Myc-induced liver tumors in transgenic zebrafish can regress in tp53 null mutation

10.1371/journal.pone.0117249PLoS ONE101e011724

Neoplasia and Neoplasm Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design

During the past decade the zebrafish has emerged as a leading model for mechanistic cancer research due to its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost-effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast to other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type (wt) lines of zebrafish. So far a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues in not available for zebrafish as it is for other intensely studied laboratory animal species. This manuscript confirms that as in other species diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wt lines of zebrafish exhibit elevated neoplasm incidences and neoplasm associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation.Keywords: Zebrafish, Hepatocyte megalocytosis, Diet, Non-protocol induced variation, Naturally occurring carcinogen, Danio rerio, Husbandry, NeoplasiaKeywords: Zebrafish, Hepatocyte megalocytosis, Diet, Non-protocol induced variation, Naturally occurring carcinogen, Danio rerio, Husbandry, Neoplasi

Myc-Induced Liver Tumors in Transgenic Zebrafish Can Regress in tp53 Null Mutation

Hepatocellular carcinoma (HCC) is currently one of the top lethal cancers with an increasing trend. Deregulation of MYC in HCC is frequently detected and always correlated with poor prognosis. As the zebrafish genome contains two differentially expressed zebrafish myc orthologs, myca and mycb, it remains unclear about the oncogenicity of the two zebrafish myc genes. In the present study, we developed two transgenic zebrafish lines to overexpress myca and mycb respectively in the liver using a mifepristone-inducible system and found that both myc genes were oncogenic. Moreover, the transgenic expression of myca in hepatocytes caused robust liver tumors with several distinct phenotypes of variable severity. ~5% of myca transgenic fish developing multinodular HCC with cirrhosis after 8 months of induced myca expression. Apoptosis was also observed with myca expression; introduction of homozygous tp53[superscript -/-] mutation into the myca transgenic fish reduced apoptosis and accelerated tumor progression. The malignant status of hepatocytes was dependent on continued expression of myca; withdrawal of the mifepristone inducer resulted in a rapid regression of liver tumors, and the tumor regression occurred even in the tp53[superscript -/-] mutation background. Thus, our data demonstrated the robust oncogenicity of zebrafish myca and the requirement of sustained Myc overexpression for maintenance of the liver tumor phenotype in this transgenic model. Furthermore, tumor regression is independent of the function of Tp53

Development of a conditional liver tumor model by mifepristone-inducible Cre recombination to control oncogenic kras(V12) expression in transgenic zebrafish

Here we report a new transgenic expression system by combination of liver-specific expression, mifepristone induction and Cre-loxP recombination to conditionally control the expression of oncogenic kras[superscript]V12. This transgenic system allowed expression of kras[superscript]V12 specifically in the liver by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system. We found that liver tumors were generally induced from multiple foci due to incomplete Cre-loxP recombination, thus mimicking naturally occurring human tumors resulting from one or a few mutated cells and clonal proliferation to form nodules. Similar to our earlier studies by both constitutive and inducible expression of the kras[superscript]V12 oncogene, hepatocellular carcinoma (HCC) is the main type of liver tumor induced by kras[superscript]V12 expression. Moreover, mixed tumors with hepatocellular adenoma and hepatoblastoma (HB) were also frequently observed. Molecular analyses also indicated similar increase of phosphorylated ERK1/2 in all types of liver tumors, but nuclear localization of β–catenin, a sign of malignant transformation, was found only in HCC and HB. Taken together, our new transgenic system reported in this study allows transgenic kras[superscript]V12 expression specifically in the zebrafish liver only by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Nature Publishing Group. The published article can be found at: http://www.nature.com/articles/srep1955

Primary Intestinal and Vertebral Chordomas in Laboratory Zebrafish (Danio rerio)

Chordomas are uncommon neoplasms arising from notochord remnants, most commonly occurring in the axial skeleton. Extraskeletal soft tissue chordomas are rare primary tumors, and primary intestinal chordomas have not been reported. Herein we report 24 cases of spontaneous primary intestinal chordomas in zebrafish, as well as 9 spontaneous vertebral chordomas. Both intestinal and vertebral tumors showed invasive behavior, although more commonly in the latter. In all cases of primary intestinal chordomas, there was no axial or peripheral skeletal or other non-visceral involvement. Although uncommon, intestinal chordomas represent a unique background lesion in aged zebrafish.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American College of Veterinary Pathologists and published by Sage Publications. It can be found at: http://intl-vet.sagepub.com/.Keywords: Zebrafish, Chordomas, Vertebral, Intestina

