evaluated by flow cytometry, western blot, and subcutaneous xenografts mice. TNIK expression was upregulated in PTC tissues. TNIK knockdown could suppress cell proliferation and tumor growth in no matter cell models or nude mice. The transcriptome analysis, GO enrichment analysis, and GSEA analysis results indicated TNIK was highly correlated with cytoskeleton, cell motility, and Wnt pathways. The mechanistic studies demonstrated that TNIK regulated cytoskeleton remodeling and promoted cell migration. NCB-0846 significantly inhibited TNIK kinase activity, induced cell apoptosis, and activated apoptosis-related proteins in a dose-dependent manner. In addition, NCB-0846 inhibited tumor growth in tumor-bearing mice. In summary, we proposed a novel regulatory mechanism in which TNIK-mediated cytoskeleton remodeling and cell migration to regulate tumor progression in PTC. TNIK is a therapeutic target in PTC and NCB-0846 would act as a novel targeted drug for PTC therapy.

Impact of the COVID-19 pandemic on the demographic and disease burden of pediatric malignant solid tumors in China: a single-center, cross-sectional study.

Background: With the development of the novel coronavirus disease 2019 (COVID-19), China implemented measures in an attempt to control the infection rate. We conducted a single-center, cross-sectional study to ascertain the impact of the COVID-19 pandemic on the equitable availability of medical resources for children diagnosed with malignant solid tumors in China. Methods: Data on the demographics, clinical characteristics, and medical expenses of 876 patients diagnosed with neuroblastoma, rhabdomyosarcoma (RMS), Wilms tumor, hepatoblastoma (HB), Ewing sarcoma (ES), and central nervous system (CNS) tumors from 2019 to 2021, during the COVID-19 pandemic, were retrospectively collected from the National Center for Children's Health. The Pearson χ2 test and Mann-Whitney test were performed to analyze the differences among variables. Results: Except for the regional origin of children with tumors during the epidemic, no significant differences were found in the demographic or clinical characteristics of patients at initial diagnosis. The number of patients from northern China and northeastern China who attended Beijing Children's Hospital (BCH) increased after the outbreak of COVID-19 (P=0.001). There was no significant alteration observed in the frequency of hospitalizations per individual per annum (P=0.641) or the mean expense incurred per individual per hospitalization (P=0.361). In addition, the medical insurance coverage rate of real-time settlement increased year by year. Conclusions: After the COVID-19 outbreak, the origin of patients with solid tumor who visited BCH was concentrated in the northern region of China. COVID-19 had no impact on the other demographic factors, clinical characteristics, or economic burden of patients with pediatric malignant solid tumors.

Translational practice of fluorescence in situ hybridisation to identify neuroblastic tumours with TERT rearrangements.

Recently, telomerase reverse transcriptase (TERT) gene rearrangements have been identified in neuroblastoma (NB), the typical pathological type of neuroblastic tumours (NTs); however, the prevalence of TERT rearrangements in other types of NT remains unknown. This study aimed to develop a practical method for detecting TERT defects and to evaluate the clinical relevance of TERT rearrangements as a biomarker for NT prognosis. A TERT break-apart probe for fluorescence in situ hybridisation (FISH) was designed, optimised, and applied to assess the genomic status of TERT in Chinese children with NTs at the Beijing Children's Hospital from 2016 to 2019. Clinical, histological, and genetic characteristics of TERT-rearranged NTs were further addressed. Genomic TERT rearrangements could be effectively detected by FISH and were mutually exclusive with MYCN amplification. TERT rearrangements were identified in 6.0% (38/633) of NTs overall, but 12.4% (31/250) in high-risk patients. TERT rearrangements identified a subtype of aggressive NTs with the characteristics of Stage 3/4, high-risk category, over 18 months old, and presenting all histological subtypes of NB and ganglioneuroblastoma nodular. Moreover, TERT rearrangements were significantly associated with elevated TERT expression levels and decreased survival chances. Multivariable analysis confirmed that it was an independent prognostic marker for NTs. FISH is an easily applicable method for evaluating TERT defects, which define a subgroup of NTs with unfavourable prognosis. TERT rearrangements would contribute to characterising NT molecular signatures in clinical practice.

