AQP5 enriches for stem cells and cancer origins in the distal stomach.

LGR5 marks resident adult epithelial stem cells at the gland base in the mouse pyloric stomach1, but the identity of the equivalent human stem cell population remains unknown owing to a lack of surface markers that facilitate its prospective isolation and validation. In mouse models of intestinal cancer, LGR5+ intestinal stem cells are major sources of cancer following hyperactivation of the WNT pathway2. However, the contribution of pyloric LGR5+ stem cells to gastric cancer following dysregulation of the WNT pathway-a frequent event in gastric cancer in humans3-is unknown. Here we use comparative profiling of LGR5+ stem cell populations along the mouse gastrointestinal tract to identify, and then functionally validate, the membrane protein AQP5 as a marker that enriches for mouse and human adult pyloric stem cells. We show that stem cells within the AQP5+ compartment are a source of WNT-driven, invasive gastric cancer in vivo, using newly generated Aqp5-creERT2 mouse models. Additionally, tumour-resident AQP5+ cells can selectively initiate organoid growth in vitro, which indicates that this population contains potential cancer stem cells. In humans, AQP5 is frequently expressed in primary intestinal and diffuse subtypes of gastric cancer (and in metastases of these subtypes), and often displays altered cellular localization compared with healthy tissue. These newly identified markers and mouse models will be an invaluable resource for deciphering the early formation of gastric cancer, and for isolating and characterizing human-stomach stem cells as a prerequisite for harnessing the regenerative-medicine potential of these cells in the clinic.

H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase.

AIMS: Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. METHODS: Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. RESULTS: H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. CONCLUSION: Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.

Metabolic pathway analyses identify proline biosynthesis pathway as a promoter of liver tumorigenesis.

BACKGROUND & AIM: Under the regulation of various oncogenic pathways, cancer cells undergo adaptive metabolic programming to maintain specific metabolic states that support their uncontrolled proliferation. As it has been difficult to directly and effectively inhibit oncogenic signaling cascades with pharmaceutical compounds, focusing on the downstream metabolic pathways that enable indefinite growth may provide therapeutic opportunities. Thus, we sought to characterize metabolic changes in hepatocellular carcinoma (HCC) development and identify metabolic targets required for tumorigenesis. METHODS: We compared gene expression profiles of Morris Hepatoma (MH3924a) and DEN (diethylnitrosamine)-induced HCC models to those of liver tissues from normal and rapidly regenerating liver models, and performed gain- and loss-of-function studies of the identified gene targets for their roles in cancer cell proliferation in vitro and in vivo. RESULTS: The proline biosynthetic enzyme PYCR1 (pyrroline-5-carboxylate reductase 1) was identified as one of the most upregulated genes in the HCC models. Knockdown of PYCR1 potently reduced cell proliferation of multiple HCC cell lines in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of the HCC cell lines. Importantly, PYCR1 expression was not elevated in the regenerating liver, and KD or overexpression of PYCR1 had no effect on proliferation of non-cancerous cells. Besides PYCR1, we found that additional proline biosynthetic enzymes, such as ALDH18A1, were upregulated in HCC models and also regulated HCC cell proliferation. Clinical data demonstrated that PYCR1 expression was increased in HCC, correlated with tumor grade, and was an independent predictor of clinical outcome. CONCLUSION: Enhanced expression of proline biosynthetic enzymes promotes HCC cell proliferation. Inhibition of PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC. LAY SUMMARY: Even with the recently approved immunotherapies against liver cancer, currently available medications show limited clinical benefits or efficacy in the majority of patients. As such, it remains a top priority to discover new targets for effective liver cancer treatment. Here, we identify a critical role for the proline biosynthetic pathway in liver cancer development, and demonstrate that targeting key proteins in the pathway, namely PYCR1 and ALDH18A1, may be a novel therapeutic strategy for liver cancer.

Tiled array-based sequencing identifies enrichment of loss-of-function variants in the highly homologous filaggrin gene in African-American children with severe atopic dermatitis.

Filaggrin (FLG) loss-of-function (LOF) variants are a major risk factor for the common inflammatory skin disease, atopic dermatitis (AD) and are often population-specific. African-American (AA) children are disproportionately affected with AD, often later developing asthma and/or allergic rhinitis and comprise an atopy health disparity group for which the role of FLG LOF is not well known. Discovery of FLG LOF using exome sequencing is challenging given the known difficulties for accurate short-read alignment to FLG's high homology repeat variation. Here, we employed an array-based sequencing approach to tile across each FLG repeat and discover FLG LOF in a well-characterized cohort of AA children with moderate-to-severe AD. Five FLG LOF were identified in 23% of our cohort. Two novel FLG LOF singletons, c.488delG and p.S3101*, were discovered as well as p.R501*, p.R826* and p.S3316* previously reported for AD. p.S3316* (rs149484917) is likely an African ancestral FLG LOF, reported in African individuals in ExAC (Exome Aggregation Consortium), Exome Variant Server (ESP), and 4 African 1000G population databases (ESN, MSL, ASW, and ACB). The proportion of FLG LOF (11.5%) among the total FLG alleles in our cohort was significantly higher in comparisons with FLG LOF reported for African individuals in ExAC (2.5%; P = 4.3 × 10-4 ) and ESP (1.7%; P = 3.5 × 10-5 ) suggesting a disease-enrichment effect for FLG LOF. Our results demonstrate the utility of array-based sequencing in discovering FLG LOF, including novel and population-specific, which are of higher prevalence in our AA severe AD group than previously reported.

