Reversion of tumor hepatocytes to normal hepatocytes during liver tumor regression in an oncogene-expressing transgenic zebrafish model.
ABSTRACT: Tumors are frequently dependent on primary oncogenes to maintain their malignant properties (known as 'oncogene addiction'). We have previously established several inducible hepatocellular carcinoma (HCC) models in zebrafish by transgenic expression of an oncogene. These tumor models are strongly oncogene addicted, as the induced and histologically proven liver tumors regress after suppression of oncogene expression by removal of a chemical inducer. However, the question of whether the liver tumor cells are eliminated or revert to normal cells remains unanswered. In the present study, we generated a novel Cre/loxP transgenic zebrafish line, Tg(fabp10: loxP-EGFP-stop-loxP-DsRed; TRE: CreERT2) (abbreviated to CreER), in order to trace tumor cell lineage during tumor regression after crossing with the xmrk (activated EGFR homolog) oncogene transgenic line, Tg(fabp10: rtTA; TRE: xmrk; krt4: EGFP) We found that, during HCC regression, restored normal liver contained both reverted tumor hepatocytes (RFP+) and newly differentiated hepatocytes (GFP+). RNA sequencing (RNA-seq) analyses of the RFP+ and GFP+ hepatocyte populations after tumor regression confirmed the conversion of tumor cells to normal hepatocytes, as most of the genes and pathways that were deregulated in the tumor stages were found to have normal regulation in the tumor-reverted hepatocytes. Thus, our lineage-tracing studies demonstrated the potential for transformed tumor cells to revert to normal cells after suppression of expression of a primary oncogene. This observation may provide a basis for the development of a therapeutic approach targeting addicted oncogenes or oncogenic pathways.
Project description:Previously three oncogene transgenic zebrafish lines with inducible expression of xmrk, kras or Myc in the liver have been generated and these transgenic lines develop oncogene-addicted liver tumors upon chemical induction. In the current study, comparative transcriptomic approaches were used to examine the correlation of the three induced transgenic liver cancers with human liver cancers. RNA profiles from the three zebrafish tumors indicated relatively small overlaps of significantly deregulated genes and biological pathways. Nevertheless, the three transgenic tumor signatures all showed significant correlation with advanced or very advanced human hepatocellular carcinoma (HCC). Interestingly, molecular signature from each oncogene-induced zebrafish liver tumor correlated with only a small subset of human HCC samples (24-29%) and there were conserved up-regulated pathways between the zebrafish and correlated human HCC subgroup. The three zebrafish liver cancer models together represented nearly half (47.2%) of human HCCs while some human HCCs showed significant correlation with more than one signature defined from the three oncogene-addicted zebrafish tumors. In contrast, commonly deregulated genes (21 up and 16 down) in the three zebrafish tumor models generally showed accordant deregulation in the majority of human HCCs, suggesting that these genes might be more consistently deregulated in a broad range of human HCCs with different molecular mechanisms and thus serve as common diagnosis markers and therapeutic targets. Thus, these transgenic zebrafish models with well-defined oncogene-induced tumors are valuable tools for molecular classification of human HCCs and for understanding of molecular drivers in hepatocarcinogenesis in each human HCC subgroup.
Project description:Melanoma development in interspecific hybrids of Xiphophorus is induced by the overexpression of the mutationally activated receptor tyrosine kinase Xmrk in pigment cells. Based on the melanocyte specificity of the transcriptional upregulation, a pigment cell-specific promoter region was postulated for xmrk, the activity of which is controlled in healthy purebred fish by the molecularly still unidentified regulator locus R. However, as yet the xmrk promoter region is still poorly characterized. In order to contribute to a better understanding of xmrk expression regulation, we performed a functional analysis of the entire putative gene regulatory region of the oncogene using conventional plasmid-based reporter systems as well as a newly established method employing BAC-derived luciferase reporter constructs in melanoma and non-melanoma cell lines. Using the melanocyte-specific mitfa promoter as control, we could demonstrate that our in vitro system is able to reliably monitor regulation of transcription through cell type-specific regulatory sequences. We found that sequences within 200kb flanking the xmrk oncogene do not lead to any specific transcriptional activation in melanoma compared to control cells. Hence, xmrk reporter constructs fail to faithfully reproduce the endogenous transcriptional regulation of the oncogene. Our data therefore strongly indicate that the melanocyte-specific transcription of xmrk is not the consequence of pigment cell-specific cis-regulatory elements in the promoter region. This hints at additional regulatory mechanisms involved in transcriptional control of the oncogene, thereby suggesting a key role for epigenetic mechanisms in oncogenic xmrk overexpression and thereby in tumor development in Xiphophorus.
