Blocking PD-L1-PD-1 improves senescence surveillance and ageing phenotypes.

The accumulation of senescent cells is a major cause of age-related inflammation and predisposes to a variety of age-related diseases1. However, little is known about the molecular basis underlying this accumulation and its potential as a target to ameliorate the ageing process. Here we show that senescent cells heterogeneously express the immune checkpoint protein programmed death-ligand 1 (PD-L1) and that PD-L1+ senescent cells accumulate with age in vivo. PD-L1- cells are sensitive to T cell surveillance, whereas PD-L1+ cells are resistant, even in the presence of senescence-associated secretory phenotypes (SASP). Single-cell analysis of p16+ cells in vivo revealed that PD-L1 expression correlated with higher levels of SASP. Consistent with this, administration of programmed cell death protein 1 (PD-1) antibody to naturally ageing mice or a mouse model with normal livers or induced nonalcoholic steatohepatitis reduces the total number of p16+ cells in vivo as well as the PD-L1+ population in an activated CD8+ T cell-dependent manner, ameliorating various ageing-related phenotypes. These results suggest that the heterogeneous expression of PD-L1 has an important role in the accumulation of senescent cells and inflammation associated with ageing, and the elimination of PD-L1+ senescent cells by immune checkpoint blockade may be a promising strategy for anti-ageing therapy.

Aberrant monocytopoiesis drives granuloma development in sarcoidosis.

In sarcoidosis, granulomas develop in multiple organs including the liver and lungs. Although mechanistic target of rapamycin complex 1 (mTORC1) activation in macrophages drives granuloma development in sarcoidosis by enhancing macrophage proliferation, little is known about the macrophage subsets that proliferate and mature into granuloma macrophages. Here, we show that aberrantly increased monocytopoiesis gives rise to granulomas in a sarcoidosis model, in which Tsc2, a negative regulator of mTORC1, is conditionally deleted in CSF1R-expressing macrophages (Tsc2csf1rΔ mice). In Tsc2csf1rΔ mice, common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), common monocyte progenitors / monocyte progenitors (cMoPs / MPs), inducible monocyte progenitors (iMoPs), and Ly6Cint CX3CR1low CD14- immature monocytes (iMOs), but not monocyte-dendritic cell progenitors (MDPs) and common dendritic cell progenitors (CDPs), accumulated and proliferated in the spleen. Consistent with this, monocytes, neutrophils, and neutrophil-like monocytes increased in the spleens of Tsc2csf1rΔ mice, whereas dendritic cells did not. The adoptive transfer of splenic iMOs into wild-type mice gave rise to granulomas in the liver and lungs. In these target organs, iMOs matured into Ly6Chi classical monocytes/macrophages (cMOs). Giant macrophages (gMAs) also accumulated in the liver and lungs, which were similar to granuloma macrophages in expression of cell surface markers such as MerTK and SLAMF7. Furthermore, the gMA-specific genes were expressed in human macrophages from sarcoidosis skin lesions. These results suggest that mTORC1 drives granuloma development by promoting the proliferation of monocyte/neutrophil progenitors and iMOs predominantly in the spleen, and that proliferating iMOs mature into cMOs and then gMAs to give rise to granuloma after migration into the liver and lungs in sarcoidosis.

Genome-wide analysis of DNA methylation in pseudomyxoma peritonei originated from appendiceal neoplasms.

INTRODUCTION: Pseudomyxoma peritonei (PMP) is a disease characterized by progressive accumulation of intraperitoneal mucinous ascites produced by neoplasms in the abdominal cavity. Since the prognosis of patients with PMP remain unsatisfactory, the development of effective therapeutic drug(s) is a matter of pressing concern. Genetic analyses of PMP have clarified the frequent activation of GNAS and/or KRAS. However, the involvement of global epigenetic alterations in PMPs has not been reported. METHODS: To clarify the genetic background of the 15 PMP tumors, we performed genetic analysis using AmpliSeq Cancer HotSpot Panel v2. We further investigated global DNA methylation in the 15 tumors and eight non-cancerous colonic epithelial cells using Methylation EPIC array BeadChip (Infinium 850k) containing a total of 865,918 probes. RESULTS: This is the first report of comprehensive DNA methylation profiles of PMPs in the world. We clarified that the 15 PMPs could be classified into at least two epigenotypes, unique methylation epigenotype (UME) and normal-like methylation epigenotype (NLME), and that genes associated with neuronal development and synaptic signaling may be involved in the development of PMPs. In addition, we identified a set of hypermethylation marker genes such as HOXD1 and TSPYL5 in the 15 PMPs. CONCLUSIONS: These findings may help the understanding of the molecular mechanism(s) of PMP and contribute to the development of therapeutic strategies for this life-threatening disease.