BPTF in bone marrow provides a potential progression biomarker regulated by TFAP4 through the PI3K/AKT pathway in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children, which is highly prone to bone marrow (BM) metastasis. BM can monitor early signs of mild disease and metastasis. Existing biomarkers are insufficient for the diagnosis and treatment of NB. Bromodomain PHD finger transcription factor (BPTF) is an important subunit of the chromatin-remodeling complex that is closely associated with tumors. Here, we evaluated whether BPTF in BM plays an important role in predicting NB progression, and explore the molecular mechanism of BPTF in NB. METHODS: The clinical relevance of the BPTF was predicted in the GEO (GSE62564) and TARGET database. The biological function of BPTF in NB was investigated by constructing cell lines and employing BPTF inhibitor AU1. Western blot was used to determine the changes of BPTF, TFAP4, PI3K/AKT signaling and Epithelial-mesenchymal transition (EMT) related markers. A total of 109 children with newly diagnosed NB in Beijing Children's Hospital from January 2018 to March 2021 were included in this study. RT-PCR was used to measure the BPTF and TFAP4 expression in BM. The cut-off level was set at the median value of BPTF expression levels. RESULTS: Databases suggested that BPTF expression was higher in NB and was significantly associated with stage and grade. Proliferation and migration of NB cells were slowed down when BPTF was silenced. Mechanistically, TFAP4 could positively regulate BPTF and promotes EMT process through activating the PI3K/AKT signaling pathway. Moreover, detection of the newly diagnosed BM specimens showed that BPTF expression was significantly higher in high-risk group, stage IV group and BM metastasis group. Children with high BPTF at initial diagnosis were considered to have high risk for disease progression and recurrence. BPTF is an independent risk factor for predicting NB progression. CONCLUSIONS: A novel and convenient BPTF-targeted humoral detection that can prompt minimal residual and predict NB progression in the early stages of the disease were identified. BPTF inhibitor AU1 is expected to become a new targeted drug for NB therapy. It's also reveal previously unknown mechanisms of BPTF in NB cell proliferation and metastasis through TFAP4 and PI3K/AKT pathways.

TAF1D promotes proliferation by transcriptionally activating G2/M phase-related genes in MYCN-amplified neuroblastoma.

High-risk neuroblastoma (HR-NB) is an aggressive childhood cancer that responds poorly to currently available therapies and is associated with only about a 50% 5-year survival rate. MYCN amplification is a critical driver of these aggressive tumors, but so far there have not been any approved treatments to effectively treat HR-NB by targeting MYCN or its downstream effectors. Thus, the identification of novel molecular targets and therapeutic strategies to treat children diagnosed with HR-NB represents an urgent unmet medical need. Here, we conducted a targeted siRNA screening and identified TATA box-binding protein-associated factor RNA polymerase I subunit D, TAF1D, as a critical regulator of the cell cycle and proliferation in HR-NB cells. Analysis of three independent primary NB cohorts determined that high TAF1D expression correlated with MYCN-amplified, high-risk disease and poor clinical outcomes. TAF1D knockdown more robustly inhibited cell proliferation in MYCN-amplified NB cells compared with MYCN-non-amplified NB cells, as well as suppressed colony formation and inhibited tumor growth in a xenograft mouse model of MYCN-amplified NB. RNA-seq analysis revealed that TAF1D knockdown downregulates the expression of genes associated with the G2/M transition, including the master cell-cycle regulator, cell-cycle-dependent kinase 1 (CDK1), resulting in cell-cycle arrest at G2/M. Our findings demonstrate that TAF1D is a key oncogenic regulator of MYCN-amplified HR-NB and suggest that therapeutic targeting of TAF1D may be a viable strategy to treat HR-NB patients by blocking cell-cycle progression and the proliferation of tumor cells.

Predictive Significance of Enhanced Level of Angiogenesis and Tissue Neutrophils for Antrochoanal Polyps Recurrence in Children.

