CASP8AP2

Gene Summary

Gene:CASP8AP2; caspase 8 associated protein 2
Aliases: CED-4, FLASH, RIP25
Location:6q15
Summary:This protein is highly similar to FLASH, a mouse apoptotic protein identified by its interaction with the death-effector domain (DED) of caspase 8. Studies of FLASH protein suggested that this protein may be a component of the death-inducing signaling complex that includes Fas receptor, Fas-binding adapter FADD, and caspase 8, and plays a regulatory role in Fas-mediated apoptosis. Alternative splicing results in multiple transcript variants encoding the same protein.[provided by RefSeq, Nov 2008]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:CASP8-associated protein 2
Source:NCBIAccessed: 16 March, 2017

Ontology:

What does this gene/protein do?
Show (15)

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 16 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Apoptosis Regulatory Proteins
  • Single Nucleotide Polymorphism
  • Cancer Gene Expression Regulation
  • Gene Expression Profiling
  • Apoptosis
  • Neoplasm Proteins
  • Zinc Finger E-box-Binding Homeobox 1
  • Computational Biology
  • Transcription
  • RTPCR
  • Childhood Cancer
  • Recurrence
  • Promoter Regions
  • Gene Expression
  • Messenger RNA
  • Acute Lymphocytic Leukaemia
  • Residual Disease
  • Chromosome Deletion
  • Mesenchymal Stromal Cells
  • Mutation
  • Calcium-Binding Proteins
  • Frameshift Mutation
  • Adolescents
  • NOTCH1 Receptor
  • CASP8AP2
  • Single-Stranded Conformational Polymorphism
  • bcl-2-Associated X Protein
  • Translocation
  • Histones
  • Polycomb-Group Proteins
  • Transcriptome
  • Cluster Analysis
  • DNA Methylation
  • Ubiquitin-Protein Ligases
  • Colorectal Cancer
  • Infant
  • Biomarkers, Tumor
  • Sequence Deletion
  • Loss of Heterozygosity
  • Chromosome 6
Tag cloud generated 16 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (3)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CASP8AP2 (cancer-related)

Zhuang K, Yan Y, Zhang X, et al.
Gastrin promotes the metastasis of gastric carcinoma through the β-catenin/TCF-4 pathway.
Oncol Rep. 2016; 36(3):1369-76 [PubMed] Related Publications
Gastric cancer is the most common epithelial malignancy and the second leading cause of cancer-related death worldwide; metastasis is a crucial factor in the progression of gastric cancer. The present study applied gastrin-17 amide (G-17) in SGC7901 cells. The results showed that G-17 promoted the cell cycle by accelerating the G0/G1 phase and by increasing the cell proliferation rate by binding to the gastrin receptor. The migratory and invasive abilities of the SGC7901 cells were increased by G-17. The expression levels of matrix metalloproteinase (MMP)-7, MMP-9 and vascular endothelial growth factor (VEGF) were enhanced by G-17 as well. Moreover, G-17 caused the overexpression of β-catenin and TCF-4. G-17 also caused a preferential cytoplasmic and nuclear localization of β-catenin with a high TOP-FLASH activity. Finally, axin reduced the migratory and invasive abilities of the SGC7901 cells, and inhibited the expression of β-catenin, TCF-4, MMP-7, MMP-9 and VEGF; these effects were counteracted by adding G-17. In summary, the present study confirmed the proliferation and metastasis-promoting role of G-17 via binding to the gastrin receptor, and the β-catenin/TCF-4 pathway was found to be essential for mediating G-17-induced metastasis in gastric cancer. These results may provide a novel gene target for the treatment of gastric cancer.

Zhou F, Huo J, Liu Y, et al.
Elevated glucose levels impair the WNT/β-catenin pathway via the activation of the hexosamine biosynthesis pathway in endometrial cancer.
J Steroid Biochem Mol Biol. 2016; 159:19-25 [PubMed] Related Publications
Endometrial cancer (EC) is one of the most common gynecological malignancies in the world. Associations between fasting glucose levels (greater than 5.6mmol/L) and the risk of cancer fatality have been reported. However, the underlying link between glucose metabolic disease and EC remains unclear. In the present study, we explored the influence of elevated glucose levels on the WNT/β-catenin pathway in EC. Previous studies have suggested that elevated concentrations of glucose can drive the hexosamine biosynthesis pathway (HBP) flux, thereby enhancing the O-GlcNAc modification of proteins. Here, we cultured EC cell lines, AN3CA and HEC-1-B, with various concentrations of glucose. Results showed that when treated with high levels of glucose, both lines showed increased expression of β-catenin and O-GlcNAcylation levels; however, these effects could be abolished by the HBP inhibitors, Azaserine and 6-Diazo-5-oxo-l-norleucine, and be restored by glucosamine. Moreover the AN3CA and HEC-1-B cells that were cultured with or without PUGNAc, an inhibitor of the O-GlcNAcase, showed that PUGNAc increased β-catenin levels. The results suggest that elevated glucose levels increase β-catenin expression via the activation of the HBP in EC cells. Subcellular fractionation experiments showed that AN3CA cells had a higher expression of intranuclear β-catenin in high glucose medium. Furthermore, TOP/FOP-Flash and RT-PCR results showed that glucose-induced increased expression of β-catenin triggered the transcription of target genes. In conclusion, elevated glucose levels, via HBP, increase the O-GlcNAcylation level, thereby inducing the over expression of β-catenin and subsequent transcription of the target genes in EC cells.

