Targeting the histone demethylase PHF8-mediated PKCα-Src-PTEN axis in HER2-negative gastric cancer.

Targeted treatments for advanced gastric cancer (GC) are needed, particularly for HER2-negative GC, which represents the majority of cases (80 to 88%). In this study, in silico analyses of the lysine histone demethylases (KDMs) involved in diverse biological processes and diseases revealed that PHD finger protein 8 (PHF8, KDM7B) was significantly associated with poor clinical outcome in HER2-negative GC. The depletion of PHF8 significantly reduced cancer progression in GC cells and in mouse xenografts. PHF8 regulated genes involved in cell migration/motility based on a microarray analysis. Of note, PHF8 interacted with c-Jun on the promoter of PRKCA which encodes PKCα. The depletion of PHF8 or PKCα greatly up-regulated PTEN expression, which could be rescued by ectopic expression of a PKCα expression vector or an active Src. These suggest that PTEN destabilization occurs mainly via the PKCα-Src axis. GC cells treated with midostaurin or bosutinib significantly suppressed migration in vitro and in zebrafish models. Immunohistochemical analyses of PHF8, PKCα, and PTEN showed a positive correlation between PHF8 and PKCα but negative correlations between PHF8 and PTEN and between PKCα and PTEN. Moreover, high PHF8-PKCα expression was significantly correlated with worse prognosis. Together, our results suggest that the PKCα-Src-PTEN pathway regulated by PHF8/c-Jun is a potential prognostic/therapeutic target in HER2-negative advanced GC.

WNK1-OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy.

Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53-/-] and [HBx,src,p53-/-,RPIA], while ppp2r1bb is downregulated in [tert x p53-/-]. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [HBx,src,p53-/-,RPIA] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1ß expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated ß-galactosidase activity in tert transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1-OSR1-PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1-OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1-OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.

Suppression of Ribose-5-Phosphate Isomerase a Induces ROS to Activate Autophagy, Apoptosis, and Cellular Senescence in Lung Cancer.

Ribose-5-phosphate isomerase A (RPIA) regulates tumorigenesis in liver and colorectal cancer. However, the role of RPIA in lung cancer remains obscure. Here we report that the suppression of RPIA diminishes cellular proliferation and activates autophagy, apoptosis, and cellular senescence in lung cancer cells. First, we detected that RPIA protein was increased in the human lung cancer versus adjust normal tissue via tissue array. Next, the knockdown of RPIA in lung cancer cells displayed autophagic vacuoles, enhanced acridine orange staining, GFP-LC3 punctae, accumulated autophagosomes, and showed elevated levels of LC3-II and reduced levels of p62, together suggesting that the suppression of RPIA stimulates autophagy in lung cancer cells. In addition, decreased RPIA expression induced apoptosis by increasing levels of Bax, cleaved PARP and caspase-3 and apoptotic cells. Moreover, RPIA knockdown triggered cellular senescence and increased p53 and p21 levels in lung cancer cells. Importantly, RPIA knockdown elevated reactive oxygen species (ROS) levels. Treatment of ROS scavenger N-acetyl-L-cysteine (NAC) reverts the activation of autophagy, apoptosis and cellular senescence by RPIA knockdown in lung cancer cells. In conclusion, RPIA knockdown induces ROS levels to activate autophagy, apoptosis, and cellular senescence in lung cancer cells. Our study sheds new light on RPIA suppression in lung cancer therapy.

L-Carnitine ameliorates congenital myopathy in a tropomyosin 3 de novo mutation transgenic zebrafish.

BACKGROUND: Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown. METHODS: Histopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated. RESULTS: While TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment. CONCLUSIONS: These results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating ß-catenin via a novel C-terminal domain.