Below-Ground Attack by the Root Knot Nematode Meloidogyne graminicola Predisposes Rice to Blast Disease.

Magnaporthe oryzae (rice blast) and the root-knot nematode Meloidogyne graminicola are causing two of the most important pathogenic diseases jeopardizing rice production. Here, we show that root-knot nematode infestation on rice roots leads to important above-ground changes in plant immunity gene expression, which is correlated with significantly enhanced susceptibility to blast disease. A detailed metabolic analysis of oxidative stress responses and hormonal balances demonstrates that the above-ground tissues have a disturbed oxidative stress level, with accumulation of H2O2, as well as hormonal disturbances. Moreover, double infection experiments on an oxidative stress mutant and an auxin-deficient rice line indicate that the accumulation of auxin in the above-ground tissue is at least partly responsible for the blast-promoting effect of root-knot nematode infection.

Identification and validation of WISP1 as an epigenetic regulator of metastasis in oral squamous cell carcinoma.

Lymph node (LN) metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC) patients. However, in approximately one third of OSCC patients nodal metastases remain undetected, and thus are not adequately treated. Therefore, clinical assessment of LN metastasis needs to be improved. The purpose of this study was to identify DNA methylation biomarkers to predict LN metastases in OSCC. Genome wide methylation assessment was performed on six OSCC with (N+) and six without LN metastases (N0). Differentially methylated sequences were selected based on the likelihood of differential methylation and validated using an independent OSCC cohort as well as OSCC from The Cancer Genome Atlas (TCGA). Expression of WISP1 using immunohistochemistry was analyzed on a large OSCC cohort (n = 204). MethylCap-Seq analysis revealed 268 differentially methylated markers. WISP1 was the highest ranking annotated gene that showed hypomethylation in the N+ group. Bisulfite pyrosequencing confirmed significant hypomethylation within the WISP1 promoter region in N+ OSCC (P = 0.03) and showed an association between WISP1 hypomethylation and high WISP1 expression (P = 0.01). Both these results were confirmed using 148 OSCC retrieved from the TCGA database. In a large OSCC cohort, high WISP1 expression was associated with LN metastasis (P = 0.05), disease-specific survival (P = 0.022), and regional disease-free survival (P = 0.027). These data suggest that WISP1 expression is regulated by methylation and WISP1 hypomethylation contributes to LN metastasis in OSCC. WISP1 is a potential biomarker to predict the presence of LN metastases.

SNP-guided identification of monoallelic DNA-methylation events from enrichment-based sequencing data.

Monoallelic gene expression is typically initiated early in the development of an organism. Dysregulation of monoallelic gene expression has already been linked to several non-Mendelian inherited genetic disorders. In humans, DNA-methylation is deemed to be an important regulator of monoallelic gene expression, but only few examples are known. One important reason is that current, cost-affordable truly genome-wide methods to assess DNA-methylation are based on sequencing post-enrichment. Here, we present a new methodology based on classical population genetic theory, i.e. the Hardy-Weinberg theorem, that combines methylomic data from MethylCap-seq with associated SNP profiles to identify monoallelically methylated loci. Applied on 334 MethylCap-seq samples of very diverse origin, this resulted in the identification of 80 genomic regions featured by monoallelic DNA-methylation. Of these 80 loci, 49 are located in genic regions of which 25 have already been linked to imprinting. Further analysis revealed statistically significant enrichment of these loci in promoter regions, further establishing the relevance and usefulness of the method. Additional validation was done using both 14 whole-genome bisulfite sequencing data sets and 16 mRNA-seq data sets. Importantly, the developed approach can be easily applied to other enrichment-based sequencing technologies, like the ChIP-seq-based identification of monoallelic histone modifications.

Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots.

