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Achaete‐scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet‐On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, colony‐formation, migration, invasion, and sphere‐formation assays. Quantitative reverse‐transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial‐mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal‐epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non‐induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway‐, EMT‐, and stemness‐related genes. ASCL2 activation may therefore be involved in the progression of HB. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single‐cell next‐generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2+CD90+CD45−CD235a−DAPI− cells were isolated as neuroblastoma CTCs using fluorescence‐activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single‐cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single‐cell RNA sequencing, angiogenesis‐related and cell cycle‐related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle‐related and proliferation‐related genes were differentially upregulated compared with the other group. In conclusion, next‐generation sequencing of CTCs at single‐cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB. [ABSTRACT FROM AUTHOR]

Aim: Most hepatoblastoma patients undergo pre/postoperative cisplatin treatment. Approximately 20% patients are cisplatin resistant, and show poor prognosis and high recurrence rates. However, some cisplatin‐sensitive patients show early recurrence. We consider that a small population of cisplatin‐resistant cells may remain after preoperative chemotherapy. Previous studies showed a correlation between DNA hypermethylation and hepatoblastoma progression. Here, we examined whether DNA hypermethylation was related to cisplatin resistance and could be a potential indicator for cisplatin as postoperative chemotherapy. Methods: We extracted DNA from 43 resected hepatoblastoma tumors. Methylation array analyses were performed in 11 samples, including six cisplatin‐sensitive and five cisplatin‐resistant samples. We also performed cDNA microarray analysis in parental and cisplatin‐resistant HuH6 cells. Through comparison of the datasets, we selected the strongest correlated cisplatin‐resistant candidate gene. Using bisulfite pyrosequencing, the candidate gene methylation level was assessed in 38 cisplatin‐sensitive patients after checking its usefulness as a substitute modality of methylation array. Correlations between the methylation status and clinical data were analyzed. Results: CSF3R was the strongest correlated variable. Bisulfite pyrosequencing analysis also confirmed CSF3R was significantly hypermethylated in cisplatin‐resistant patients. Among the 38 cisplatin‐sensitive patients, recurrence curves showed that the CSF3R high methylation patients had significantly higher recurrence than CSF3R low methylation patients. The recurrence curve of methylation high patients was similar to that of cisplatin‐resistant patients. Conclusions: Our findings suggested that CSF3R hypermethylation was related to cisplatin resistance in HB patients and could be a predictor of postoperative chemotherapy, and indicate that CSF3R high methylation patients should be treated with non‐CDDP regimens. [ABSTRACT FROM AUTHOR]

Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics‐based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type. Single circulating tumor cells analyses were carried out using a live single cell mass spectrometry system integrated with a microfluidics‐based cells enrichment technique. Potential biomarkers specific to circulating tumor cells or each cancer type (gastric colorectal cancer) were annotated, suggesting the usability of the developed method. [ABSTRACT FROM AUTHOR]

Hepatoblastoma ( HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector nuclease ( TALEN)-mediated genome editing is a powerful technique for analyzing gene functions in various cells and organisms. At target loci, TALENs can not only introduce short insertions and deletions, but also yield large deletions through the use of two TALEN pairs. Here, we report easy and efficient methods for enrichment of cells with TALEN-induced mutations and large deletions. First, we established the fluorescence-activated sorting of TALEN-induced deletions ( FAST-id) system that enabled fluorescence-activated cell sorting-mediated enrichment of cells with TALEN-induced mutations. In the FAST-id system, either EGFP or mCherry and TALENs were co-expressed. Using dual fluorescence selection, both left and right TALEN-expressing cells were easily concentrated, resulting in enrichment of TALEN-mediated mutated cells. Next, to apply the FAST-id system to enrichment of cells with large deletions, we developed the fast unification of separate endonucleases ( FUSE) method for assembly of two TALENs into a single expression vector. Using the FUSE method, we easily obtained a TALEN pair-expressing plasmid driven by a single promoter. By combining the FAST-id system and FUSE method, cells with large deletions were efficiently enriched. To the best of our knowledge, this is the first report of enrichment of cells with TALEN-induced large deletions. [ABSTRACT FROM AUTHOR]