Altered metabolism is one of the hallmarks of cancers. Deregulation of ribose-5-phosphate isomerase A (RPIA) in the pentose phosphate pathway (PPP) is known to promote tumorigenesis in liver, lung, and breast tissues. Yet, the molecular mechanism of RPIA-mediated colorectal cancer (CRC) is unknown. Our study demonstrates a noncanonical function of RPIA in CRC. Data from the mRNAs of 80 patients' CRC tissues and paired nontumor tissues and protein levels, as well as a CRC tissue array, indicate RPIA is significantly elevated in CRC. RPIA modulates cell proliferation and oncogenicity via activation of ß-catenin in colon cancer cell lines. Unlike its role in PPP in which RPIA functions within the cytosol, RPIA enters the nucleus to form a complex with the adenomatous polyposis coli (APC) and ß-catenin. This association protects ß-catenin by preventing its phosphorylation, ubiquitination, and subsequent degradation. The C-terminus of RPIA (amino acids 290 to 311), a region distinct from its enzymatic domain, is necessary for RPIA-mediated tumorigenesis. Consistent with results in vitro, RPIA increases the expression of ß-catenin and its target genes, and induces tumorigenesis in gut-specific promotor-carrying RPIA transgenic zebrafish. Together, we demonstrate a novel function of RPIA in CRC formation in which RPIA enters the nucleus and stabilizes ß-catenin activity and suggests that RPIA might be a biomarker for targeted therapy and prognosis.

WNK1-OSR1/SPAK KINASE CASCADE IS IMPORTANT FOR ANGIOGENESIS.

WNK [with-no-lysine (K)] kinases are a family of four members of serine and threonine kinases that regulate renal Na+ and K+ transport. Mutations of WNK1 and WNK4 cause a hereditary hypertensive and hyperkalemic disease known as pseudohypoaldosteronism type II (PHA2). Unlike other WNK isoforms, WNK1 is ubiquitously expressed and regulates many other cellular processes outside the kidney. Oxidative stress response kinase (OSR1) and related STE 20/SPS1-related proline alanine-rich kinase (SPAK) are downstream kinases of WNK kinases. To examine the role of WNK kinase cascade in vivo, we generated global Wnk1-deleted mice and found that Wnk1-ablated mice die in utero from embryonic angiogenesis and cardiac developmental defects. Endothelial-specific Wnk1 deletion reveals that angiogenesis defect is due to WNK1 requirement in endothelium. We further showed that global and endothelial-deletion of Osr1 phenocopies Wnk1 deletion. Furthermore, expression of a catalytic constitutively active Osr1 transgene rescues angiogenesis defects and embryonic lethality of Wnk1-ablated mice. In zebrafish, Wnk1 knockdown causes similar angiogenesis defects to Vegf2 (Flk1) knockdown and that expression of WNK1 partially rescues Flk1 angiogenesis defects. The results indicate that WNK1 is downstream of VEGF signaling cascade. T-lymphocytes isolated from Wnk1-null mice exhibit migration defects. Inhibition of WNK1-OSR1 downstream target Na-K-2Cl cotransporter NKCC1 mimics migration defect of WNK1-deficient T-lymphocytes. Thus, WNK1-OSR1/SPAK cascade is important for angiogenesis. Regulation of ion homeostasis and cell volume may underlie the mechanism for WNK1 regulation of endothelial cell migration and angiogenesis.

Ribose-5-phosphate isomerase A overexpression promotes liver cancer development in transgenic zebrafish via activation of ERK and ß-catenin pathways.

Dysregulation of the enzymes involved in the pentose phosphate pathway (PPP) is known to promote tumorigenesis. Our recent study demonstrated that ribose-5-phosphate isomerase (RPIA), a key regulator of the PPP, regulates hepatoma cell proliferation and colony formation. Our studies in zebrafish reveal that RPIA-mediated hepatocarcinogenesis requires extracellular signal-regulated kinase (ERK) and ß-catenin signaling. To further investigate RPIA-mediated hepatocarcinogenesis, two independent lines of transgenic zebrafish expressing human RPIA in the liver were generated. These studies reveal that RPIA overexpression triggers lipogenic factor/enzyme expression, steatosis, fibrosis and proliferation of the liver. In addition, the severity of fibrosis and the extent of proliferation are positively correlated with RPIA expression levels. Furthermore, RPIA-mediated induction of hepatocellular carcinoma (HCC) requires the ERK and ß-catenin signaling pathway but is not dependent upon transaldolase levels. Our study presents a mechanism for RPIA-mediated hepatocarcinogenesis and suggests that RPIA represents a valuable therapeutic target for the treatment of HCC.