Galani BR, Sahuc ME, Sass G, et al.
Khaya grandifoliola C.DC: a potential source of active ingredients against hepatitis C virus in vitro.
Arch Virol. 2016; 161(5):1169-81 [PubMed] Related Publications
In this study, we examined the antiviral properties of Khaya grandifoliola C.DC (Meliaceae) on the hepatitis C virus (HCV) life cycle in vitro and identified some of the chemical constituents contained in the fraction with the most antiviral activity. Dried bark powder was extracted by maceration in a methylene chloride/methanol (MCM) system (50:50; v/v) and separated on silica gel by flash chromatography. Infection and replication rates in Huh-7 cells were investigated by luciferase reporter assay and indirect immunofluorescence assay using subgenomic replicons, HCV pseudotyped particles, and cell-culture-derived HCV (HCVcc), respectively. Cell viability was assessed by MTT assay, and cellular gene expression was analysed by qRT-PCR. The chemical composition of the fraction with the most antiviral activity was analysed by coupled gas chromatography and mass spectrometry (GC-MS). Five fractions of different polarities (F0-F100) were obtained from the MCM extract. One fraction (KgF25) showed the strongest antiviral effect on LucUbiNeoET replicons at nontoxic concentrations. Tested at 100 µg/mL, KgF25 had a high inhibitory effect on HCV replication, comparable to that of 0.01 µM daclatasvir or 1 µM telaprevir. This fraction also inhibited HCVcc infection by mostly targeting the entry step. KgF25 inhibited HCV entry in a pan-genotypic manner by directly inactivating free viral particles. Its antiviral effects were mediated by the transcriptional upregulation of the haem oxygenase-1 gene and interferon antiviral response. Three constituents, namely, benzene, 1,1'-(oxydiethylidene)bis (1), carbamic acid, (4-methylphenyl)-, 1-phenyl (2), and 6-phenyl, 4-(1'-oxyethylphenyl) hexene (3), were identified from the active fraction KgF25 by GC-MS. Khaya grandifoliola contains ingredients capable of acting on different steps of the HCV life cycle.

Gao Y, Lu X
Decreased expression of MEG3 contributes to retinoblastoma progression and affects retinoblastoma cell growth by regulating the activity of Wnt/β-catenin pathway.
Tumour Biol. 2016; 37(2):1461-9 [PubMed] Related Publications
The aberrant expression of MEG3 has been found in some types of cancers; however, little is known concerning the function of MEG3 in retinoblastoma. To elucidate the roles of MEG3 in retinoblastoma, MEG3 expression was quantified in 63 retinoblastoma samples and corresponding nontumor tissues in this work. Moreover, retinoblastoma cell lines were transfected with pcDNA3.1-MEG3 or si-MEG3, after which proliferation, apoptosis, and expression of β-catenin were assayed. TOP-Flash reporter assay was also used to investigate the activity of the Wnt/β-catenin pathway. The results showed that MEG3 was downregulated in retinoblastoma tissues, and the level of MEG3 was negatively associated with IIRC stages and nodal or distant metastasis. More importantly, Kaplan-Meier survival analysis demonstrated that patients with low MEG3 expression had poorer survival and multivariate Cox regression analysis revealed that MEG3 was an independent prognostic factor in retinoblastoma patients. We also observed that MEG3 expression can be modulated by DNA methylation by using 5-aza-CdR treatment. In addition, overexpression of MEG3 suppressed proliferation, promoted apoptosis, and influences the activity of the Wnt/β-catenin pathway in retinoblastoma cell lines. Furthermore, we found that Wnt/β-catenin pathway activator rescued the anticancer effect of MEG3 in retinoblastoma. In conclusion, our study for the first time demonstrated that MEG3 was a tumor suppressor by negatively regulating the activity of the Wnt/β-catenin pathway in the progression of retinoblastoma and might serve as a prognostic biomarker and molecular therapeutic target.

Ma Y, Zhu B, Liu X, et al.
Inhibition of oleandrin on the proliferation show and invasion of osteosarcoma cells in vitro by suppressing Wnt/β-catenin signaling pathway.
J Exp Clin Cancer Res. 2015; 34:115 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Osteosarcoma (OS) is a high-grade bone sarcoma with early metastasis potential, and the clinical chemotherapy drugs that are currently used for its treatment have some limitations. Recently, several studies have reported the selective antitumor effect of oleandrin on various tumor cells. In this study, we aimed to evaluate the effects and underlying mechanisms of oleandrin on OS cells.
METHODS: The effect of oleandrin on the proliferation, morphology, and apoptosis of U2OS and SaOS-2 cells were analyzed in vitro. The activity of the Wnt/β-catenin signaling pathway was determined using a dual luciferase assay. Semi-quantitative RT-PCR and western blot assays were performed to evaluate the mRNA and total protein expression of the downstream target genes. Changes of β-catenin in intracellular localization were also explored using a western blot after separating the nucleus and cytoplasm proteins. The MMP-2 and MMP-9 enzymatic activities were determined using gelatin zymography.
RESULTS: Oleandrin significantly inhibited the proliferation and invasion of OS cells in vitro, and induced their apoptosis. After treatment with oleandrin, the TOP/FOP flash ratio in OS cells was noticeably decreased, which indicated that the Wnt/β-catenin signaling pathway was repressed. The expression of related Wnt target genes and total β-catenin was downregulated, and a reduced nuclear β-catenin level by oleandrin was observed as well. In addition, oleandrin suppressed the activities of MMP-2 and MMP-9.
CONCLUSIONS: Oleandrin, in vitro, exerted a strong antitumor effect on human OS cells by suppressing the Wnt/β-catenin signaling pathway, which interfered with the proliferation and invasion of OS cells, as well as induced cells apoptosis. Moreover, the expression and activities of MMP-2 and MMP-9 were downregulated by oleandrin, which contributed to the cells' lower invasiveness.