Project description:The liver and pancreas are two major digestive organs, and among the different cell types in them, hepatocytes and the insulin-producing ? cells have roles in both health and diseases. Accordingly, clinicians and researchers are very interested in the mechanisms underlying the development and regeneration of liver and pancreatic ? cells. Gene and enhancer traps such as the Tol2 transposon-based system are useful for identifying genes potentially involved in developmental processes in the zebrafish model. Here, we developed a strategy that combines a Tol2-mediated enhancer trap and the Cre/loxP system by using loxP-flanked reporters driven by ? cell- or hepatocyte-specific promoters and the upstream activating sequence (UAS)-driving Cre. Two double-transgenic reporter lines, Tg(ins:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus) and Tg(fabp10:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus), were established to label pancreatic ? cells and hepatocytes, respectively. These two double-transgenic lines were each crossed with the Tol2-enhancer trap founder lines to screen for and identify genes expressed in the ? cell and hepatocytes during development. This trap system coupled with application of nitroreductase (NTR)/metronidazole (Mtz)-mediated cell ablation could identify genes expressed during regeneration. Of note, pilot enhancer traps captured transiently and weakly expressed genes such as rab3da and ensab with higher efficiencies than traditional enhancer trap systems. In conclusion, through permanent genetic labeling by Cre/loxP, this improved Tol2-mediated enhancer trap system provides a promising method to identify transiently or weakly expressed, but potentially important, genes during development and regeneration.
Project description:The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus.
Project description:Previously we have generated inducible liver tumor models by transgenic expression of Myc or xmrk (activated EGFR homolog) oncogenes in zebrafish. To investigate the interaction of the two oncogenes, we crossed the two transgenic lines and observed more severe and faster hepatocarcinogenesis in Myc/xmrk double transgenic zebrafish than either single transgenic fish. RNA-Seq analyses revealed distinct changes in many molecular pathways among the three types of liver tumors. In particular, we found dramatic alteration of cancer metabolism based on the uniquely enriched pathways in the Myc/xmrk tumors. Critical glycolytic genes including hk2, pkm and ldha were significantly up-regulated in Myc/xmrk tumors but not in either single oncogene-induced tumors, suggesting a potential Warburg effect. In RT-qPCR analyses, the specific pkm2 isoformin Warburg effect was found to be highly enriched in the Myc/xmrk tumors but not in Myc or xmrk tumors, consistent with the observations in many human cancers with Warburg effect. Moreover, the splicing factor genes (hnrnpa1, ptbp1a, ptbp1b and sfrs3b) responsible for generating the pkm isoform were also greatly up-regulated in the Myc/xmrk tumors. As Pkm2 isoform is generally inactive and causes incomplete glycolysis to favor anabolism and tumor growth, by treatment with a Pkm2-specific activator, TEPP-46, we further demonstrated that activation of Pkm2 suppressed the growth of oncogenic liver as well as proliferation of liver cells. Collectively, our Myc/xmrk zebrafish model suggests synergetic effect of EGFR and MYC in triggering Warburg effect in the HCC formation and may provide a promising in vivo model for Warburg effect.
Project description:Three-dimensional (3D) bioprinting of living structures with cell-laden biomaterials has been achieved in vitro, however, some cell-cell interactions are limited by the existing hydrogel. To better mimic tumor microenvironment, self-assembled multicellular heterogeneous brain tumor fibers have been fabricated by a custom-made coaxial extrusion 3D bioprinting system, with high viability, proliferative activity and efficient tumor-stromal interactions. Therein, in order to further verify the sufficient interactions between tumor cells and stroma MSCs, CRE-LOXP switch gene system which contained GSCs transfected with "LOXP-STOP-LOXP-RFP" genes and MSCs transfected with "CRE recombinase" gene was used. Results showed that tumor-stroma cells interacted with each other and fused, the transcription of RFP was higher than that of 2D culture model and control group with cells mixed directly into alginate, respectively. RFP expression was observed only in the cell fibers but not in the control group under confocal microscope. In conclusion, coaxial 3D bioprinted multicellular self-assembled heterogeneous tumor tissue-like fibers provided preferable 3D models for studying tumor microenvironment in vitro, especially for tumor-stromal interactions.