Toll-like receptor 9 and 21 have different ligand recognition profiles and cooperatively mediate activity of CpG-oligodeoxynucleotides in zebrafish.

CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli currently under investigation as antimicrobial agents for different species. Toll-like receptor (TLR) 9 and TLR21 are the cellular receptors of CpG-ODN in mammals and chickens, respectively. The avian genomes lack TLR9, whereas mammalian genomes lack TLR21. Although fish contain both of these genes, the biological functions of fish TLR9 and TLR21 have not been investigated previously. In this study, we comparatively investigated zebrafish TLR9 (zebTLR9) and TLR21 (zebTLR21). The two TLRs have similar expression profiles in zebrafish. They are expressed during early development stages and are preferentially expressed in innate immune function-related organs in adult fish. Results from cell-based activation assays indicate that these two zebrafish TLRs are functional, responding to CpG-ODN but not to other TLR ligands. zebTLR9 broadly recognized CpG-ODN with different CpG motifs, but CpG-ODN with GACGTT or AACGTT had better activity to this TLR. In contrast, zebTLR21 responded preferentially to CpG-ODN with GTCGTT motifs. The distinctive ligand recognition profiles of these two TLRs were determined by their ectodomains. Activation of these two TLRs by CpG-ODN occurred inside the cells and was modulated by UNC93B1. The biological functions of these two TLRs were further investigated. The CpG-ODNs that activate both zebTLR9 and zebTLR21 were more potent than others that activate only zebTLR9 in the activation of cytokine productions and were more bactericidal in zebrafish. These results suggest that zebTLR9 and zebTLR21 cooperatively mediate the antimicrobial activities of CpG-ODN. Overall, this study provides a molecular basis for the activities of CpG-ODN in fish.

Ribose-5-phosphate isomerase A regulates hepatocarcinogenesis via PP2A and ERK signaling.

The deregulated nonoxidative pentose phosphate pathway (PPP) is known to promote oncogenesis, but the molecular mechanism remains unknown. Here, we report that human ribose-5-phosphate isomerase A (RPIA) plays a role in human hepatocellular carcinoma (HCC). A significant increase in RPIA expression was detected both in tumor biopsies of HCC patients and in a liver cancer tissue array. Importantly, the clinicopathological analysis indicated that RPIA mRNA levels were highly correlated with clinical stage, grade, tumor size, types, invasion and alpha-fetoprotein levels in the HCC patients. In addition, we demonstrated that the ability of RPIA to regulate cell proliferation and colony formation in different liver cancer cell lines required ERK signaling as well as the negative modulation of PP2A activity and that the effects of RPIA could be modulated by the addition of either a PP2A inhibitor or activator. Furthermore, the xenograft studies in nude mice revealed that the modulation of RPIA in liver cancer cells regulated tumor growth and that NIH3T3 cells overexpressing RPIA exhibited increased proliferation, enhanced colony formation, elevated levels of p-ERK1/2 and accelerated tumor growth. This study provides new insight into the molecular mechanisms by which RPIA overexpression can induce oncogenesis in HCC. Furthermore, it suggests that RPIA can be a good prognosis biomarker and a potential target for HCC therapy.

Creatine and L-carnitine attenuate muscular laminopathy in the LMNA mutation transgenic zebrafish.

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify L-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest L-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.

The Comprehensive Effects of Carassius auratus Complex Formula against Lipid Accumulation, Hepatocarcinogenesis, and COVID-19 Pathogenesis via Stabilized G-Quadruplex and Reduced Cell Senescence.