BACKGROUND: The pathologic features and potential predictive biomarkers for recurrence of antrochoanal polyps (ACPs) in children are not fully understood. OBJECTIVES: To identify the pathologic differences between recurrent and nonrecurrent group and to explore potential clinical markers which predict recurrence of ACPs in children. MATERIAL AND METHODS: A total of 11 recurrent and 21 nonrecurrent ACPs children were enrolled into this retrospect study. Clinical basic information was collected before the first surgery. The counts of vessels were evaluated by hematoxylin-eosin (HE) staining, and CD34 was detected by immunohistochemistry. Meanwhile, the percentage of each tissue inflammatory cells (eosinophils, neutrophils, lymphocytes, and plasma cells) was assessed by HE staining. RESULTS: No statistical significance was observed between the 2 groups in the basic clinical features. Moreover, both the counts of blood vessels and the tissue neutrophils percentage were enhanced significantly in group with ACPs recurrence (P < .05). According to the receiver operating characteristic curves, the area under the curve for the counts of blood vessels and tissue neutrophils percentage in the prediction of ACPs' recurrence was 0.779 (P = .0105) and 0.989 (P < .0001) respectively. CONCLUSIONS AND SIGNIFICANCE: It was concluded that the counts of blood vessels and the percentage of tissue neutrophils appeared to be potential excellent predictors of ACPs recurrence in children.

Primary cardiac CIC-rearranged undifferentiated sarcoma in an infant.

INTRODUCTION: Cardiac neoplasms are particularly rare in children, and the majority of these tumors are benign. Approximately 10% of cardiac neoplasms are malignant, including soft tissue sarcomas and lymphomas. Cardiac tumors could also be metastases. Primitive EWSR1-negative round or spindle cell undifferentiated sarcoma harboring CIC gene translocation is a highly aggressive malignancy mainly occurring in soft tissues. However, it has not yet been described in the heart. CASE PRESENTATION: We report a sarcoma that arose from the right ventricle in a 1-year-old girl. Histologically, it was composed of closely arranged small round or oval undifferentiated cells with fibrovascular separation, hyaline degeneration, and geographical necrosis. Immunohistochemically, the neoplastic cells exhibited focal membrane positivity for CD99 and diffuse positivity for WT1 and ETV4. Fluorescent in situ hybridization analysis showed EWSR1-negative but CIC-positive split signals. The breakpoint was also confirmed by whole genome sequencing. CONCLUSION: Based on morphological, immunohistochemical and molecular findings, this cardiac mass was diagnosed as CIC-rearranged sarcoma.

Loss of the wild-type KRAS allele promotes pancreatic cancer progression through functional activation of YAP1.

Human pancreatic ductal adenocarcinoma (PDAC) harboring one KRAS mutant allele often displays increasing genomic loss of the remaining wild-type (WT) allele (known as LOH at KRAS) as tumors progress to metastasis, yet the molecular ramification of this WT allelic loss is unknown. In this study, we showed that the restoration of WT KRAS expression in human PDAC cell lines with LOH at KRAS significantly attenuated the malignancy of PDAC cells both in vitro and in vivo, demonstrating a tumor-suppressive role of the WT KRAS allele. Through RNA-Seq, we identified the HIPPO signaling pathway to be positively regulated by WT KRAS in PDAC cells. In accordance with this observation, PDAC cells with LOH at KRAS exhibited increased nuclear localization and activation of transcriptional co-activator YAP1. Mechanistically, we discovered that WT KRAS expression sequestered YAP1 from the nucleus, through enhanced 14-3-3zeta interaction with phosphorylated YAP1 at S127. Consistently, expression of a constitutively-active YAP1 mutant in PDAC cells bypassed the growth inhibitory effects of WT KRAS. In patient samples, we found that the YAP1-activation genes were significantly upregulated in tumors with LOH at KRAS, and YAP1 nuclear localization predicted poor survival for PDAC patients. Collectively, our results reveal that the WT allelic loss leads to functional activation of YAP1 and enhanced tumor malignancy, which explains the selection advantage of the tumor cells with LOH at KRAS during pancreatic cancer clonal evolution and progression to metastasis, and should be taken into consideration in future therapeutic strategies targeting KRAS.

A Novel Germline Compound Heterozygous Mutation of BRCA2 Gene Associated With Familial Peripheral Neuroblastic Tumors in Two Siblings.