Thomas LN, Merrimen J, Bell DG, et al.
Prolactin- and testosterone-induced carboxypeptidase-D correlates with increased nitrotyrosines and Ki67 in prostate cancer.
Prostate. 2015; 75(15):1726-36 [PubMed] Related Publications
BACKGROUND: Carboxypeptidase-D (CPD) cleaves C-terminal arginine for conversion to nitric oxide (NO) by nitric oxide synthase (NOS). Prolactin (PRL) and androgens stimulate CPD gene transcription and expression, which increases intracellular production of NO to promote viability of prostate cancer (PCa) cells in vitro. The current study evaluated whether hormonal upregulation of CPD and NO promote PCa cell viabilty in vivo, by correlating changes in expression of CPD and nitrotyrosine residues (products of NO action) with proliferation marker Ki67 and associated proteins during PCa development and progression.
METHODS: Fresh prostate tissues, obtained from 40 men with benign prostatic hyperplasia (BPH) or PCa, were flash-frozen at the time of surgery and used for RT-qPCR analysis of CPD, androgen receptor (AR), PRL receptor (PRLR), eNOS, and Ki67 levels. Archival paraffin-embedded tissues from 113 men with BPH or PCa were used for immunohistochemical (IHC) analysis of CPD, nitrotyrosines, phospho-Stat5 (for activated PRLR), AR, eNOS/iNOS, and Ki67.
RESULTS: RT-qPCR and IHC analyses showed strong AR and PRLR expression in benign and malignant prostates. CPD mRNA levels increased ∼threefold in PCa compared to BPH, which corresponded to a twofold increase in Ki67 mRNA levels. IHC analysis showed a progressive increase in CPD from 11.4 ± 2.1% in benign to 21.8 ± 3.2% in low-grade (P = 0.007), 40.7 ± 4.0% in high-grade (P < 0.0001) and 50.0 ± 9.5% in castration-recurrent PCa (P < 0.0001). Immunostaining for nitrotyrosines and Ki67 mirrored these increases during PCa progression. CPD, nitrotyrosines, and Ki67 tended to co-localize, as did phospho-Stat5.
CONCLUSIONS: CPD, nitrotyrosine, and Ki67 levels were higher in PCa than in benign and tended to co-localize, along with phospho-Stat5. The strong correlation in expression of these proteins in benign and malignant prostate tissues, combined with abundant AR and PRLR, supports in vitro evidence that the CPD-Arg-NO pathway is involved in the regulation of PCa cell proliferation. It further highlights a role for PRL in the development and progression of PCa.

Dong Z, Zhou L, Han N, et al.
Wnt/β-catenin pathway involvement in ionizing radiation-induced invasion of U87 glioblastoma cells.
Strahlenther Onkol. 2015; 191(8):672-80 [PubMed] Related Publications
BACKGROUND: Radiotherapy has been reported to promote the invasion of glioblastoma cells; however, the underlying mechanisms remain unclear. Here, we investigated the role of the Wnt/β-catenin pathway in radiation-induced invasion of glioblastoma cells.
METHODS: U87 cells were irradiated with 3 Gy or sham irradiated in the presence or absence of the Wnt/β-catenin pathway inhibitor XAV 939. Cell invasion was determined by an xCELLigence real-time cell analyser and matrigel invasion assays. The intracellular distribution of β-catenin in U87 cells with or without irradiation was examined by immunofluorescence and Western blotting of nuclear fractions. We next investigated the effect of irradiation on Wnt/β-catenin pathway activity using TOP/FOP flash luciferase assays and quantitative polymerase chain reaction analysis of β-catenin target genes. The expression levels and activities of two target genes, matrix metalloproteinase (MMP)-2 and MMP-9, were examined further by Western blotting and zymography.
RESULTS: U87 cell invasiveness was increased significantly by ionizing radiation. Interestingly, ionizing radiation induced nuclear translocation and accumulation of β-catenin. Moreover, we found increased β-catenin/TCF transcriptional activities, followed by up-regulation of downstream genes in the Wnt/β-catenin pathway in irradiated U87 cells. Importantly, inhibition of the Wnt/β-catenin pathway by XAV 939, which promotes degradation of β-catenin, significantly abrogated the pro-invasion effects of irradiation. Mechanistically, XAV 939 suppressed ionizing radiation-triggered up-regulation of MMP-2 and MMP-9, and inhibited the activities of these gelatinases.
CONCLUSION: Our data demonstrate a pivotal role of the Wnt/β-catenin pathway in ionizing radiation-induced invasion of glioblastoma cells, and suggest that targeting β-catenin is a promising therapeutic approach to overcoming glioma radioresistance.

Li F, Wang T, Tang S
SOX14 promotes proliferation and invasion of cervical cancer cells through Wnt/β-catenin pathway.
Int J Clin Exp Pathol. 2015; 8(2):1698-704 [PubMed] Free Access to Full Article Related Publications
SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although SOX14 expression profile and function during development was revealed in various animal model systems, the role of this gene during tumor progression is totally unknown. In this study, the expression of SOX14 increases in four cervical cancer cell lines (HeLa, Caski, HT-3 and SiHa) as revealed by real-time PCR and Western blot analyses. Through knocking down or overexpressing SOX14 in SiHa and HeLa cells, the expression level of SOX14 was found to be positively related to cell proliferation and invasion in vitro. Moreover, the TOP-Flash reporter assay and Western blot for β-catenin genes of the Wnt/β-catenin pathway, indicated that SOX14 significantly activated Wnt/β-catenin signaling. Further study showed that the blockage of Wnt/β-catenin pathway by knocking down β-catenin resulted in a significant inhibition of cell proliferation and invasion capacity induced by SOX14. To summarize, these results demonstrate that SOX14 can promote proliferation and invasion capacity of cervical cancer cells by activating the Wnt/β-catenin pathway.