Carassius auratus complex formula (CACF) is a traditional Chinese medicine known for its antidiabetic effects. Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide, and there are currently no effective therapies for advanced HCC. This study explores the comprehensive effects and possible mechanisms of CACF on HCC. The results show that CACF reduces the viability of hepatoma cells in vitro, while benefiting normal hepatocytes. In addition, CACF inhibits hepatoma cell growth in a zebrafish xenotransplantation model and decreases lipid accumulation, represses inflammation and cell proliferation markers in fatty acid translocase (CD36) transgenic zebrafish, and inhibits the expression of cell proliferation and ß-catenin downstream targets in telomerase (tert) transgenic zebrafish models. Ingenuity Pathway Analysis reveals that CACF exerts multiple functions, including reduction of inflammation and inhibition of lipid transporter and PPAR signaling pathway. Surprisingly, CACF also regulates the expression of genes and reduces coronavirus infection and pathogenesis in a zebrafish model. CACF treatment is validated to regulate the expression of genes for anti-coronavirus activity. Mechanistically, CACF stabilizes G-quadruplex and reduces cell senescence associated ß-galactosidase activity. In summary, CACF may be a promising therapeutic agent with multiple functions including anticancer, anti-inflammation, and anti-microorganisms in a zebrafish model.

Reduced Ribose-5-Phosphate Isomerase A-1 Expression in Specific Neurons and Time Points Promotes Longevity in Caenorhabditis elegans.

Deregulation of redox homeostasis is often associated with an accelerated aging process. Ribose-5-phosphate isomerase A (RPIA) mediates redox homeostasis in the pentose phosphate pathway (PPP). Our previous study demonstrated that Rpi knockdown boosts the healthspan in Drosophila. However, whether the knockdown of rpia-1, the Rpi ortholog in Caenorhabditis elegans, can improve the healthspan in C. elegans remains unknown. Here, we report that spatially and temporally limited knockdown of rpia-1 prolongs lifespan and improves the healthspan in C. elegans, reflecting the evolutionarily conserved phenotypes observed in Drosophila. Ubiquitous and pan-neuronal knockdown of rpia-1 both enhance tolerance to oxidative stress, reduce polyglutamine aggregation, and improve the deteriorated body bending rate caused by polyglutamine aggregation. Additionally, rpia-1 knockdown temporally in the post-developmental stage and spatially in the neuron display enhanced lifespan. Specifically, rpia-1 knockdown in glutamatergic or cholinergic neurons is sufficient to increase lifespan. Importantly, the lifespan extension by rpia-1 knockdown requires the activation of autophagy and AMPK pathways and reduced TOR signaling. Moreover, the RNA-seq data support our experimental findings and reveal potential novel downstream targets. Together, our data disclose the specific spatial and temporal conditions and the molecular mechanisms for rpia-1 knockdown-mediated longevity in C. elegans. These findings may help the understanding and improvement of longevity in humans.

Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model.

Hepatitis B virus X antigen plays an important role in the development of human hepatocellular carcinoma (HCC). The key regulators controlling the temporal downstream gene expression for HCC progression remains unknown. In this study, we took advantage of systems biology approach and analyzed the microarray data of the HBx transgenic mouse as a screening process to identify the differentially expressed genes and applied the software Pathway Studio to identify potential pathways and regulators involved in HCC. Using subnetwork enrichment analysis, we identified five common regulator genes: EDN1, BMP7, BMP4, SPIB and SRC. Upregulation of the common regulators was validated in the other independent HBx transgenic mouse lines. Furthermore, we verified the correlation of their RNA expression levels by using the human HCC samples, and their protein levels by using the human liver disease tissue arrays. EDN1, bone morphogenetic protein (BMP) 4 and BMP7 were upregulated in cirrhosis, BMP4, BMP7 and SRC were further upregulated in hepatocellular or cholangiocellular carcinoma samples. The trend of increasing expression of the common regulators correlates well with the progression of human liver cancer. Overexpression of the common regulators increases the cell viability, promotes migration and invasiveness and enhances the colony formation ability in Hep3B cells. Our approach allows us to identify the critical genes in hepatocarcinogenesis in an HBx-induced mouse model. The validation of the gene expressions in the liver cancer of human patients and their cellular function assays suggests that the identified common regulators may serve as useful molecular targets for the early-stage diagnosis or therapy for HCC.

An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish.