One of the reasons for the progressive yield decline observed in aerobic rice production is the rapid build-up of populations of the rice root knot nematode Meloidogyne graminicola. These nematodes induce specialized feeding cells inside root tissue, called giant cells. By injecting effectors in and sipping metabolites out of these cells, they reprogramme normal cell development and deprive the plant of its nutrients. In this research we have studied the transcriptome of giant cells in rice, after isolation of these cells by laser-capture microdissection. The expression profiles revealed a general induction of primary metabolism inside the giant cells. Although the roots were shielded from light induction, we detected a remarkable induction of genes involved in chloroplast biogenesis and tetrapyrrole synthesis. The presence of chloroplast-like structures inside these dark-grown cells was confirmed by confocal microscopy. On the other hand, genes involved in secondary metabolism and more specifically, the majority of defence-related genes were strongly suppressed in the giant cells. In addition, significant induction of transcripts involved in epigenetic processes was detected inside these cells 7 days after infection.

No shorter telomeres in subjects with a family history of cardiovascular disease in the Asklepios study.

OBJECTIVE: Shorter telomere length is associated with the occurrence of cardiovascular events, but the question of causality is complicated by the intertwined effects of inheritance, aging, and lifestyle factors on both telomere length and cardiovascular disease (CVD). Some studies indicated that healthy offspring of coronary artery disease patients exhibited shorter telomeres than subjects without a family history. Importantly, this result would imply that inheritance of shorter telomeres is a primary abnormality associated with an increased risk of CVD, the so-called Telomere Hypothesis of CVD. Therefore, we aimed at further validating the latter results in the large, population-representative Asklepios Study. METHODS AND RESULTS: Peripheral blood leukocyte telomere length was measured using telomere restriction fragment analysis in the young to middle-aged (≈ 35-55 years old) Asklepios study population, free from overt CVD, and could be successfully combined with data from the Asklepios Family History Database for 2136 subjects. No shorter telomere length could be found in healthy subjects with a family history of CVD compared with those without. CONCLUSIONS: These findings cast serious doubt on the hypothesis that telomere length is shorter in families with an increased risk of CVD and do not support the Telomere Hypothesis of CVD.

Transcriptional reprogramming by root knot and migratory nematode infection in rice.

Rice is one of the most important staple crops worldwide, but its yield is compromised by different pathogens, including plant-parasitic nematodes. In this study we have characterized specific and general responses of rice (Oryza sativa) roots challenged with two endoparasitic nematodes with very different modes of action. Local transcriptional changes in rice roots upon root knot (Meloidogyne graminicola) and root rot nematode (RRN, Hirschmanniella oryzae) infection were studied at two time points (3 and 7 d after infection, dai), using mRNA-seq. Our results confirm that root knot nematodes (RKNs), which feed as sedentary endoparasites, stimulate metabolic pathways in the root, and enhance nutrient transport towards the induced root gall. The migratory RRNs, on the other hand, induce programmed cell death and oxidative stress, and obstruct the normal metabolic activity of the root. While RRN infection causes up-regulation of biotic stress-related genes early in the infection, the sedentary RKNs suppress the local defense pathways (e.g. salicylic acid and ethylene pathways). Interestingly, hormone pathways mainly involved in plant development were strongly induced (gibberellin) or repressed (cytokinin) at 3 dai. These results uncover previously unrecognized nematode-induced expression profiles related to their specific infection strategy.

Transcriptome analysis of rice mature root tissue and root tips in early development by massive parallel sequencing.

Despite the major physiological dissimilarities between mature root regions and their tips, differences in their gene expression profiles remain largely unexplored. In this research, the transcriptome of rice (Oryza sativa L. subsp. japonica) mature root tissue and root tips was monitored using mRNA-Seq at two time points. Almost 50 million 76 bp reads were mapped onto the rice genome sequence, expression patterns for different tissues and time points were investigated, and at least 1106 novel transcriptionally active regions (nTARs) expressed in rice root tissue were detected. More than 30 000 genes were found to be expressed in rice roots, among which were 1761 root-enriched and 306 tip-enriched transcripts. Mature root tissue appears to respond more strongly to external stimuli than tips, showing a higher expression of, for instance, auxin-responsive and abscisic acid-responsive genes, as well as the phenylpropanoid pathway and photosynthesis upon light. The root tip-enriched transcripts are mainly involved in mitochondrial electron transport, organelle development, secondary metabolism, DNA replication and metabolism, translation, and cellular component organization. During root maturation, genes involved in cell wall biosynthesis and modification, response to oxidative stress, and secondary metabolism were activated. For some nTARs, a potential role in root development can be put forward based on homology to genes involved in CLAVATA signalling, cell cycle regulators, and hormone signalling. A subset of differentially expressed genes and novel transcripts was confirmed using (quantitative) reverse transcription-PCR. These results uncover previously unrecognized tissue-specific expression profiles and provide an interesting starting point to study the different regulation of transcribed regions of these tissues.