Gonzalez BD, Jim HS, Donovan KA, et al.
Course and Moderators of Hot Flash Interference during Androgen Deprivation Therapy for Prostate Cancer: A Matched Comparison.
J Urol. 2015; 194(3):690-5 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Many men receiving androgen deprivation therapy for prostate cancer experience hot flashes. This study aimed to describe the course of hot flash interference with time in androgen deprivation therapy recipients relative to matched prostate cancer and cancer-free controls from before the start of androgen deprivation therapy to 12 months later. We also examined demographic, clinical and genetic predictors of the impact of androgen deprivation therapy on hot flash interference.
MATERIALS AND METHODS: Three groups were examined, including 60 patients with prostate cancer recruited before or within 21 days of starting androgen deprivation therapy, 83 age and education matched patients with prostate cancer treated with prostatectomy only, and 86 age and education matched men with no history of cancer. Participants provided blood samples and completed the Hot Flash Related Daily Interference Scale at baseline as well as 6 and 12 months later.
RESULTS: Androgen deprivation therapy recipients reported increasing hot flash interference with time relative to controls (p <0.001). Group differences were evident at 6 and 12 months (all p <0.001) with androgen deprivation therapy recipients reporting greater hot flash interference than controls. Several genetic polymorphisms were found to predict greater increases in hot flash interference (all p <0.01), including polymorphisms on genes associated with vasoconstriction, immune function, neurotransmission and circadian rhythms. Androgen deprivation therapy recipients who were younger and had a lower body mass index at baseline also showed greater increases in hot flash interference with time (all p ≤0.01).
CONCLUSIONS: This study, which is to our knowledge the first to prospectively examine hot flash interference in androgen deprivation therapy recipients, reveals that those with certain genetic polymorphisms, younger age and lower body mass index had greater increases in hot flash interference with time relative to controls.

Choi MR, Gwak M, Yoo NJ, Lee SH
Regional Bias of Intratumoral Genetic Heterogeneity of Apoptosis-Related Genes BAX, APAF1, and FLASH in Colon Cancers with High Microsatellite Instability.
Dig Dis Sci. 2015; 60(6):1674-9 [PubMed] Related Publications
BACKGROUND: Apoptosis inactivation and intratumoral heterogeneity (ITH) are common features of cancers, including colorectal cancer (CRC). Inactivation of apoptosis prolongs cancer cell survival, and ITH may contribute to CRC progression.
AIM: To examine the presence and extent of mutational ITH in the pro-apoptotic genes APAF1, BAX, and FLASH and the association of mutational ITH with pathologic parameters of CRC.
METHODS: The ITH of mutations in the mononucleotide repeats of APAF1, BAX and FLASH in different tumors were analyzed in 16 cases of CRC with high microsatellite instability (MSI-H) and 41 cases of CRC with stable MSI/low MSI (MSS/MSI-L) by single-strand conformation polymorphism and DNA sequencing analyses.
RESULTS: Frameshift mutations of APAF1, BAX, and FLASH were identified in 19, 31, and 6 % of CRC with MSI-H, respectively, but also in cases of CRC with MSS/MSI-L. All but one CRC with a mutation (8/9) harbored regional ITH of the APAF1, BAX and FLASH frameshift mutations. ITH, however, was not associated with histopathologic features of CRC with MSI-H, suggesting that ITH might not be related to development of the MSI-H phenotype itself, but rather to disease progression.
CONCLUSIONS: Our results indicate that the APAF1, BAX, and FLASH genes not only harbor frameshift mutations but also demonstrate mutational ITH, which together might play a role in the tumorigenesis of CRC with MSI-H by affecting the apoptosis of cancer cells. Our data also suggest that multiregional mutation analysis is needed for a better evaluation of the mutation status in CRC.

Cui L, Gao C, Zhang RD, et al.
Low expressions of ARS2 and CASP8AP2 predict relapse and poor prognosis in pediatric acute lymphoblastic leukemia patients treated on China CCLG-ALL 2008 protocol.
Leuk Res. 2015; 39(2):115-23 [PubMed] Related Publications
ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.

Chen Q, Cao HZ, Zheng PS
LGR5 promotes the proliferation and tumor formation of cervical cancer cells through the Wnt/β-catenin signaling pathway.
Oncotarget. 2014; 5(19):9092-105 [PubMed] Free Access to Full Article Related Publications
Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a seven transmembrane receptor known as a potential stem cell marker for intestinal crypts and hair follicles, has recently been found to be overexpressed in some types of human cancers. However, the role of LGR5 in cervical cancer remains unclear. In this study, the expression of LGR5 gradually increases from normal cervix to cervical cancer in situ and to cervical cancers as revealed by immunohistochemistry and western blot analyses. Through knocking down or overexpressing LGR5 in SiHa and HeLa cells, the expression level of LGR5 was found to be positively related to cell proliferation in vitro and to tumor formation in vivo. Further investigation indicated that LGR5 protein could significantly promote the acceleration of cell cycle. Moreover, the TOP-Flash reporter assay and western blot for β-catenin, cyclinD1, and c-myc proteins, target genes of the Wnt/β-catenin pathway, indicated that LGR5 significantly activated Wnt/β-catenin signaling. Additionally, the blockage of Wnt/β-catenin pathway resulted in a significant inhibition of cell proliferation induced by LGR5. Taken together, these results demonstrate that LGR5 can promote proliferation and tumor formation in cervical cancer cells by activating the Wnt/β-catenin pathway.