Histopathologic Alterations Associated with Global Gene Expression Due to Chronic Dietary TCDD Exposure in Juvenile Zebrafish

The goal of this project was to investigate the effects and possible developmental disease implication of chronic dietary TCDD exposure on global gene expression anchored to histopathologic analysis in juvenile zebrafish by functional genomic, histopathologic and analytic chemistry methods. Specifically, juvenile zebrafish were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb, and fish were sampled following 0, 7, 14, 28 and 42 d after initiation of the exposure. TCDD accumulated in a dose- and time-dependent manner and 100 ppb TCDD caused TCDD accumulation in female (15.49 ppb) and male (18.04 ppb) fish at 28 d post exposure. Dietary TCDD caused multiple lesions in liver, kidney, intestine and ovary of zebrafish and functional dysregulation such as depletion of glycogen in liver, retrobulbar edema, degeneration of nasal neurosensory epithelium, underdevelopment of intestine, and diminution in the fraction of ovarian follicles containing vitellogenic oocytes. Importantly, lesions in nasal epithelium and evidence of endocrine disruption based on alternatively spliced vasa transcripts are two novel and significant results of this study. Microarray gene expression analysis comparing vehicle control to dietary TCDD revealed dysregulated genes involved in pathways associated with cardiac necrosis/cell death, cardiac fibrosis, renal necrosis/cell death and liver necrosis/cell death. These baseline toxicological effects provide evidence for the potential mechanisms of developmental dysfunctions induced by TCDD and vasa as a biomarker for ovarian developmental disruption

Pathological and Behavioral Manifestations of the “Cayuga Syndrome,” a Thiamine Deficiency in Larval Landlocked Atlantic Salmon

The “Cayuga syndrome” is a maternally transmitted, naturally occurring thiamine deficiency that causes 100% mortality of larval landlocked Atlantic salmon Salmo salar in several of New York's Finger Lakes, Results of multiyear studies to qualify and quantify the neurobehavioral and gross pathological signs of this condition are described, Affected sac fry became weak and ataxic and responded atypically to stimuli 1–2 weeks before death. Quantitative assays of stimulus-provoked swimming revealed a significant neuropathy whereby the sac fry exhibited abnormal thigmotactic and phototactic behaviors. Gross lesions observed in Cayuga sac fry included yolk-sac opacities, subcutaneous edema, vitelline hemorrhage or congestion, pericardial edema, retrobulbar edema, branchial congestion, foreshortened maxillae, hydrocephalus, and occasional caudal fin deformities, Lesion frequency in progeny differed significantly between dam source. Yolk conversion efficiency was decreased at least 1 week before death, suggesting that the bioenergetics of the fish was compromised and thereby supporting the thiamine residue and treatment data reported elsewhere, Comparisons with coagulated-yolk, blue-sac and swim-up syndromes are presented, The pathological signs of the Cayuga syndrome represent a unique departure from the lesions induced by toxicants or pathogens in other piscine models, and for the first time profile the profound effects of thiamine deficiency on cardiovascular and neurological systems of larval fish.This is the publisher’s final pdf. The published article is copyrighted by the American Fisheries Society and can be found at: http://www.tandfonline.com/toc/uahh20/current#.UgvziXfAF8G

Naturally Occurring Thiamine Deficiency Causing Reproductive Failure in Finger Lakes Atlantic Salmon and Great Lakes Lake Trout

A maternally transmitted, noninfectious disease known as the Cayuga syndrome caused 100% mortality in larval offspring of wild-caught landlocked Atlantic salmon Salmo salar from several of New York's Finger Lakes. Survival of lake trout Salvelinus namaycush from Lakes Erie and Ontario was also impaired, but not until yolk absorption was nearly complete; moreover, mortality was greatly reduced relative to that of the salmon (range: 5–87%). Tissue concentrations of thiamine hydrochloride were severely reduced in these salmonid fish relative to unaffected control stocks. Afflicted Atlantic salmon treated with thiamine by yolk-sac injection or by bath immersion recovered completely from the Cayuga syndrome, as evidenced by the quantified reversal of abnormal swimming behaviors only 2 d after treatment and by the excellent survival (>95%) of the treated Atlantic salmon through 1.5 months of feeding. These data represent the first evidence of a vitamin deficiency causing the complete reproductive failure of an animal population in nature. These lethal vitamin deficiencies are presumably caused by a diet of alewives Alosa pseudoharengus, nonnative forage fishes of the herring family that exhibit high thiaminase activity.This is the publisher’s final pdf. The published article is copyrighted by the American Fisheries Society and can be found at: http://www.tandfonline.com/toc/utaf20/current#.Ug5s9XfAF8E