OBJECTIVES: To investigate the genetic variants that are responsible for peripheral neuroblastic tumors (PNTs) oncogenesis in one family case. MATERIALS AND METHODS: One family was recruited, including the healthy parents, sister affected by neuroblastoma (NB), and brother who suffered from ganglioneuroma (GN). Whole-genome sequencing (WGS) of germline DNA from all the family members and RNA-seq of tumor RNA from the siblings were performed. Mutants were validated by Sanger sequencing and co-IP was performed to assess the impact of the mutant on chemosensitivity in the SH-SY5Y cell line. RESULTS: A novel compound heterozygous mutation of BRCA2 was locked as the cause of carcinogenesis. One allele was BRCA2-S871X (stop-gain) from the siblings' mother, the other was BRCA2-N372H (missense) from their father. This novel compound heterozygous mutations of the BRCA2 gene associated with PNTs by disordering DNA damage and response (DDR) signal pathway. Moreover, chemosensitivity was reduced in the NB cell line due to the BRCA2-N372H mutant. CONCLUSION: In summary, these results revealed a novel germline compound heterozygous mutation of the BRCA2 gene associated with familial PNTs.

DCX and CRABP2 are candidate genes for differential diagnosis between pre-chemotherapy embryonic and alveolar rhabdomyosarcoma in pediatric patients.

IMPORTANCE: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. More than 90% of cases are classified as embryonic RMS (ERMS) or alveolar RMS (ARMS). ERMS has a worse prognosis than ARMS. Early differential diagnosis is of paramount importance for optimization of treatment. OBJECTIVE: To identify genes that are differentially expressed between ARMS and ERMS, which can be used for accurate rhabdomyosarcoma classification. METHODS: Three Gene Expression Omnibus datasets composed of ARMS and ERMS samples were screened and 35 differentially expressed genes (DEGs) were identified. Receiver operating characteristic curve analysis and area under the curve analysis was performed for these 35 DEGs and seven candidate genes with the best differential expression scores between ARMS and ERMS were determined. The expression of these seven candidate genes was validated by immunohistochemical analysis of pre-chemotherapy ARMS and ERMS specimens. RESULTS: The levels of DCX and CRABP2 were confirmed to be remarkably different between paraffin-embedded ARMS and ERMS tissues, while EGFR abundance was only marginally different between these two RMS subtypes. INTERPRETATION: DCX and CRABP2 are potential biomarkers for distinguishing ARMS from ERMS in pre-chemotherapy pediatric patients.

Clinical implications of TPO and AOX1 in pediatric papillary thyroid carcinoma.

BACKGROUND: Thyroid carcinoma is a common pediatric head and neck cancer, of which papillary thyroid cancer (PTC) is the most common type. Previously, we found that thyroid peroxidase (TPO) and aldehyde oxidase 1 (AOX1) were differentially expressed in PTC. This study explored the clinical importance of TPO and AOX1 in the diagnosis and prognosis of PTC in children. METHODS: Both TPO and AOX1 expression in PTC were analyzed using datasets from Gene Expression Omnibus (GEO). TPO and AOX1 protein levels in plasma from patients with PTC and non-tumor controls were detected via enzyme-linked immunosorbent assay (ELISA). The diagnostic accuracy of TPO and AOX1 was assessed using receiver operating characteristic (ROC) curve analysis. The association between gene expression levels and patient survival was explored using the Kaplan-Meier plotter online database. RESULTS: The results revealed that TPO and AOX1 expression was significantly downregulated in four independent datasets (GSE33630, GSE27155, GSE3678, and GSE3467). TPO and AOX1 protein levels in blood plasma were significantly decreased in patients with PTC. Quantitative analysis demonstrated that TPO and AOX1 levels in plasma had satisfactory predictive performance and the ability to discriminate PTC from healthy samples. Prognostic analysis demonstrated that low levels of TPO and AOX1 were markedly associated with poor survival in patients with PTC. CONCLUSIONS: In summary, these results implied that TPO and AOX1 could serve as novel biomarkers for the diagnosis and prognosis of pediatric PTC.

MYC-associated protein X binding with the variant rs72780850 in RNA helicase DEAD box 1 for susceptibility to neuroblastoma.