Scholtysik R, Kreuz M, Hummel M, et al.
Characterization of genomic imbalances in diffuse large B-cell lymphoma by detailed SNP-chip analysis.
Int J Cancer. 2015; 136(5):1033-42 [PubMed] Related Publications
The pathogenesis of diffuse large B-cell lymphomas (DLBCL) is only partly understood. We analyzed 148 DLBCL by single nucleotide polymorphism (SNP)-chips to characterize genomic imbalances. Seventy-nine cases were of the germinal center B-cell like (GCB) type of DLBCL, 49 of the activated B-cell like (ABC) subtype and 20 were unclassified DLBCL. Twenty-four regions of recurrent genomic gains and 38 regions of recurrent genomic losses were identified over the whole cohort, with a median of 25 imbalances per case for ABC-DLBCL and 19 per case for GCB-DLBCL. Several recurrent copy number changes showed differential frequencies in the GCB- and ABC-DLBCL subgroups, including gains of HDAC7A predominantly in GCB-DLBCL (38% of cases) and losses of BACH2 and CASP8AP2 predominantly in ABC-DLBCL (35%), hinting at disparate pathogenetic mechanisms in these entities. Correlating gene expression and copy number revealed a strong gene dosage effect in all tumors, with 34% of probesets showing a concordant expression change in affected regions. Two new potential tumor suppressor genes emerging from the analysis, CASP3 and IL5RA, were sequenced in ten and 16 candidate cases, respectively. However, no mutations were found, pointing to a potential haploinsufficiency effect of these genes, considering their reduced expression in cases with deletions. Our study thus describes differences and similarities in the landscape of genomic aberrations in the DLBCL subgroups in a large collection of cases, confirming already known targets, but also discovering novel copy number changes with possible pathogenetic relevance.

Edmonds MD, Eischen CM
Differences in miRNA expression in early stage lung adenocarcinomas that did and did not relapse.
PLoS One. 2014; 9(7):e101802 [PubMed] Free Access to Full Article Related Publications
Relapse of adenocarcinoma, the most common non-small cell lung cancer (NSCLC), is a major clinical challenge to improving survival. To gain insight into the early molecular events that contribute to lung adenocarcinoma relapse, and taking into consideration potential cell type specificity, we used stringent criteria for sample selection. We measured miRNA expression only from flash frozen stage I lung adenocarcinomas, excluding other NSCLC subtypes. We compared miRNA expression in lung adenocarcinomas that relapsed within two years to those that did not relapse within three years after surgical resection prior to adjuvant therapy. The most significant differences in mRNA expression for recurrent tumors compared to non-recurrent tumors were decreases in miR-106b*, -187, -205, -449b, -774* and increases in miR-151-3p, let-7b, miR-215, -520b, and -512-3p. A unique comparison between adjacent normal lung tissue from relapse and non-relapse groups revealed dramatically different miRNA expression, suggesting dysregulation of miRNA in the environment around the tumor. To assess patient-to-patient variability, miRNA levels in the tumors were normalized to levels in matched adjacent normal lung tissue. This analysis revealed a different set of significantly altered miRNA in tumors that recurred compared to tumors that did not. Together our analyses elucidated miRNA not previously linked to lung adenocarcinoma that likely have important roles in its development and progression. Our results also highlight the differences in miRNA expression in normal lung tissue in adenocarcinomas that do and do not recur. Most notably, our data identified those miRNA that distinguish early stage tumors likely to relapse prior to treatment and miRNA that could be further studied for use as biomarkers for prognosis, patient monitoring, and/or treatment decisions.

Yang Z, Zhang M, Xu K, et al.
Knockdown of YAP1 inhibits the proliferation of osteosarcoma cells in vitro and in vivo.
Oncol Rep. 2014; 32(3):1265-72 [PubMed] Related Publications
Yes-associated protein 1 (YAP1) is a candidate oncogene that is involved in tumorigenesis and progression of many malignant tumors. Recently, many studies have revealed that YAP1 is highly expressed in human osteosarcoma. To investigate the role of YAP1 in osteosarcoma tumorigenesis, the expression of YAP1 in the osteosarcoma cell lines (MG-63 and HOS) was knocked down by small hairpin RNA (shRNA), and the cell proliferation and colony formation assay showed that knockdown of YAP1 significantly suppressed the cell proliferation and colony formation of osteosarcoma cells. Subsequently, cell cycle distribution was analyzed by flow cytometry, and the results showed an accumulation of YAP1-knockdown cells in the G0/G1 phase, suggesting that YAP1 knockdown results in the arrest of cell cycle progression. Additionally, the knockdown of YAP1 also inhibited the tumorsphere formation in vitro and the growth of xenograft tumors in vivo. Therefore, these data suggest that YAP1 knockdown inhibits the proliferation of osteosarcoma cells. However, the mechanism of action was unclear. Further investigation showed that in the YAP1-knockdown MG-63 and HOS cells, the level of cylinD1 and c-myc expression, target genes of the Wnt signaling pathway and TOP-Flash reporter activity were all significantly decreased, which indicated that the inhibitory effect of YAP1 knockdown on osteosarcoma might be associated with the Wnt signaling pathway. Taken together, our results demonstrated that YAP1 is an important regulator of osteosarcoma tumorigenesis and knockdown of YAP1 would be a novel therapeutic strategy for osteosarcoma.

Johnson H, White FM
Quantitative analysis of signaling networks across differentially embedded tumors highlights interpatient heterogeneity in human glioblastoma.
J Proteome Res. 2014; 13(11):4581-93 [PubMed] Free Access to Full Article Related Publications
Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor, with a dismal mean survival even with the current standard of care. Although in vitro cell systems can provide mechanistic insight into the regulatory networks governing GBM cell proliferation and migration, clinical samples provide a more physiologically relevant view of oncogenic signaling networks. However, clinical samples are not widely available and may be embedded for histopathologic analysis. With the goal of accurately identifying activated signaling networks in GBM tumor samples, we investigated the impact of embedding in optimal cutting temperature (OCT) compound followed by flash freezing in LN2 vs immediate flash freezing (iFF) in LN2 on protein expression and phosphorylation-mediated signaling networks. Quantitative proteomic and phosphoproteomic analysis of 8 pairs of tumor specimens revealed minimal impact of the different sample processing strategies and highlighted the large interpatient heterogeneity present in these tumors. Correlation analyses of the differentially processed tumor sections identified activated signaling networks present in selected tumors and revealed the differential expression of transcription, translation, and degradation associated proteins. This study demonstrates the capability of quantitative mass spectrometry for identification of in vivo oncogenic signaling networks from human tumor specimens that were either OCT-embedded or immediately flash-frozen.