Identification of odontogenic ameloblast associated as a novel target gene of the Wnt/ß-catenin signaling pathway.

The Wnt/ß-catenin signaling pathway plays a key role in development and carcinogenesis. Although some target genes of this signaling have been identified in various tissues and neoplasms, the comprehensive understanding of the target genes and their roles in the development of human cancer, including hepatoma and colorectal cancer remain to be fully elucidated. In this study, we searched for genes regulated by the Wnt signaling in liver cancer using HuH-7 hepatoma cells. A comparison of the expression profiles between cells expressing an active form of mutant ß-catenin and cells expressing enhanced green fluorescent protein (EGFP) identified seven genes upregulated by the mutant ß-catenin gene (CTNNB1). Among the seven genes, we focused in this study on ODAM, odontogenic, ameloblast associated, as a novel target gene. Interestingly, its expression was frequently upregulated in hepatocellular carcinoma, colorectal adenocarcinoma, and hepatoblastoma. We additionally identified a distant enhancer region that was associated with the ß-catenin/TCF7L2 complex. Further analyses revealed that ODAM plays an important role in the regulation of the cell cycle, DNA synthesis, and cell proliferation. These data may be useful for clarification of the main molecular mechanism(s) underlying these cancers.

Genetic analysis of low-grade adenosquamous carcinoma of the breast progressing to high-grade metaplastic carcinoma.

PURPOSE: Low-grade adenosquamous carcinoma (LGASC) is a rare type of metaplastic carcinoma of the breast (MBC) with an indolent clinical course. A few LGASC cases with high-grade transformation have been reported; however, the genetics underlying malignant progression of LGASC remain unclear. METHODS: We performed whole-genome sequencing analysis on five MBCs from four patients, including one case with matching primary LGASC and a lymph node metastatic tumor consisting of high-grade MBC with a predominant metaplastic squamous cell carcinoma component (MSC) that progressed from LGASC and three cases of independent de novo MSC. RESULTS: Unlike de novo MSC, LGASC and its associated MSC showed no TP53 mutation and tended to contain fewer structural variants than de novo MSC. Both LGASC and its associated MSC harbored the common GNAS c.C2530T:p.Arg844Cys mutation, which was more frequently detected in the cancer cell fraction of MSC. MSC associated with LGASC showed additional pathogenic deletions of multiple tumor-suppressor genes, such as KMT2D and BTG1. Copy number analysis revealed potential 18q loss of heterozygosity in both LGASC and associated MSC. The frequency of SMAD4::DCC fusion due to deletions increased with progression to MSC; however, chimeric proteins were not detected. SMAD4 protein expression was already decreased at the LGASC stage due to unknown mechanisms. CONCLUSION: Not only LGASC but also its associated high-grade MBC may be genetically different from de novo high-grade MBC. Progression from LGASC to high-grade MBC may involve the concentration of driver mutations caused by clonal selection and inactivation of tumor-suppressor genes.

Impact of neoadjuvant therapy on gut microbiome in patients with resectable/borderline resectable pancreatic ductal adenocarcinoma.