An evolutionarily conserved subcircuit (kernel) dedicated to a specific developmental function is found at the top of the gene regulatory networks (GRNs) hierarchy. Here we comprehensively demonstrate that a pan-deuterostome endoderm specification kernel exists in zebrafish. We analyzed interactions among gata5, gata6, otx2 and prdm1a using specific morpholino knockdowns and measured the gene expression profiles by quantitative real-time RT-PCR and in situ hybridization. The mRNA rescue experiment validated the specificity of the morpholino knockdown. We found that the interactions among gata5, gata6, otx2 and prdm1a determine the initial specification of the zebrafish endoderm. Although otx2 can activate both gata5 and gata6, and the prdm1a/krox homologue also activates some endoderm transcription factors, a feedback loop from Gata to otx2 and prdm1a is missing. Furthermore, we found the positive regulation between gata5 and gata6 to further lock-on the mesendoderm specification by the Gata family. Chromatin immunoprecipitation was used to further validate the recruitment of Otx2 to the gata5 and gata6 loci. Functional assays revealed that module B of gata6 and the basal promoter of gata5 drive the gene at the mesendoderm, and mutational analysis demonstrated that Otx2 and Gata5/6 contribute to reporter gene activation. This is the first direct evidence for evolutionarily conserved endoderm specification across echinoderms and vertebrates.

WNK1 kinase signaling in metastasis and angiogenesis.

With-no-lysine kinases (WNKs) are a novel family of serine/threonine protein kinases participating in ion homeostasis via the WNK-OSR1/SPAK-NKCC cascade. Recent studies of WNK1 have revealed that its related signaling pathways mediated tumor-induced angiogenesis and carcinogenesis and uncovered novel roles of WNK1 in endothelial cell migration and proliferation, tumor cell proliferation, and metastasis. Herein, we review the functions of WNK1 in cancer metastasis and angiogenesis and propose WNK1 targeting as an anti-cancer strategy.

Low concentrations of 4-ABP promote liver carcinogenesis in human liver cells and a zebrafish model.

4-Aminobiphenyl (4-ABP) is a human bladder cancer carcinogen found in the manufacture of azo dyes and the composition of cigarette smoke in the environment. To determine whether low concentrations of 4-ABP induced or promote liver carcinogenesis and investigate the underlying mechanism, we have established the liver cell carcinogenesis model in human liver cell lines and zebrafish to evaluate liver cancer development associated with long-term exposure to low concentrations of 4-ABP. Results show that repeated 4-ABP exposure promoted cellular proliferation and migration via the involvement of ROS in Ras/MEK/ERK pathway in vitro. Also, 4-ABP (1, 10, and 100 nM) induces hepatocellular carcinoma (HCC) formation in HBx, Src (p53-/-) transgenic zebrafish at four months of age and in wild-type zebrafish at seven months of age. In addition, we observed a correlation between the Ras-ERK pathway and 4-ABP-induced HCC in vitro and in vivo. Our finding suggests low concentrations of 4-ABP repeated exposure is a potential risk factor for liver cancer. To our knowledge, this is the first report on the promotion of liver carcinogenesis in human liver cells and zebrafish following 4-ABP exposure.

Liver development and cancer formation in zebrafish.

Liver is the largest organ in the human body, and it regulates many physiological processes. Many studies on liver development in different model organisms have demonstrated that the mechanism of hepatogenesis is conserved in vertebrates. The identification of the genes and regulatory pathways involved in liver formation provides a basis for the diagnosis of liver diseases and therapeutic interventions. Hepatocellular carcinoma is the third leading cause of mortality worldwide. In the last decade, genetic alterations, which include the gain and loss of DNA, as well as mutations and epigenomic changes, have been identified as important factors in liver cancer. Many genetic pathways are dysregulated during carcinogenesis. Here, we review the gene regulatory networks that underlie liver organogenesis and the dysregulation of these pathways in liver cancer. The genes and pathways involved in hepatogenesis and liver cancer are largely conserved between zebrafish and humans, making this an ideal model organism for the study of this disease. A better understanding of liver development may aid in the development of new diagnostic and therapeutic approaches to liver cancer.