Bianchi A, Dufort S, Fortin PY, et al.
In vivo MRI for effective non-invasive detection and follow-up of an orthotopic mouse model of lung cancer.
NMR Biomed. 2014; 27(8):971-9 [PubMed] Related Publications
One of the main reasons for the dismal prognosis of lung cancer is related to the late diagnosis of this pathology. In this study, we evaluated the potential of optimized lung MRI techniques as a completely non-invasive approach for non-small-cell lung cancer (NSCLC) MRI in vivo detection and follow-up in a mouse model of lung adenocarcinoma expressing the luciferase gene. Bioluminescent lung tumour cells were orthotopically implanted in immuno-deficient mice. Ultra-short echo-time (UTE) MRI free-breathing acquisitions were compared with standard gradient-echo lung MRI (FLASH) using both respiratory-gated and free-breathing protocols. The MRI findings were validated against bioluminescence imaging (BLI) and gold-standard histopathology analysis. Adenocarcinoma-like pathological tissue was successfully identified in all the mice with gated-FLASH and non-gated UTE MRI, and good tumour co-localization was found between MRI, BLI and histological analyses. An excellent or good correlation was found between the measured bioluminescent signal and the total tumour volumes quantified with UTE MRI or gated-FLASH MRI, respectively. No significant correlation was found when the tumours were segmented on non-gated MR FLASH images. MRI was shown to be a powerful imaging tool able to detect, quantify and longitudinally monitor the development of sub-millimetric NSCLCs. To our knowledge, this is the first study which proves the feasibility of a completely non-invasive MRI quantitative detection of lung adenocarcinoma in freely breathing mice. The absence of ionizing radiation and the high-resolution of MRI, along with the complete non-invasiveness and good reproducibility of the proposed non-gated protocol, make this imaging tool ideal for direct translational applications.

Dragoi AM, Swiss R, Gao B, Agaisse H
Novel strategies to enforce an epithelial phenotype in mesenchymal cells.
Cancer Res. 2014; 74(14):3659-72 [PubMed] Free Access to Full Article Related Publications
E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definition of several known regulators of E-cadherin expression, including ZEB1, HDAC1, and MMP14. We identified three new regulators (FLASH, CASP7, and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. In addition, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a posttranscriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through posttranscriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT.

Xing J, Cao G, Fu C
HMGA1 interacts with β-catenin to positively regulate Wnt/β-catenin signaling in colorectal cancer cells.
Pathol Oncol Res. 2014; 20(4):847-51 [PubMed] Related Publications
The high mobility group A1 (HMGA1) protein plays an important role in numerous biological processes, such as embryogenesis, cell proliferation, differentiation, apoptosis and carcinogenesis. Wnt/β-catenin signaling pathway plays a key role in development and cancer. Although previous reports have shown HMGA1 protein level can be induced by Wnt/β-catenin signaling pathway, however, the specific mechanism of HMGA1 on regulating Wnt/β-catenin signaling remains unclear. Here, we reported that HMGA1 interacted with β-catenin by using coimmunoprecipitation approach with exogenous and endogenous protein samples. HMGA1 positively regulated Wnt/β-catenin signaling, as determined by that HMGA1 increased the TOP-FLASH activity in a dose-dependent manner and β-catenin downstream target gene expression. Moreover, HMGA1 induced proliferation of colorectal cancer cells. Mechanistically, HMGA1 increased the β-catenin-TCF4 complex formation. Importantly, there was a correlation between HMGA1 and β-catenin expression in human colorectal cancer tissues. In summary, HMGA1 positively regulates Wnt/β-catenin signaling through interacting with β-catenin, which leads to increase the β-catenin-TCF4 complex formation. This suggests that targeting HMGA1 may be a useful therapeutic option in clinical application.

Juárez-Velázquez R, Reyes-León A, Salas-Labadía C, et al.
Significance of CASP8AP2 and H2AFZ expression in survival and risk of relapse in children with acute lymphoblastic leukemia.
Leuk Lymphoma. 2014; 55(10):2305-11 [PubMed] Related Publications
Novel biomarkers for risk refinement and stratification in childhood acute lymphoblastic leukemia (ALL) are needed to optimize treatment results. We studied the expression of CASP8AP2 and H2AFZ associated with relapse and survival in bone marrow samples from newly diagnosed children with ALL. We found: (a) an increased risk for early relapse in those patients with low expression of CASP8AP2 (odds ratio [OR] 3.93, 95% confidence interval [CI] 1.40-11.02, p < 0.05) confirming its usefulness as a predictive risk marker, although H2AFZ did not present the same effect; (b) patients with low expressions of CASP8AP2 and H2AFZ had inferior survival rates (p < 0.001); (c) the predictive values regarding low expressions of H2AFZ and CASP8AP2 and high white blood cell count suggest that these features could help to identify more accurately patients at greater risk of relapse.

Li ZG, Jiao Y, Li WJ, et al.
Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia.
Leuk Res. 2013; 37(10):1287-93 [PubMed] Related Publications
DNA hypermethylation of Caspase 8 associated protein 2 (CASP8AP2) and its role in childhood acute lymphoblastic leukemia (ALL) is unclear. We analyzed methylation status of CpG sites upstream of CASP8AP2 gene in 86 children with ALL by bisulfite sequencing and quantitative PCR. Methylation percentage of two CpG sites at positions of -1189 and -1176 was inversely correlated with mRNA expression (Spearman correlation: -0.333, P=0.002). High methylation was associated with the existence of minimal residual disease (MRD) at day 78 (P=0.035), The patients in high methylation group had a poor treatment outcome. The combination of methylation level and MRD at day 33 might improve current risk stratification.

