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Oncoprints showing steatosis-associated CIS gene amplifications, deep deletions, and mutations occurring in at least 5% of all(S5), steatosis-associated(S6), and alcohol-associated(S7) TCGA HCC cases

Supplementary tables detailing qRT-PCR primer sequences (S1), IHC antibodies (S2), and parameters for histological stain quantification (S3-6) described in methods.

Wnt/beta-catenin(S11) and PKA/cAMP(S12) signaling alterations in liver cancer, and transgene expression in mouse livers after PKA/shp53 injection(S13)

Tables of mice(S7); CIS genes(S8); insertions(S9); human HCC CIS gene alterations(S10); steatosis-enriched CIS(S11); pathways altered in HCC(S12-18)

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAFV600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/RAC1/PAK signaling axis capable of driving resistance to both current and next-generation BRAFis. However, we show that the SRC inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance.Significance:A simple, rapid, and flexible genetic screening approach identifies genes that drive resistance to MAPK inhibitors when overexpressed in human melanoma cells.

Supplemental figures showing more detailed analysis of the genetic screen results (S1-S3), additional experiments showing biological validation of major candidate genes (S4-S7), and characterization of spontaneous Vemurafenib resistance in A375 cells (S8).

This file contains 4 supplemental tables.

Supplementary Figure S1 shows FOXA1 and FOXA2 repression in steatotic mouse livers

Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis–associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Cancer Res; 77(23); 6576–88. ©2017 AACR.

Transposon insertion profiles from animals with tumors sharing 0-2(S2), 3(S3), or >3(S4) insertions

Schematic of transposon insertions in Nat10(S9) and transgene expression in livers of Fah-deficient mice after hydrodynamic injection with Nat10/shp53(S10)

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAFV600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/RAC1/PAK signaling axis capable of driving resistance to both current and next-generation BRAFis. However, we show that the SRC inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance. Significance: A simple, rapid, and flexible genetic screening approach identifies genes that drive resistance to MAPK inhibitors when overexpressed in human melanoma cells.

SUMMARY Given its role as the source of definitive hematopoietic cells, we sought to determine whether mutations initiated in the hemogenic endothelium would yield hematopoietic abnormalities or malignancies. Here, we find that endothelium-specific transposon mutagenesis in mice promotes hematopoietic pathologies that are both myeloid and lymphoid in nature. Frequently mutated genes included previously recognized cancer drivers and additional candidates, such as Pi4ka, a lipid kinase whose mutation was found to promote myeloid and erythroid dysfunction. Subsequent validation experiments showed that targeted inactivation of the Pi4ka catalytic domain or reduction in mRNA expression inhibited myeloid and erythroid cell differentiation in vitro and promoted anemia in vivo through a mechanism involving deregulation of AKT, MAPK, SRC, and JAK-STAT signaling. Finally, we provide evidence linking PI4KAP2, previously considered a pseudogene, to human myeloid and erythroid leukemia., Graphical abstract

Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis–associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Cancer Res; 77(23); 6576–88. ©2017 AACR.

Phenotypes are rarely consistent across genetic backgrounds and environments, but instead vary in many ways depending on allelic variants, unlinked genes, epigenetic factors, and environmental exposures. In the extreme, individuals carrying the same causal DNA sequence variant but on different backgrounds can be classified as having distinct conditions. Similarly, some individuals that carry disease alleles are nevertheless healthy despite affected family members in the same environment. These genetic background effects often result from the action of so-called “modifier genes” that modulate the phenotypic manifestation of target genes in an epistatic manner. While complicating the prospects for gene discovery and the feasibility of mechanistic studies, such effects are opportunities to gain a deeper understanding of gene interaction networks that provide organismal form and function as well as resilience to perturbation. Here, we review the principles of modifier genetics and assess progress in studies of modifier genes and their targets in both simple and complex traits. We propose that modifier effects emerge from gene interaction networks whose structure and function vary with genetic background and argue that these effects can be exploited as safe and effective ways to prevent, stabilize, and reverse disease and dysfunction.