Project description:The mechanisms by which cancer evolves and persists in natural systems have been difficult to ascertain. In the Xiphophorus melanoma model, a functional oncogene (Xiphophorus melanoma receptor kinase Xmrk) has been maintained for several million years despite being deleterious and in an extremely unstable genomic region. Melanomas in Xiphophorus spp. fishes (platyfishes and swordtails) have been investigated since the 1920s, and, yet, positive selection that could explain the maintenance of Xmrk has not been found. Here, we show that Xiphophorus cortezi females from two populations prefer males with the spotted caudal (Sc) melanin pattern, which is associated with the presence of the Xmrk oncogene and serves as the site of melanoma formation within this species. Moreover, X. cortezi females prefer males with an enhanced Sc to males with a reduced Sc pattern. RT-PCR analysis confirms tissue-specific Xmrk expression within the Sc pattern in X. cortezi. Because of the association of Xmrk with the Sc pigment pattern and the fact that melanoma formation augments this visual signal, sexual selection appears to be maintaining this oncogene because of a mating preference for Sc, as well as the exaggeration of this male trait. At the individual level, decreases in viability and fecundity because of Xmrk and subsequent melanoma formation may be mitigated via increases in mate acquisition. At the population level, maintenance of this oncogene appears to be under frequency dependent selection, as we detected female preference for males without Sc in a third population that had higher frequencies of Sc in females.
Project description:Development of spontaneous melanoma in Xiphophorus interspecies backcross hybrid progeny, (X. hellerii?×?[X. maculatus Jp 163 A?×?X. hellerii]) is due to Mendelian segregation of a oncogene (xmrk) and a molecularly uncharacterized locus, called R(Diff), on LG5. R(Diff) is thought to suppresses the activity of xmrk in healthy X. maculatus Jp 163 A parental species that rarely develop melanoma. To better understand the molecular genetics of R(Diff), we utilized RNA-Seq to study allele-specific gene expression of spontaneous melanoma tumors and corresponding normal skin samples derived from 15 first generation backcross (BC1 ) hybrids and 13 fifth generation (BC5 ) hybrids. Allele-specific expression was determined for all genes and assigned to parental allele inheritance for each backcross hybrid individual. Results showed that genes residing in a 5.81 Mbp region on LG5 were exclusively expressed from the X. hellerii alleles in tumor-bearing BC1 hybrids. This observation indicates this region is consistently homozygous for X. hellerii alleles in tumor bearing animals, and therefore defines this region to be the R(Diff) locus. The R(Diff) locus harbors 164 gene models and includes the previously characterized R(Diff) candidate, cdkn2x. Twenty-one genes in the R(Diff) region show differential expression in the tumor samples compared to normal skin tissue. These results further characterize the R(Diff) locus and suggest tumor suppression may require a multigenic region rather than a single gene variant. Differences in gene expression between tumor and normal skin tissue in this region may indicate interactions among several genes are required for backcross hybrid melanoma development.
Project description:The X and Y chromosomes of the platyfish (Xiphophorus maculatus) contain a region that encodes several important traits, including the determination of sex, pigment pattern formation, and predisposition to develop malignant melanoma. Several sex-chromosomal crossovers were identified in this region. As the melanoma-inducing oncogene Xmrk is the only molecularly identified constituent, its genomic organization on both sex chromosomes was analyzed in detail. Using X and Y allele-specific sequence differences a high proportion of the crossovers was found to be intragenic in the oncogene Xmrk, concentrating in the extracellular domain-encoding region. The genetic and molecular data allowed establishment of an order of loci over approximately 0.6 cM. It further revealed a sequence located within several kilobases of the extracellular domain-encoding region of Xmrk that regulates overexpression of the oncogene.
Project description:Because Ras signaling is frequently activated by major hepatocellular carcinoma etiological factors, a transgenic zebrafish constitutively expressing the kras(V12) oncogene in the liver was previously generated by our laboratory. Although this model depicted and uncovered the conservation between zebrafish and human liver tumorigenesis, the low tumor incidence and early mortality limit its use for further studies of tumor progression and inhibition. Here, we employed a mifepristone-inducible transgenic system to achieve inducible kras(V12) expression in the liver. The system consisted of two transgenic lines: the liver-driver line had a liver-specific fabp10 promoter to produce the LexPR chimeric transactivator, and the Ras-effector line contained a LexA-binding site to control EGFP-kras(V12) expression. In double-transgenic zebrafish (driver-effector) embryos and adults, we demonstrated mifepristone-inducible EGFP-kras(V12) expression in the liver. Robust and homogeneous liver tumors developed in 100% of double-transgenic fish after 1 month of induction and the tumors progressed from hyperplasia by 1 week post-treatment (wpt) to carcinoma by 4 wpt. Strikingly, liver tumorigenesis was found to be 'addicted' to Ras signaling for tumor maintenance, because mifepristone withdrawal led to tumor regression via cell death in transgenic fish. We further demonstrated the potential use of the transparent EGFP-kras(V12) larvae in inhibitor treatments to suppress Ras-driven liver tumorigenesis by targeting its downstream effectors, including the Raf-MEK-ERK and PI3K-AKT-mTOR pathways. Collectively, this mifepristone-inducible and reversible kras(V12) transgenic system offers a novel model for understanding hepatocarcinogenesis and a high-throughput screening platform for anti-cancer drugs.