EZH2 is a key component of hepatoblastoma tumor cell growth.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) catalyzes the trimethylation of histone H3 at lysine 27 via the polycomb recessive complex 2 (PRC2) and plays a time-specific role in normal fetal liver development. EZH2 is overexpressed in hepatoblastoma (HB), an embryonal tumor. EZH2 can also promote tumorigenesis via a noncanonical, PRC2-independent mechanism via proto-oncogenic, direct protein interaction, including ß-catenin. We hypothesize that the pathological activation of EZH2 contributes to HB propagation in a PRC2-independent manner. METHODS AND RESULTS: We demonstrate that EZH2 promotes proliferation in HB tumor-derived cell lines through interaction with ß-catenin. Although aberrant EZH2 expression occurs, we determine that both canonical and noncanonical EZH2 signaling occurs based on specific gene-expression patterns and interaction with SUZ12, a PRC2 component, and ß-catenin. Silencing and inhibition of EZH2 reduce primary HB cell proliferation. CONCLUSIONS: EZH2 overexpression promotes HB cell proliferation, with both canonical and noncanonical function detected. However, because EZH2 directly interacts with ß-catenin in human tumors and EZH2 overexpression is not equal to SUZ12, it seems that a noncanonical mechanism is contributing to HB pathogenesis. Further mechanistic studies are necessary to elucidate potential pathogenic downstream mechanisms and translational potential of EZH2 inhibitors for the treatment of HB.

DHA Supplementation Attenuates Inflammation-Associated Gene Expression in the Mammary Gland of Lactating Mothers Who Deliver Preterm.

BACKGROUND: In a randomized trial of DHA supplementation to lactating mothers who delivered preterm, there were significant increases in DHA status in the mother and her infant. OBJECTIVES: Our objective here was to characterize the mammary gland transcriptomes from the above study. We hypothesized that proinflammatory gene expression would be attenuated in the increased DHA group compared with the standard DHA group. METHODS: In the original trial, mothers delivering at <29 wk gestation at the University of Cincinnati Medical Center and intending to express their milk were randomly assigned to supplementation with 200 mg/d DHA (standard group: STD) or 1000 mg/d DHA (experimental group: EXP) within 7 d of delivery. Here, we conducted RNA-seq transcriptome analysis of n = 5 EXP and n = 4 STD extracellular mammary mRNA samples extracted from the fat layer of milk samples obtained 4 wk postenrollment. Transcripts were assessed for differential expression (false discovery rate adjusted P value <0.05) and clustering between EXP compared with STD groups. Ontological analysis of all differentially expressed genes (DEGs) was performed with Toppcluster. RESULTS: There were 409 DEGs. We observed 5 main groups of biological processes that were upregulated, including those associated with improved immune regulation and management of oxidative stress; and 3 main groups of biological processes that were downregulated, including 1 associated with immune dysregulation. For example, we observed upregulation of inflammation-inhibiting genes including NFKB inhibitor alpha (NFKBIA; fold-change (FC), adjusted P value: FC = 1.70, P = 0.007) and interleukin-18 binding protein (IL18BP: FC = 2.2, adjusted P = 0.02); and downregulation of proinflammatory genes including interleukin 7 receptor (IL7R: FC = -1.9, adjusted P = 0.02) and interleukin 1 receptor like 1 (IL1RL1: FC = -13.0, adjusted P = 0.02). CONCLUSIONS: Increased DHA supplementation during lactation can modulate the expression of inflammation-related genes within the mammary gland. This might translate to milk composition with a more optimal inflammasome profile. Future research with a larger clinical trial and greater interrogation of clinical outcomes is warranted.

Multiple calcium sources are required for intracellular calcium mobilization during gastric organoid epithelial repair.

Calcium (Ca2+ ) is a known accelerator for gastric wound repair. We have demonstrated in vivo and in vitro that intracellular Ca2+ increases in the gastric epithelial cells directly adjacent to a damaged cell, and that this Ca2+ rise is essential for the cellular migration that rapidly repairs the epithelium (restitution). While intracellular Ca2+ has been shown to be an important signaling factor during epithelial restitution, the source from which this intracellular Ca2+ originates remains unclear. Using gastric organoids derived from mice transgenic for a genetically encoded Ca2+ indicator, we sought to investigate the potential sources of intracellular Ca2+ mobilization. During confocal imaging, photodamage (PD) was induced to 1-2 gastric organoid epithelial cells and epithelial restitution measured simultaneously with changes in intracellular Ca2+ (measured as FRET/CFP ratio in migrating cells adjacent to the damaged area). Inhibition of voltage-gated Ca2+ channels (verapamil, 10 µM) or store-operated calcium entry (YM58483, 20 µM) resulted in delayed repair and dampened intracellular Ca2+ response. Furthermore, inhibition of phospholipase C (U73122, 10 µM) or inositol trisphosphate receptor (2-APB, 50 µM) likewise resulted in delayed repair and dampened Ca2+ response. Results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ mobilization that stimulates repair.

Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids.