Neuroblastoma (NB) is one of the most common malignant tumors in children, with variable clinical behaviors and a 15% death rate of all malignancies in childhood. However, genetic susceptibility to sporadic NB in Han Chinese patients is largely unknown. To identify genetic risk factors for NB, we performed an association study on 357 NB patients and 738 control subjects among Han Chinese children. We focused on DEAD box 1 (DDX1), a putative RNA helicase, which is involved in NB carcinogenesis. The potential association of DDX1 polymorphisms with NB has not been discovered. Our results demonstrate that rs72780850 (NM_004939.2:c.-1555T>C) located in the DDX1 promoter region is significantly associated with higher expression of DDX1 transcript and increased NB risk (odds ratio=1.64, 95% confidence interval=1.03%-2.60%, P=0.004), especially in aggressive NB compared with ganglioneuroma and ganglioneuroblastoma in a dominant model (TC+CC vs. TT). Furthermore, the MYC-associated protein X (MAX) transcription factor showed stronger binding affinity to the DDX1 rs 72780850 CC allele compared with the TT allele, explaining the molecular mechanism of the increased NB risk caused by the rs72780850 polymorphism. Our results highlight the involvement of regulatory genetic variants of DDX1 in NB.

Downregulated NORAD in neuroblastoma promotes cell proliferation via chromosomal instability and predicts poor prognosis.

Increasing evidence suggests that long non-coding RNAs (lncRNAs) are involved in neuroblastoma (NB) pathogenesis. The aim of this study was to elucidate the roles and underlying mechanism of non-coding RNA activated by DNA damage (NORAD) in childhood NB. Both public data and clinical specimens were used to determine NORAD expression. Colony formation, cell proliferation and wound healing assays were performed to evaluate NORAD effects on proliferation and migration of SH-SY5Y and SK-N-BE(2) cells. Flow cytometry was used to examine the cell cycle changes. The expression of genes and proteins involved in chromosomal instability was determined by qRT-PCR and western blotting, respectively. Our results showed that low NORAD expression correlated with advanced tumor stage, high risk and MYCN amplification in both public data and clinical samples. Kaplan-Meier analysis indicated that patients with low NORAD expression had poor survival outcomes. Functional research showed that NORAD knockdown promoted cell proliferation and migration, and arrested the cell cycle at the G2/M phase. Moreover, the expression of the DNA damage sensor, PARP1, increased after NORAD knockdown, indicating a potential contribution of NORAD to DNA damage repair. NORAD silencing also affected the expression of genes and proteins related to sister chromatid cohesion and segregation, which are involved in chromosomal instability and consequent aneuploidy. These results suggest that NORAD may serve as a tumor suppressor in NB pathogenesis and progression. Thus, NORAD is a potential therapeutic target and a promising prognostic marker for NB patients.

Two novel mutations of PAX3 and SOX10 were characterized as genetic causes of Waardenburg Syndrome.

BACKGROUND: The objective of this study was to investigate the genetic causes of two probands diagnosed as Waardenburg syndrome (WS type I and IV) from two unrelated Chinese families. METHODS: PAX3 and SOX10 were the main pathogenic genes for WS type I (WS I) and IV (WS IV), respectively; all coding exons of these genes were sequenced on the two probands and their family members. Luciferase reporter assay and co-immunoprecipitation (CO-IP) were conducted to verify potential functional outcomes of the novel mutations. RESULTS: The first proband is a 9 years old girl diagnosed with WS I. A novel PAX3 heterozygous mutation of c.372-373delGA (p.N125fs) was identified, which results in a frameshift and truncation of PAX3 protein. In family II, a 2 years old girl was diagnosed with WS IV, and Sanger sequencing revealed a de novo SOX10 mutation of c.1114insTGGGGCCCCCACACTACACCGAC (p.Q372fs), a frameshift mutation that extends the amino acid chain of SOX10 protein. Functional studies indicated that the novel mutation of SOX10 had no effects on the interaction of SOX10 and PAX3, but reduced transactivate capacity of melanocyte inducing transcription factor (MITF) promoter. Both PAX3 and SOX10 mutation-induced defects of MITF transcription might contribute to the WS pathogenesis. CONCLUSION: We revealed a novel mutation in PAX3 and a de novo mutation in SOX10, which might account for the underlying pathogenesis of WS. This study expands the database of both PAX10 and PAX3 mutations and improves our understanding of the causes of WS.