Patil JR, Jayaprakasha GK, Kim J, et al.
5-Geranyloxy-7-methoxycoumarin inhibits colon cancer (SW480) cells growth by inducing apoptosis.
Planta Med. 2013; 79(3-4):219-26 [PubMed] Related Publications
For the first time, three coumarins were isolated from the hexane extract of limes (Citrus aurantifolia) and purified by flash chromatography. The structures were identified by NMR (1D, 2D) and mass spectral analyses as 5-geranyloxy-7-methoxycoumarin, limettin, and isopimpinellin. These compounds inhibited human colon cancer (SW-480) cell proliferation, with 5-geranyloxy-7-methoxycoumarin showing the highest inhibition activity (67 %) at 25 µM. Suppression of SW480 cell proliferation by 5-geranyloxy-7-methoxycoumarin was associated with induction of apoptosis, as evidenced by annexin V staining and DNA fragmentation. In addition, 5-geranyloxy-7-methoxycoumarin arrested cells at the G0/G1 phase, and induction of apoptosis was demonstrated through the activation of tumour suppressor gene p53, caspase8/3, regulation of Bcl2, and inhibition of p38 MAPK phosphorylation. These findings suggest that 5-geranyloxy-7-methoxycoumarin has potential as a cancer preventive agent.

Diaz RJ, Guduk M, Romagnuolo R, et al.
High-resolution whole-genome analysis of skull base chordomas implicates FHIT loss in chordoma pathogenesis.
Neoplasia. 2012; 14(9):788-98 [PubMed] Free Access to Full Article Related Publications
Chordoma is a rare tumor arising in the sacrum, clivus, or vertebrae. It is often not completely resectable and shows a high incidence of recurrence and progression with shortened patient survival and impaired quality of life. Chemotherapeutic options are limited to investigational therapies at present. Therefore, adjuvant therapy for control of tumor recurrence and progression is of great interest, especially in skull base lesions where complete tumor resection is often not possible because of the proximity of cranial nerves. To understand the extent of genetic instability and associated chromosomal and gene losses or gains in skull base chordoma, we undertook whole-genome single-nucleotide polymorphism microarray analysis of flash frozen surgical chordoma specimens, 21 from the clivus and 1 from C1 to C2 vertebrae. We confirm the presence of a deletion at 9p involving CDKN2A, CDKN2B, and MTAP but at a much lower rate (22%) than previously reported for sacral chordoma. At a similar frequency (21%), we found aneuploidy of chromosome 3. Tissue microarray immunohistochemistry demonstrated absent or reduced fragile histidine triad (FHIT) protein expression in 98% of sacral chordomas and 67%of skull base chordomas. Our data suggest that chromosome 3 aneuploidy and epigenetic regulation of FHIT contribute to loss of the FHIT tumor suppressor in chordoma. The finding that FHIT is lost in a majority of chordomas provides new insight into chordoma pathogenesis and points to a potential new therapeutic target for this challenging neoplasm.

Morenos L, Saffery R, Mechinaud F, et al.
Evaluation of microRNA expression in patient bone marrow aspirate slides.
PLoS One. 2012; 7(8):e42951 [PubMed] Free Access to Full Article Related Publications
Like formalin fixed paraffin embedded (FFPE) tissues, archived bone marrow aspirate slides are an abundant and untapped resource of biospecimens that could enable retrospective molecular studies of disease. Historically, RNA obtained from slides is limited in utility because of their low quality and highly fragmented nature. MicroRNAs are small (≈ 22 nt) non-coding RNA that regulate gene expression, and are speculated to preserve well in FFPE tissue. Here we investigate the use of archived bone marrow aspirate slides for miRNA expression analysis in paediatric leukaemia. After determining the optimal method of miRNA extraction, we used TaqMan qRT-PCR to identify reference miRNA for normalisation of other miRNA species. We found hsa-miR-16 and hsa-miR-26b to be the most stably expressed between lymphoblastoid cell lines, primary bone marrow aspirates and archived samples. We found the average fold change in expression of hsa-miR-26b and two miRNA reportedly dysregulated in leukaemia (hsa-miR-128a, hsa-miR-223) was <0.5 between matching archived slide and bone marrow aspirates. Differential expression of hsa-miR-128a and hsa-miR-223 was observed between leukaemic and non-leukaemic bone marrow from archived slides or flash frozen bone marrow. The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role miRNA play in the development and long term outcome of hematologic, as well as non-hematologic, diseases.

Chen YL, Ko CJ, Lin PY, et al.
Clustered DNA methylation changes in polycomb target genes in early-stage liver cancer.
Biochem Biophys Res Commun. 2012; 425(2):290-6 [PubMed] Related Publications
Polycomb-group proteins mark specific chromatin conformations in embryonic and somatic stem cells that are critical for maintenance of their "stemness". These proteins also mark altered chromatin modifications identified in various cancers. In normal differentiated cells or advanced cancerous cells, these polycomb-associated loci are frequently associated with increased DNA methylation. It has thus been hypothesized that changes in DNA methylation status within polycomb-associated loci may dictate cell fate and that abnormal methylation within these loci may be associated with tumor development. To assess this, we examined the methylation states of four polycomb target loci -Trip10, Casp8AP2, ENSA, and ZNF484 - in liver cancer. These four targets were selected because their methylation levels are increased during mesenchymal stem cell-to-liver differentiation. We found that these four loci were hypomethylated in most early-stage liver cancer specimens. For comparison, two non-polycomb tumor suppressor genes, HIC1 and RassF1A, were also examined. Whereas the methylation level of HIC1 did not differ significantly between normal and tumor samples, RassF1A was significantly hypermethylated in liver tumor samples. Unsupervised clustering analysis classified the methylation changes within polycomb and non-polycomb targets to be independent, indicating independent epigenetic evolution. Thus, pre-deposited polycomb marks within somatic stem cells may contribute to the determination of methylation changes during hepatic tumorigenesis.