Human organoid systems recapitulate in vivo organ architecture yet fail to capture complex pathologies such as inflammation and fibrosis. Here, using 11 different healthy and diseased pluripotent stem cell lines, we developed a reproducible method to derive multi-cellular human liver organoids composed of hepatocyte-, stellate-, and Kupffer-like cells that exhibit transcriptomic resemblance to in vivo-derived tissues. Under free fatty acid treatment, organoids, but not reaggregated cocultured spheroids, recapitulated key features of steatohepatitis, including steatosis, inflammation, and fibrosis phenotypes in a successive manner. Interestingly, an organoid-level biophysical readout with atomic force microscopy demonstrated that organoid stiffening reflects the fibrosis severity. Furthermore, organoids from patients with genetic dysfunction of lysosomal acid lipase phenocopied severe steatohepatitis, rescued by FXR agonism-mediated reactive oxygen species suppression. The presented key methodology and preliminary results offer a new approach for studying a personalized basis for inflammation and fibrosis in humans, thus facilitating the discovery of effective treatments.

De novo formation of the biliary system by TGFß-mediated hepatocyte transdifferentiation.

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, in the presence of a fully developed scaffold and niche1. Here we show that transdifferentiation of hepatocytes in the mouse liver can build a structure that failed to form in development-the biliary system in a mouse model that mimics the hepatic phenotype of human Alagille syndrome (ALGS)2. In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, that is, incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system3-6. In contrast to bile duct development7-9, we show that de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signalling. We identify TGFß signalling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. Furthermore, we show that TGFß signalling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodelling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for the treatment of ALGS and other cholestatic liver diseases.

HGFL-mediated RON signaling supports breast cancer stem cell phenotypes via activation of non-canonical ß-catenin signaling.

Breast cancer stem cells (BCSCs), which drive tumor progression, recurrence, and metastasis, are considered a major challenge for breast cancer treatments, thus the discovery of novel pathways regulating BCSC maintenance remains essential to develop new strategies to effectively target this population and combat disease mortality. The HGFL-RON signaling is overexpressed in human breast cancers and is associated with increased breast cancer progression, metastasis, and poor prognosis. Here, we report that overexpression of RON/MST1R and HGFL/MST1 in cell lines and primary tumors increases BCSC self-renewal, numbers, and tumorigenic potential after syngeneic transplantation. Transcriptome analyses also reveal that the HGFL-RON signaling pathway regulates additional BCSC functions and supports an immunosuppressive microenvironment to stimulate tumor formation and progression. Moreover, we show that genetic and chemical downregulation of HGFL-RON signaling disrupts BCSC phenotypes and tumor growth by suppressing the RON-mediated phosphorylation/activation of ß-CATENIN/CTNNB1 and its effector NF-κB/RELA. These studies indicate that HGFL-RON signaling regulates BCSC phenotypes and the tumor microenvironment to drive tumorigenesis and present HGFL/RON as novel therapeutic targets to effectively eradicate BCSCs in patients.

Epithelial Regeneration After Gastric Ulceration Causes Prolonged Cell-Type Alterations.

BACKGROUND & AIMS: The peptic ulcer heals through a complex process, although the ulcer relapse often occurs several years later after healing. Our hypothesis is that even after visual evidence of healing of gastric ulceration, the regenerated epithelium is aberrant for an extended interval, increasing susceptibility of the regenerated epithelium to damage and further diseases. METHODS: Gastric ulcers were induced in mice by serosal topical application of acetic acid. RESULTS: Gastric ulcers induced by acetic acid visually healed within 30 days. However, regenerated epithelial architecture was poor. The gene profile of regenerated tissue was abnormal, indicating increased stem/progenitor cells, deficient differentiated gastric cell types, and deranged cell homeostasis. Despite up-regulation of PDX1 in the regenerated epithelium, no mature antral cell type was observed. Four months after healing, the regenerated epithelium lacks parietal cells, trefoil factor 2 (TFF2) and (sex-determining region Y)-box 9 (SOX9) remain up-regulated deep in the gastric gland, and the Na/H exchanger 2 (a TFF2 effector in gastric healing) remains down-regulated. Gastric ulcer healing was strongly delayed in TFF2 knockout mice, and re-epithelialization was accompanied with mucous metaplasia. After Helicobacter pylori inoculum 30 days after ulceration, we observed that the gastric ulcer selectively relapses at the same site where it originally was induced. Follow-up evaluation at 8 months showed that the relapsed ulcer was not healed in H pylori-infected tissues. CONCLUSIONS: These findings show that this macroscopically regenerated epithelium has prolonged abnormal cell distribution and is differentially susceptible to subsequent damage by H pylori.

IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival.

The chromatin-binding DEK protein was recently reported to promote the growth of HPV+ and HPV- head and neck squamous cell carcinomas (HNSCCs). Relevant cellular and molecular mechanism(s) controlled by DEK in HNSCC remain poorly understood. While DEK is known to regulate specific transcriptional targets, global DEK-dependent gene networks in HNSCC are unknown. To identify DEK transcriptional signatures we performed RNA-Sequencing (RNA-Seq) in HNSCC cell lines that were either proficient or deficient for DEK. Bioinformatic analyses and subsequent validation revealed that IRAK1, a regulator of inflammatory signaling, and IRAK1-dependent regulatory networks were significantly repressed upon DEK knockdown in HNSCC. According to TCGA data, 14% of HNSCC specimens overexpressed IRAK1, thus supporting possible oncogenic functions. Furthermore, genetic or pharmacologic inhibition of IRAK1 in HNSCC cell lines was sufficient to attenuate downstream signaling such as ERK1/2 and to induce HNSCC cell death by apoptosis. Finally, targeting DEK and IRAK1 simultaneously enhanced cell death as compared to targeting either alone. Our findings reveal that IRAK1 promotes cell survival and is an attractive therapeutic target in HNSCC cells. Thus, we propose a model wherein IRAK1 stimulates tumor signaling and phenotypes both independently and in conjunction with DEK.