BACKGROUND: /Objectives: Effects of chemotherapy on gut microbiota have been reported in various carcinomas. The current study aimed to evaluate the changes in the gut microbiota before and after neoadjuvant chemotherapy (NAC) in patients with resectable (R) and borderline resectable (BR) pancreatic ductal adenocarcinoma (PDAC) and understand their clinical implications. METHODS: Twenty patients diagnosed with R/BR-PDAC were included in this study. Stool samples were collected at two points, before and after NAC, for microbiota analysis using 16S ribosomal RNA (16S rRNA) gene sequences. RESULTS: Of the 20 patients, 18 (90%) were treated with gemcitabine plus S-1 as NAC, and the remaining patients received gemcitabine plus nab-paclitaxel and a fluorouracil, leucovorin, irinotecan, and oxaliplatin combination. No significant differences were observed in the α- and ß-diversity before and after NAC. Bacterial diversity was not associated with Evans classification (histological grade of tumor destruction by NAC) or postoperative complications. The relative abundance of Actinobacteria phylum after NAC was significantly lower than that before NAC (P = 0.02). At the genus level, the relative abundance of Bifidobacterium before NAC in patients with Evans grade 2 disease was significantly higher than that in patients with Evans grade 1 disease (P = 0.03). Patients with Evans grade 2 lost significantly more Bifidobacterium than patients with Evans grade 1 (P = 0.01). CONCLUSIONS: The diversity of gut microbiota was neither decreased by NAC for R/BR-PDAC nor associated with postoperative complications. Lower incidence of Bifidobacterium genus before NAC may be associated with a lower pathological response to NAC.

Functional Restoration of Bacteriomes and Viromes by Fecal Microbiota Transplantation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. METHODS: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. RESULTS: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. CONCLUSIONS: The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.

Halcyon: an accurate basecaller exploiting an encoder-decoder model with monotonic attention.

MOTIVATION: In recent years, nanopore sequencing technology has enabled inexpensive long-read sequencing, which promises reads longer than a few thousand bases. Such long-read sequences contribute to the precise detection of structural variations and accurate haplotype phasing. However, deciphering precise DNA sequences from noisy and complicated nanopore raw signals remains a crucial demand for downstream analyses based on higher-quality nanopore sequencing, although various basecallers have been introduced to date. RESULTS: To address this need, we developed a novel basecaller, Halcyon, that incorporates neural-network techniques frequently used in the field of machine translation. Our model employs monotonic-attention mechanisms to learn semantic correspondences between nucleotides and signal levels without any pre-segmentation against input signals. We evaluated performance with a human whole-genome sequencing dataset and demonstrated that Halcyon outperformed existing third-party basecallers and achieved competitive performance against the latest Oxford Nanopore Technologies' basecallers. AVAILABILITYAND IMPLEMENTATION: The source code (halcyon) can be found at https://github.com/relastle/halcyon.

Robust parameter design of human induced pluripotent stem cell differentiation protocols defines lineage-specific induction of anterior-posterior gut tube endodermal cells.

Tissues and cells derived from pluripotent stem cells (PSC) are likely to become widely used in disease modeling, drug screening, and regenerative medicine. For these applications, the in vitro PSC differentiation process must be elaborately investigated and controlled to reliably obtain the desired end products. However, because traditional experimental methods, such as one factor at a time or brute-force approaches, are impractical for detailed screening of complex PSC cultivation conditions, more strategic and effective screening based on statistical design of experiments (DOE) ought to be indispensable. Among various DOE approaches, we regard robust parameter design (RPD) as particularly suited for differentiation protocol optimization due to its suitability for multifactorial screening. We confirmed the adaptability of RPD for investigating human induced PSC lineage specification toward anterior-posterior gut tube endodermal cells and clarified both the contribution of each cell signaling pathway and the effect of cell signaling condition alteration on marker RNA expression levels, while increasing the efficiency of the screening in 243-fold (18 vs 4374) compared with that of a brute-force approach. Specific induction of anterior foregut, hepatic, pancreatic, or mid-hindgut cells was achieved using seven iPSC strains with the optimal culture protocols established on the basis of RPD analysis. RPD has the potential to enable efficient construction and optimization of PSC differentiation protocols, and its use is recommended from fundamental research to mass production of PSC-derived products.

Application of targeted nanopore sequencing for the screening and determination of structural variants in patients with Lynch syndrome.

Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.

Discovery of chemical probes that suppress Wnt/ß-catenin signaling through high-throughput screening.