Functional analysis of the evolutionarily conserved cis-regulatory elements on the sox17 gene in zebrafish.

The Sox17 is an important transcription factor for endodermal cells (Danio rerio). According to the predictions of the GRNs, based on perturbation experiments and literature search, the sox17 gene is engaged with two other regulatory genes, sox32 and pou5f1. Nodal signaling operated on several endoderm-specific transcription factors to determine the endoderm specification. In addition, endoderm specification requires the Fgf and Bmp signaling pathways to be repressed in the cells which will become endoderm. It is predicted that Nodal activates sox32 and works synergistically with Pou5f1 to activate sox17. Bmp represses the expression of sox17 on the ventral side and Fgf represses it on the dorsal side. The regulatory inputs of sox17 at the genomic sequence level are not known. Here, we have uncovered the relevant sox17 cis-regulatory elements, and examined the specific input predictions of the GRNs. We discovered three conserved modules, A, B, and C, with a synergistic effect among them. We revealed that the Pou5f1-binding element on the B module and the Sox32-binding element on the C module work synergistically. Furthermore, an evolutionarily non-conserved R module exhibits a repressive effect on both the ventral and dorsal side. We have directly demonstrated the structural and functional relationships of the genomic code at this key node of the endoderm GRNs in zebrafish development. This information provides new insight into the complexity of endoderm formation and serves as a valuable resource for the establishment of a complete endoderm gene regulatory network.

Developmental gene regulatory networks in the zebrafish embryo.

The genomic developmental program operates mainly through the regulated expression of genes encoding transcription factors and signaling pathways. Complex networks of regulatory genetic interactions control developmental cell specification and fates. Development in the zebrafish, Danio rerio, has been studied extensively and large amounts of experimental data, including information on spatial and temporal gene expression patterns, are available. A wide variety of maternal and zygotic regulatory factors and signaling pathways have been discovered in zebrafish, and these provide a useful starting point for reconstructing the gene regulatory networks (GRNs) underlying development. In this review, we describe in detail the genetic regulatory subcircuits responsible for dorsoanterior-ventroposterior patterning and endoderm formation. We describe a number of regulatory motifs, which appear to act as the functional building blocks of the GRNs. Different positive feedback loops drive the ventral and dorsal specification processes. Mutual exclusivity in dorsal-ventral polarity in zebrafish is governed by intra-cellular cross-inhibiting GRN motifs, including vent/dharma and tll1/chordin. The dorsal-ventral axis seems to be determined by competition between two maternally driven positive-feedback loops (one operating on Dharma, the other on Bmp). This is the first systematic approach aimed at developing an integrated model of the GRNs underlying zebrafish development. Comparison of GRNs' organizational motifs between different species will provide insights into developmental specification and its evolution. The online version of the zebrafish GRNs can be found at http://www.zebrafishGRNs.org.

Complexity of cis-regulatory organization of six3a during forebrain and eye development in zebrafish.

BACKGROUND: Six3a belongs to the SIX family of homeodomain proteins and is expressed in the most anterior neural plate at the beginning of neurogenesis in various species. Though the function of Six3a as a crucial regulator of eye and forebrain development has been thoroughly investigated, the transcriptional regulation of six3a is not well understood. RESULTS: To elucidate the transcriptional regulation of six3a, we performed an in vivo reporter assay. Alignment of the 21-kb region surrounding the zebrafish six3a gene with the analogous region from different species identified several conserved non-coding modules. Transgenesis in zebrafish identified two enhancer elements and one suppressor. The D module drives the GFP reporter in the forebrain and eyes at an early stage, while the A module is responsible for the later expression. The A module also works as a repressor suppressing ectopic expression from the D module. Mutational analysis further minimized the A module to four highly conserved elements and the D module to three elements. Using electrophoresis mobility shift assays, we also provided evidence for the presence of DNA-binding proteins in embryonic nuclear extracts. The transcription factors that may occupy those highly conserved elements were also predicted. CONCLUSION: This study provides a comprehensive view of six3a transcription regulation during brain and eye development and offers an opportunity to establish the gene regulatory networks underlying neurogenesis in zebrafish.