Crosstalk of Ras and Rho: activation of RhoA abates Kras-induced liver tumorigenesis in transgenic zebrafish models

RAS and Rho small GTPases are key molecular switches that control cell dynamics, cell growth and tissue development through their distinct signaling pathways. While much has been learnt about their individual functions in both cell and animal models, the physiological and pathophysiological consequences of their signaling crosstalk in multi-cellular context in vivo remain largely unknown, especially in liver development and liver tumorigenesis. Furthermore, the roles of RhoA in RAS-mediated transformation and their crosstalk in vitro remain highly controversial. When challenged with carcinogens, zebrafish developed liver cancer that resembles the human liver cancer both molecularly and histopathologically. Capitalizing on the growing importance and relevance of zebrafish (Danio rerio) as an alternate cancer model, we have generated liver-specific, Tet-on inducible transgenic lines expressing oncogenic Kras[superscript G12V], RhoA, constitutively-active RhoA[superscript G14V] or dominant-negative RhoA[superscript T19N]. Double transgenic lines expressing Kras[superscript G12V] with one of the three RhoA genes were also generated. Based on quantitative bioimaging and molecular markers for genetic and signaling aberrations, we showed that the induced expression of oncogenic Kras during early development led to liver enlargement and hepatocyte proliferation, associated with elevated Erk phosphorylation, Akt2-p21Cip expression and activation. Such an increase in liver size and Akt2 expression was augmented by dominant-negative RhoA[superscript T19N], but was abrogated by the constitutive-active RhoA[superscript G14V]. Consequently, induced expression of the oncogenic Kras in adult transgenic fish led to the development of hepatocellular carcinomas. Survival studies further revealed that the co-expression of dominant-negative RhoA[superscript T19N] with oncogenic Kras increased the mortality rate compared to the other single or double transgenic lines. This study represents the first in vivo investigation of the previously unappreciated signaling crosstalk between Kras and RhoA in regulating liver overgrowth and liver tumorigenesis. Our results also implicate that activating Rho could be beneficial to suppress the Kras-induced liver malignancies.Keywords: RhoA, Zebrafish, Signaling crosstalk, Akt, Ras, Hepatocellular carcinom

Reproductive Failure of Landlocked Atlantic Salmon from New York's Finger Lakes: Investigations into the Etiology and Epidemiology of the “Cayuga Syndrome”

We describe a disease syndrome that afflicts larval, landlocked Atlantic salmon Salmo salar from Cayuga Lake, one of central New York's Finger Lakes. Mortality associated with the “Cayuga syndrome” is 98–100%. Death usually occurs between 650 and 850 centigrade degree-days after fertilization, approximately 2–4 weeks before yolk resorption is complete. Although there is minor temporal variation in the onset of the Cayuga syndrome in progeny from individual females, all sac fry eventually succumb. Incubation of embryos and sac fry under constant, ambient, or reduced temperature regimens slightly alters the degree-day timing of syndrome onset, but does not improve survival. Based on mortality rate, manifestation of the Cayuga syndrome has not changed in the past 10 years, even though incubation waters of varying chemistry and temperature have been used. Mortality of the negative control stocks used for these studies never exceeded 10% from hatching to first feeding. Findings from reciprocal crossbreeding experiments indicate the problem is associated with ova only. A noninfectious etiology is indicated by the lack of consistently identifiable fish pathogens from syndrome-afflicted sac fry and by the failure to transmit the condition horizontally. Suspect contaminants were eliminated as potential causative factors. Epidemiological studies on the viability of other Finger Lakes stocks indicate that Atlantic salmon from Keuka and Seneca lakes are also afflicted (100% mortality). yet those from Skaneateles Lake are not. The cause of this syndrome appears to be nutritional.This is the publisher’s final pdf. The published article is copyrighted by the American Fisheries Society and can be found at: http://www.tandfonline.com/toc/uahh20/current#.Ug5ocnfAF8E