Aberrant activation of the Wnt/ß-catenin signaling pathway has been observed in a wide range of human tumors. Deregulation of the pathway is closely linked to various aspects of human carcinogenesis such as cell viability, regulation of cell cycle, epithelial-mesenchymal transition, and maintenance of stemness. In addition, recent studies have disclosed the involvement of Wnt signaling in immune evasion of tumor cells. The accumulation of ß-catenin in the nucleus is a common feature of cancer cells carrying defects in the pathway, which leads to the continuous activation of T-cell factor (TCF)/LEF transcription factors. Consequently, a genetic program is switched on, leading to the uncontrolled growth, prolonged survival, and acquisition of mesenchymal phenotype. As ß-catenin/TCF serves as a signaling hub for the pathway, ß-catenin/TCF-dependent transcriptional activity is a relevant readout of the pathway. To date, a wide variety of synthetic TCF/LEF reporters has been developed, and high-throughput screening (HTS) using these reporters has made significant contributions to the discovery of Wnt inhibitors. Indeed, HTS led to the identification of chemical probes targeting porcupine, a membrane bound O-acyltransferase, and CREB-binding protein, a transcriptional coactivator. This review focuses on various screening strategies for the discovery of Wnt inhibitors and their mode of action to help the creation of new concepts for assay/screening methods.

Efficacy of the novel tubulin polymerization inhibitor PTC-028 for myelodysplastic syndrome.

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.

Correction: Importance of gastric cancer for the diagnosis and surveillance of Japanese Lynch syndrome patients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of an MSI-positive colon tumor with aberrant DNA methylation in a PPAP patient.

Polymerase proofreading-associated polyposis (PPAP) is a disease caused by germline variations in the POLE and POLD1 genes that encode catalytic subunits of DNA polymerases. Studies of cancer genomes have identified somatic mutations in these genes, suggesting the importance of polymerase proofreading of DNA replication in suppressing tumorigenesis. Here, we identified a germline frameshift variation in the POLE gene (c.4191_4192delCT, p.Tyr1398*) in a case with multiple adenomatous polyps and three synchronous colon cancers. Interestingly, one of the colon cancers showed microsatellite instability-high (MSI-H) and another microsatellite stable. Immunohistochemical staining revealed that the MSI-H tumor cells lost the expression of MLH1 protein. Whole genome sequencing of the MSI-H tumor did not find pathogenic somatic mutations in mismatch repair genes but found frameshift mutations in the TET genes that catalyze 5-methylcytosine hydroxylation. Bisulfite sequencing of the tumor corroborated an increase in the number of hypermethylated regions including the MLH1 promoter. These data indicate that PPAP patients might develop MSI-positive tumors through epigenetic silencing of MLH1. These findings will contribute to comprehensive understanding of the molecular basis of tumors that involve deficiency of proofreading activity of DNA polymerases.

Importance of gastric cancer for the diagnosis and surveillance of Japanese Lynch syndrome patients.

Lynch syndrome (LS) is an autosomal dominantly inherited disease predisposed to not only colorectal cancer but also other LS-related tumors. Although the clinical and genetic characteristics of LS in Western countries have been well characterized, the information of Japanese LS is limited. As a collaborative study of Japanese Society for Cancer of the Colon and Rectum (JSCCR), we registered colorectal cancer (CRC) patients who fulfilled the modified Amsterdam II criteria including gastric cancer as an LS-related tumor. Among 4030 CRC patients initially registered in this project, 85 patients (2.1%) fulfilled the modified criteria. An additional 26 patients who met the same criteria were enrolled in the analysis. We analyzed three major responsible genes, MLH1, MSH2, and MSH6 by direct sequencing, and further performed multiplex ligation-dependent probe amplification for MLH1 and MSH2. Consequently, we identified pathogenic variants in 64 of the 111 patients comprising of 34 patients in MLH1, 28 in MSH2, and 2 in MSH6. It is of note that large structural alterations were found in 17 patients. Among the 64 patients, 11 patients would not have been enrolled in the analysis if gastric cancer were not included in the modified criteria. In addition, 10 of the 64 variant carriers (15.6%) had medical history of gastric cancer. Furthermore, the standardized incidence ratio of gastric cancer in the LS patients to the Japanese population is estimated to be as high as 20.2. These data underscore the importance of gastric cancer in the diagnosis and healthcare of Japanese LS patients.

Efficacy of liquid-based genetic diagnosis of endometrial cancer.