Bioinformatics analysis to screen key genes in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma, and its incidence has been on the increase in recent years. However, the molecular mechanism of PTC is unclear and misdiagnosis remains a major issue. Therefore, the present study aimed to investigate this mechanism, and to identify key prognostic biomarkers. Integrated analysis was used to explore differentially expressed genes (DEGs) between PTC and healthy thyroid tissue. To investigate the functions and pathways associated with DEGs, Gene Ontology, pathway and protein-protein interaction (PPI) network analyses were performed. The predictive accuracy of DEGs was evaluated using the receiver operating characteristic (ROC) curve. Based on the four microarray datasets obtained from the Gene Expression Omnibus database, namely GSE33630, GSE27155, GSE3467 and GSE3678, a total of 153 DEGs were identified, including 66 upregulated and 87 downregulated DEGs in PTC compared with controls. These DEGs were significantly enriched in cancer-related pathways and the phosphoinositide 3-kinase-AKT signaling pathway. PPI network analysis screened out key genes, including acetyl-CoA carboxylase beta, cyclin D1, BCL2, and serpin peptidase inhibitor clade A member 1, which may serve important roles in PTC pathogenesis. ROC analysis revealed that these DEGs had excellent predictive performance, thus verifying their potential for clinical diagnosis. Taken together, the findings of the present study suggest that these genes and related pathways are involved in key events of PTC progression and facilitate the identification of prognostic biomarkers.

lncRNA SNHG16 is associated with proliferation and poor prognosis of pediatric neuroblastoma.

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children, which has complex molecular mechanisms. Increasing evidence has suggested that long noncoding RNAs (lncRNAs) account for NB pathogenesis. However, the function of small nucleolar RNA host gene 16 (SNHG16) in NB is currently unclear. In the present study, publically available data and clinical specimens were employed to verify the expression of SNHG16 in NB. Colony formation, real­time cell proliferation and migration assays were performed to demonstrate the status of cellular proliferation and migration. Flow cytometry was used to examine cell cycle progression in SH­SY5Y cells, and acridine orange/ethidium bromide staining and caspase­3/7 activity measurements were applied to study cell apoptosis. To explore the underlying mechanism of SNHG16 function, an online database was used to identify potential RNA­binding proteins that bind SNHG16. The expression of SNHG16 was revealed to be in line with the clinical staging of NB, and high SNHG16 expression was positively associated with poor clinical outcome. Furthermore, SNHG16 silencing inhibited cell proliferation, repressed migration, and induced cell cycle arrest at the G0/G1 phase in SH­SY5Y cells. Additionally, apoptosis was undetectable in SH­SY5Y cells following SNHG16 silencing. Bioinformatics analysis revealed that SNHG16 regulated cell proliferation in NB through transcriptional and translational pathways. These results suggested that SNHG16 may serve important roles in the development and progression of NB, and could represent a potential target for NB therapy.

HHLA2 is a novel immune checkpoint protein in pancreatic ductal adenocarcinoma and predicts post-surgical survival.

HHLA2 is a newly identified member of the B7 immune checkpoint family, but its function and crosstalk with immune cells is not fully understood. To gain insights into the HHLA2 expression profile and to determine the clinical significance and function of HHLA2 in pancreatic cancer, we performed immunohistochemistry (IHC) analyses on tissue microarrays (TMAs) of pancreatic ductal adenocarcinoma (PDAC, n = 92) with matched peritumoral tissues as well as in cohorts of precancerous lesions: pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN). We found that HHLA2 was rarely detected in normal acinar, islet, and ductal cells but widely expressed from early pancreatic precancerous lesions to invasive PDAC. The overall HHLA2 positivity was 95% (19/20) in low grade PanIN and 70.73% (29/41) in IPMN. HHLA2 expression was detected in 77.17% (71/92) of the PDAC cases and was significantly associated with better prognosis in this cohort. Our findings suggest that HHLA2 may behave as a costimulatory ligand in pancreatic cancer, which differs from other B7 family members that are largely characterized as checkpoint inhibitors. Further investigation of the HHLA2 signaling pathway and its receptors is warranted by our data and may lead to novel therapeutic interventions.

RNA-splicing factor SART3 regulates translesion DNA synthesis.

Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance that uses specialized DNA polymerases to replicate damaged DNA. DNA polymerase η (Polη) is well known to facilitate TLS across ultraviolet (UV) irradiation and mutations in POLH are implicated in skin carcinogenesis. However, the basis for recruitment of Polη to stalled replication forks is not completely understood. In this study, we used an affinity purification approach to isolate a Polη-containing complex and have identified SART3, a pre-mRNA splicing factor, as a critical regulator to modulate the recruitment of Polη and its partner RAD18 after UV exposure. We show that SART3 interacts with Polη and RAD18 via its C-terminus. Moreover, SART3 can form homodimers to promote the Polη/RAD18 interaction and PCNA monoubiquitination, a key event in TLS. Depletion of SART3 also impairs UV-induced single-stranded DNA (ssDNA) generation and RPA focus formation, resulting in an impaired Polη recruitment and a higher mutation frequency and hypersensitivity after UV treatment. Notably, we found that several SART3 missense mutations in cancer samples lessen its stimulatory effect on PCNA monoubiquitination. Collectively, our findings establish SART3 as a novel Polη/RAD18 association regulator that protects cells from UV-induced DNA damage, which functions in a RNA binding-independent fashion.

MiR-20a-5p suppresses tumor proliferation by targeting autophagy-related gene 7 in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common malignant tumor originating from the extracranial sympathetic nervous system in children. The molecular mechanisms underlying this disease are complex, and not completely understood. METHODS: Quantitative real-time PCR (qRT-PCR) was applied to quantify the expression of miR-20a-5p and its target gene ATG7 in clinical NB tissues. The biological function of miR-20a-5p and ATG7 in SH-SY5Y cells was investigated through in vitro studies (Real-Time cell kinetic analyzer, colony formation assay, caspase-Glo 3/7 assay and western blotting). The luciferase reporter assay was conducted to verify the biological relationship between miR-20a-5p and ATG7. RESULTS: Here we found that miR-20a-5p expression was significantly downregulated whereas its target autophagy-related gene 7 (ATG7) was increased along with clinical staging of NB progression. Correlation analysis showed that miR-20a-5p had a negative correlation trend with ATG7. In SH-SY5Y cells, forced expression of miR-20a-5p suppressed ATG7 expression, autophagy initiation and cellular proliferation while promoted apoptosis, suggesting a potential association between miR-20a-5p and ATG7. Further bioinformatic target prediction combined with protein expression and luciferase reporter assay verified that miR-20a-5p inhibited ATG7 by directly binding to its 3'-UTR, confirming the involvement of miR-20a-5p in the regulation of ATG7 in NB. CONCLUSIONS: These results clarified that miR-20a-5p inhibited cell proliferation and promoted apoptosis through negative regulation of ATG7 and thus autophagy suppression in SH-SY5Y cells. Therefore, defining the context-specific roles of autophagy in NB and regulatory mechanisms involved will be critical for developing autophagy-targeted therapeutics against NB. Both miR-20a-5p and ATG7 would be potential therapeutic targets for future NB treatment.

Whole-Genome Sequencing Identifies a Novel Variation of WAS Gene Coordinating With Heterozygous Germline Mutation of APC to Enhance Hepatoblastoma Oncogenesis.

Hepatoblastoma (HB), a leading primary hepatic malignancy in children, originates from primitive hepatic stem cells. This study aimed to uncover the genetic variants that are responsible for HB oncogenesis. One family, which includes the healthy parents, and two brothers affected by HB, was recruited. Whole-genome sequencing (WGS) of germline DNA from all the family members identified two maternal variants, located within APC gene and X-linked WAS gene, which were harbored by the two brothers. The mutation of APC (rs137854573, c.C1606T, p.R536X) could result in HB carcinogenesis by activating Wnt signaling. The WAS variant (c.G3T, p.M1-P5del) could promote HB cell proliferation and inhibit T-cell-based immunity by activating PLK1 signaling and inactivating TCR signaling. Further analysis reflected that WAS deficiency might affect the antitumor activity of natural killer and dendritic cells. In summary, the obtained results imply that an APC mutant together with an X-linked WAS mutant, could lead to HB tumorigenesis by activating Wnt and PLK1 signaling, inhibiting TCR signaling, and reducing the antitumor activity of natural killer and dendritic cells.