Yang T, Cai SY, Zhang J, et al.
Krüppel-like factor 8 is a new Wnt/beta-catenin signaling target gene and regulator in hepatocellular carcinoma.
PLoS One. 2012; 7(6):e39668 [PubMed] Free Access to Full Article Related Publications
Krüppel-like factor 8 (KLF8) plays important role in cell cycle and oncogenic transformation. Here we report the mechanisms by which KLF8 crosstalks with Wnt/β-catenin signaling pathway and regulates hepatocellular carcinoma (HCC) cells proliferation. We show that overexpression of KLF8 and nucleus accumulation of β-catenin in the human HCC samples are positively correlated. More importantly, KLF8 protein levels plus nucleus accumulation of β-catenin levels were significantly elevated in high-grade HCC compared to low-grade HCC. Using HCC HepG2 cells we find that, on the one hand both protein and mRNA of KLF8 are up-regulated under Wnt3a stimulation, on the other hand overexpression of KLF8 increases the cytoplasm and nucleus accumulation of β-catenin, recruits p300 to β-catenin/T-cell factor 4 (TCF4) transcription complex, enhances TOP flash report gene transcription, and induces Wnt/β-catenin signaling target genes c-Myc, cyclin D1 and Axin1 expression. Knockdown of KLF8 using shRNA inhibits Wnt3a induced transcription of TOP flash report gene and expression of c-Myc, cyclin D1 and Axin1. Knockdown of β-catenin by shRNA rescues the enhanced HepG2 and Hep3B cells proliferation ability induced by overexpression of KLF8.

Gerard B, Sanders MA, Visscher DW, et al.
Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin.
Biochim Biophys Acta. 2012; 1823(10):1686-96 [PubMed] Free Access to Full Article Related Publications
The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces β-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify β-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in β-catenin, and characterize their breast cancer tissue expression. β-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-β-catenin and His-Rad6B deletion mutants identified amino acids 131-181 and 50-116, respectively, as necessary for their interaction. Ubiquitination assays using β-catenin deletion mutants mapped Rad6B-induced ubiquitination within β-catenin 181-422 encompassing Armadillo repeats 2-7. Lysine to arginine mutations within repeats 5-7 identified K394 as the major Rad6B ubiquitination site in vitro and in vivo, and confirmed by Rad6B ubiquitination of a β-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-β-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-β-catenin, K394R mutation caused ~50% drop in TOP/Flash activity in Wnt-silent MCF-7 cells. Consistent with these data, expression of Rad6B, itself a β-catenin/TCF transcriptional target, was also reduced in K394R-β-catenin transfected cells. Steady-state K394R-β-catenin levels are decreased compared to wild type-β-catenin. The decreased expression is not due to proteasomal degradation as treatment with MG132 failed to rescue its levels. Lymph node-positive breast carcinomas express higher levels of Rad6 protein and Rad6 activity, and K63-linked ubiquitinated β-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of β-catenin ubiquitination that may be important for the stability and activity of β-catenin in breast cancer.

Lee KD, Pai MY, Hsu CC, et al.
Targeted Casp8AP2 methylation increases drug resistance in mesenchymal stem cells and cancer cells.
Biochem Biophys Res Commun. 2012; 422(4):578-85 [PubMed] Related Publications
Casp8AP2 contains a FLASH functional domain and is critical for the formation of death complex and the relay of death signal into the cells. Genetic defects in Casp8AP2 are associated with several diseases. A CpG island within the Casp8AP2 promoter is differentially regulated during somatic stem cell differentiation, and aberrant DNA methylation within the Casp8AP2 promoter has been reported in cancers. We hypothesized that abnormal DNA methylation of Casp8AP2 promoter might contribute to prolonged cellular survival or drug resistance in cancer. The epigenetic state within the Casp8AP2 promoter was then determined in different cancer cell lines and patient samples by methylation-specific PCR. Targeted Casp8AP2 methylation within normal and tumor cells was performed to see whether methylation promoted drug resistance. We found differential Casp8AP2 methylation among the normal and tumoral samples. Global demethylation in a platinum drug-resistant human gastric cancer cell line reversed Casp8AP2 methylation and diminished drug resistance. Targeted methylation of the Casp8AP2 promoter in somatic stem cells and cancer cells increased their resistance to drugs including platinum drugs. These data demonstrate that methylation within the Casp8AP2 promoter correlates with the development of drug resistance and might serve as a biomarker and treatment target for drug resistance in cancer cells.

Park C, Han S, Lee KM, et al.
Association between CASP7 and CASP14 genetic polymorphisms and the risk of childhood leukemia.
Hum Immunol. 2012; 73(7):736-9 [PubMed] Related Publications
Current evidence suggests that apoptosis and the cell cycle system play an important role in cancer development. To identify susceptible genetic markers in these mechanisms, we did an association study in 63 patients and 148 controls. A total of 304 SNPs in 31 gene regions were selected. We evaluated an association at a gene region level by computing the minimum P-value (minP) and doing the false discovery rate (FDR) test. Both SNP and gene-based analyses presented associations with the risk of childhood leukemia for 5 genes: CASP7, CASP14, CASP8AP2, MYC, and RIPK1 (P(trend)<0.05). There were statistically significant associations for CASP7 (rs12416109 and rs3814231, P(trend) = 0.002 and 0.009, respectively, minP = 0.013, FDR = 0.042) and CASP14 (rs8110862, P(trend)<0.001, minP = 0.002, FDR = 0.027). This study suggests that genetic polymorphisms in apoptosis and cell cycle related genes might play a role in childhood leukemia development.

Reyon D, Tsai SQ, Khayter C, et al.
FLASH assembly of TALENs for high-throughput genome editing.
Nat Biotechnol. 2012; 30(5):460-5 [PubMed] Free Access to Full Article Related Publications
Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.

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