Although liquid-based cytology (LBC) has increased the sensitivity of cytological diagnosis of endometrial cancer (EC) compared with conventional smear cytology, the sensitivity of LBC for the detection of EC is between 70% and 96% and remains unsatisfactory. In the present study, we compared the efficacy of LBC with liquid-based genetic diagnosis (LBGDx) by amplicon sequencing of five genes including PTEN, PIK3CA, CTNNB1, KRAS, and TP53 in 48 LBC subjects who underwent endometrial screening. Consequently, LBC classified 15 samples as "positive or suspicious for malignancy" and the 15 were later confirmed as EC. However, LBC failed to identify five cases who were diagnosed as EC by additional transvaginal ultrasound and endometrial curettage, indicating that the sensitivity of cytology alone was 75% (15/20). LBGDx identified 11 pathogenic PTEN variants in 10 subjects, six PIK3CA variants in nine, three CTNNB1 variants in five, two KRAS variants in four, and three TP53 variants in three. Collectively, at least one pathogenic variant was identified in 19 subjects, which included 17 EC (15 endometrioid carcinoma and 2 endometrial carcinosarcomas), and one cervical adenocarcinoma. However, LBGDx did not identify any pathogenic mutations in three of the 20 EC, indicating that the sensitivity of LBGDx alone was 85% (17/20). Although five EC were negative for malignancy by LBC and three were negative for pathogenic mutations by LBGDx, the combination of LBC and LBGDx would successfully diagnose all 20 EC. These data suggested that LBGDx is a useful strategy to improve the sensitivity of screening of EC by LBC.

Regulation of tankyrase activity by a catalytic domain dimer interface.

Tankyrases (TNKS and TNKS2) are enzymes that catalyze poly-ADP-ribosylation (PARsylation) of their target proteins. Tankyrase-mediated PARsylation plays critical regulatory roles in cell signaling, particularly in the Wnt/ß-catenin pathway. The sterile alpha motif (SAM) domain in tankyrases mediates their oligomerization, which is essential for tankyrase function. The oligomerization regulates the catalytic activity of tankyrases, but the underlying mechanism is unclear. Our analyses of crystal structures of the tankyrase catalytic domain suggest that formation of a head-to-head dimer regulates the catalytic activity. Our activity assays show that residues in the catalytic domain dimer interface are important for the PARsylation activity of tankyrases both in solution and cells. The dimer is weak and may only form in the context of the SAM domain-mediated oligomers of tankyrases, consistent with the dependence of the tankyrase activity on the SAM domain.

Requirement of glycosylation machinery in TLR responses revealed by CRISPR/Cas9 screening.

The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the TLR response is not yet fully understood. In the present study, we used a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA (gRNA) library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced. Identified genes were ranked according to the count of sequence reads and the number of gRNA target sites. The screening system worked correctly, as molecules that were already known to be required for the TLR response were identified by the screening. Furthermore, this system revealed that the oligosaccharide transferase complex (OSTC) mediating co-translational glycosylation was required for TLR5, 7 and 9 responses. Protein expression of TLR5, but not an irrelevant molecule (CD44), was abolished by the lack of OSTC, suggesting the essential role of glycosylation in TLR5 protein stability. These results demonstrate that the screening system established here is able to reveal molecular mechanisms underlying the TLR response.

Identification of FERM domain-containing protein 5 as a novel target of ß-catenin/TCF7L2 complex.

Deregulation of the canonical Wnt signaling pathway plays an important role in human tumorigenesis through the accumulation of ß-catenin and subsequent transactivation of TCF7L2. Although some of the consequences associated with the accumulated ß-catenin have been clarified, the comprehensive effect of activated ß-catenin/TCF7L2 transcriptional complex on tumorigenesis remains to be elucidated. To understand the precise molecular mechanisms underlying colorectal cancer, we searched for genes regulated by the complex in colorectal tumors. We performed expression profile analysis of HCT116 and SW480 colon cancer cells treated with ß-catenin siRNAs, and ChIP-sequencing using anti-TCF7L2 antibody. Combination of these data with public microarray data of LS174 cells with a dominant-negative form of TCF7L2 identified a total of 11 candidate genes. In this paper, we focused on FERM domain-containing protein 5 (FRMD5), and confirmed that it is regulated by both ß-catenin and TCF7L2. An additional reporter assay disclosed that a region in intron1 transcriptionally regulated the expression of FRMD5. ChIP assay also corroborated that TCF7L2 associates with this region. These data suggested that FRMD5 is a novel direct target of the ß-catenin/TCF7L2 complex.

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of ß-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by ß-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when ß-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/ß-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/ß-catenin signaling pathway.