GATA6 Cooperates with EOMES/SMAD2/3 to Deploy the Gene Regulatory Network Governing Human Definitive Endoderm and Pancreas Formation.

Heterozygous de novo mutations in GATA6 are the most frequent cause of pancreatic agenesis in humans. In mice, however, a similar phenotype requires the biallelic loss of Gata6 and its paralog Gata4. To elaborate the human-specific requirements for GATA6, we chose to model GATA6 loss in vitro by combining both gene-edited and patient-derived pluripotent stem cells (hPSCs) and directed differentiation toward ß-like cells. We find that GATA6 heterozygous hPSCs show a modest reduction in definitive endoderm (DE) formation, while GATA6-null hPSCs fail to enter the DE lineage. Consistent with these results, genome-wide studies show that GATA6 binds and cooperates with EOMES/SMAD2/3 to regulate the expression of cardinal endoderm genes. The early deficit in DE is accompanied by a significant reduction in PDX1+ pancreatic progenitors and C-PEPTIDE+ ß-like cells. Taken together, our data position GATA6 as a gatekeeper to early human, but not murine, pancreatic ontogeny.

Low-dose calcipotriol can elicit wound closure, anti-microbial, and anti-neoplastic effects in epidermolysis bullosa keratinocytes.

Recessive dystrophic epidermolysis bullosa (RDEB) patients suffer from chronic and repeatedly infected wounds predisposing them to the development of aggressive and life-threatening skin cancer in these areas. Vitamin D3 is an often neglected but critical factor for wound healing. Intact skin possesses the entire enzymatic machinery required to produce active 1-alpha,25-dihydroxyvitamin D3 (calcitriol), underscoring its significance to proper skin function. Injury enhances calcitriol production, inducing the expression of calcitriol target genes including the antimicrobial peptide cathelicidin (hCAP18), an essential component of the innate immune system and an important wound healing factor. We found significantly reduced hCAP18 expression in a subset of RDEB keratinocytes which could be restored by calcipotriol treatment. Reduced scratch closure in RDEB cell monolayers was enhanced up to 2-fold by calcipotriol treatment, and the secretome of calcipotriol-treated cells additionally showed increased antimicrobial activity. Calcipotriol exhibited anti-neoplastic effects, suppressing the clonogenicity and proliferation of RDEB tumor cells. The combined wound healing, anti-microbial, and anti-neoplastic effects indicate that calcipotriol may represent a vital therapeutic option for RDEB patients which we could demonstrate in a single-patient observation study.

ENPP1 Mutation Causes Recessive Cole Disease by Altering Melanogenesis.

Cole disease is a genodermatosis of pigmentation following a strict dominant mode of inheritance. In this study, we investigated eight patients affected with an overlapping genodermatosis after recessive inheritance. The patients presented with hypo- and hyperpigmented macules over the body, resembling dyschromatosis universalis hereditaria in addition to punctuate palmoplantar keratosis. By homozygosity mapping and whole-exome sequencing, a biallelic p.Cys120Arg mutation in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was identified in all patients. We found that this mutation, like those causing dominant Cole disease, impairs homodimerization of the ENPP1 enzyme that is mediated by its two somatomedin-B-like domains. Histological analysis revealed structural and molecular changes in affected skin that were likely to originate from defective melanocytes because keratinocytes do not express ENPP1. Consistently, RNA-sequencing analysis of patient-derived primary melanocytes revealed alterations in melanocyte development and in pigmentation signaling pathways. We therefore conclude that germline ENPP1 cysteine-specific mutations, primarily affecting the melanocyte lineage, cause a clinical spectrum of dyschromatosis, in which the p.Cys120Arg allele represents a recessive and more severe form of Cole disease.

Lgr5-expressing chief cells drive epithelial regeneration and cancer in the oxyntic stomach.

The daily renewal of the corpus epithelium is fuelled by adult stem cells residing within tubular glands, but the identity of these stem cells remains controversial. Lgr5 marks homeostatic stem cells and 'reserve' stem cells in multiple tissues. Here, we report Lgr5 expression in a subpopulation of chief cells in mouse and human corpus glands. Using a non-variegated Lgr5-2A-CreERT2 mouse model, we show by lineage tracing that Lgr5-expressing chief cells do not behave as corpus stem cells during homeostasis, but are recruited to function as stem cells to effect epithelial renewal following injury by activating Wnt signalling. Ablation of Lgr5+ cells severely impairs epithelial homeostasis in the corpus, indicating an essential role for these Lgr5+ cells in maintaining the homeostatic stem cell pool. We additionally define Lgr5+ chief cells as a major cell-of-origin of gastric cancer. These findings reveal clinically relevant insights into homeostasis, repair and cancer in the corpus.

Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells.

Pluripotent stem cells have been proposed as an unlimited source of pancreatic ß cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional ß cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ ß-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.