Our previous report demonstrated a good correlation between high telomerase activity of cancer tissues and a poor prognosis of patients with colorectal cancers, except for several cases. To elucidate the additional factors that contribute to patient prognosis, the correlation among the expression levels of telomere binding proteins (TBP), the lengths of telomeres, the lengths of telomere 3′-overhang (3′-OH) and telomerase activity in 106 paired colorectal cancer and corresponding noncancerous mucosa (NCM) specimens were examined. The expression levels of eight TBP genes ( TRF1, TRF2, TIN2, TANK1, TANK2, POT1, RAP1 and TPP1) were analyzed. Among the 106 cases, 35 cases had shortened telomeres (<7 kb), 15 had shortened 3′-OH (3′-OH length ratio of cancer/NCM <0.5) and 88 were classified as telomerase-activated cancers (activity ratio of cancer/NCM >2). Comparison between NCM and cancer in each case showed that all TBP except for POT1 were downregulated in cancers. A survival analysis using a Cox proportional hazard model showed that the survival rate of the telomerase-activated cases with shortened 3′-OH and that of telomerase-inactivated cases were significantly better than that of telomerase-activated cases without 3′-OH shortening, that is, restored or maintained 3′-OH ( P = 0.018). In the telomerase-activated cancers, the length of 3′-OH was significantly correlated with the expression levels of POT1. Elongation of telomeric overhang by telomerase, which might be regulated by POT1, may contribute to the increase of malignant potential in colorectal cancers. ( Cancer Sci 2011; 102: 330-335) [ABSTRACT FROM AUTHOR]

p130Cas (Cas, rk- ssociated ubstrate) is an adaptor molecule composed of a Src homology 3 (SH3) domain, a substrate domain (SD) and a Src binding domain (SBD). The SH3 domain of Cas associates with focal adhesion kinase (FAK), but its role in cellular function has not fully been understood. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking exon 2, which encodes the SH3 domain (Cas Δexon 2). In comparison to wild-type cells, Cas exon 2Δ/Δ cells showed reduced motility, which could be due to impaired tyrosine-phosphorylation of FAK and Cas, reduced FAK/Cas/Src/CrkII binding, and also impaired localization of Cas Δexon 2 to focal adhesions on fibronectin. In addition, to analyze downstream signaling pathways regulated by Cas exon 2, we performed microarray analyses. Interestingly, we found that a deficiency of Cas exon 2 up-regulated expression of CXC Chemokine Receptor-4 and CC Chemokine Receptor-5, which may be regulated by IκBα phosphorylation. These results indicate that the SH3-encoding exon of Cas participates in cell motility, tyrosine-phosphorylation of FAK and Cas, FAK/Cas/Src/CrkII complex formation, recruitment of Cas to focal adhesions and regulation of cell motility-associated gene expression in primary fibroblasts. [ABSTRACT FROM AUTHOR]

Abstract Background : Despite the advent of effective chemotherapy, a poor prognosis has been reported for patients with metastatichepatoblastoma. To improve this prognosis, we conducted high-dose chemotherapy with autologous bone marrow rescue in patients withmetastatic hepatoblastoma. Methods and results : Three patients were treated with high-dose chemotherapy. In patient 1, high-dose chemotherapywas given after the patient’s first pulmonary relapse. Additional pulmonary metastases, which developed more than 6 monthsafter high-dose chemotherapy, were treated by multiple thoracotomy without additional chemotherapy. Patient 2 presented additionalpulmonary metastases soon after the end of the first thoracotomy and high-dose chemotherapy. Because of a decreased serum alpha-fetoproteinlevel after re-excision of the pulmonary metastases, a second round of high-dose chemotherapy was performed. In patient 3, multiplepulmonary metasteses responded to preoperative chemotherapy and disappeared according to the chest computed tomography. Intensivetreatment with a high-dose chemotherapeutic regimen was performed at the end of postoperative chemotherapy. All three patients are aliveand well, more than 6 years after receiving their diagnosis. Conclusion : The role of high-dose chemotherapy in treatment of metastatic hepatoblastoma could not be clarified,because of the small number of patients. However, the better outcome of our patients indicates that multimodal therapy, including high-dosechemotherapy, may improve the outcome of the patients with metastatic hepatoblastoma. [ABSTRACT FROM AUTHOR]