BRAF/MEK inhibition remains an important treatment option for patients with BRAF V600 mutant melanoma who show disease progression on immunotherapy; however, the majority of patients treated with BRAF/MEK (MAPKi) therapy develop MAPKi-resistant disease within two years of initiating treatment. Genomic analysis of drug-resistant melanomas has identified somatic mutations associated with resistance, including BRAF amplification or truncation and RAS mutation. In addition, other forms of acquired resistance involving adaptation and reprogramming have been described, with different studies identifying different drivers in different melanoma cell lines and patient-derived xenografts. The heterogeneity of acquired resistance mechanisms presents a major clinical challenge for identifying novel drug combinations with potential activity in significant subsets of patients. To enable the rapid identification of context-dependent drivers of MAPKi resistance, we adapted our Sleeping Beauty (SB) transposon mutagenesis for use in cell culture. SB mutagenesis in this setting identifies predominantly gain-of-function mechanisms and thus has the ability to identify both genetic mutations (such as expression of truncated proteins) and genes within alternative acquired resistance pathways whose upregulated expression drives MAPKi resistance. Validating our approach, our screen identified N-terminal truncation of BRAF—a known mechanism of vemurafenib resistance. In addition, we identified DBL family guanine exchange factors (GEFs), MCF2 and VAV1, as drivers of MAPKi resistance that we have functionally validated in multiple BRAF V600E mutant melanoma cell lines. Crucially, many DBL family GEFs are well known to be regulated by Src family kinases (SFKs), and our data indicate that GEF-driven MAPKi resistance can be blocked by combination treatment with vemurafenib and saracatinib, a selective SFK inhibitor. Expansion of resistant cells in the presence of vemurafenib or vemurafenib with cobimetinib can be reversed by switching to vemurafenib plus saracatinib. Consistently, we find that adding saracatinib converts MAPKi-mediated cytostasis into cell killing and blocks spontaneous MAPKi resistance in responsive melanoma cell lines. The GEFs signal downstream to a RAC1-PAK kinase module that is required for GEF-driven resistance. Importantly, the potential utility of both SFK inhibitors and PAK inhibitors for overcoming MAPKi resistance has been proposed, but the connection between SFK-dependent and PAK-dependent resistance mechanisms had not been obvious. We show that combined BRAF/SFK inhibition is effective in cell lines in which the GEF-driven mechanism operates, but not in cell lines in which the GEFs do not drive resistance. Our SB mutagenesis system has the ability to rapidly survey the context-dependent landscape of MAPKi resistance in any transfectable cell system, with the potential to reveal connections between seemingly disparate resistance mechanisms. This abstract is also being presented as Poster B04. Citation Format: Charlotte R. Feddersen, Jacob Schillo, Afshin Varzavand, Hayley Vaughn, Andrew Voight, Eliot Zhu, Jesse D Riordan, Christopher S. Stipp, Adam J. Dupuy. Sleeping Beauty mutagenesis reveals a Src-dependent DBL GEF family signaling mechanism driving MAPK inhibitor resistance in BRAF mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR17.

The serine/threonine protein kinase BRAF is mutated in approximately 50% of cutaneous melanomas, leading to hyperactivation of the MAPK/ERK pathway. The most common mutations, BRAFV600, can be targeted by selective kinase inhibitors, such as vemurafenib. Although initial clinical response to BRAF inhibition (BRAFi) is encouraging, 90% of patients develop drug resistance within a few months. Drug resistance can be delayed, but not prevented, by combining BRAFi with an MEK inhibitor (MEKi), such as cobimetinib. While some resistance mechanisms are known, disease progression on drug cannot be explained in all patients. We performed a gain-of-function mutagenesis screen utilizing the Sleeping Beauty transposon system to identify novel drivers of resistance in BRAFV600E mutant melanoma cells sensitive to current therapies. We chose four of the top candidates from our screen and validated the ability to drive resistance to both vemurafenib and vemurafenib-cobimetinib combination treatment in multiple melanoma cell lines. In an effort to determine the broader role of candidate vemurafenib-resistance drivers, we conducted an additional in vivo mutagenesis screen, of which genetic analysis is ongoing. Our initial cell-based screen identified two members of the Dbl family of guanine nucleotide exchange factors (GEFs), VAV1 and MCF2, as candidate drivers of vemurafenib resistance. A375 melanoma cells overexpressing VAV1 or MCF2 maintain significant growth under vemurafenib treatment, while control cells do not. Functional tests of VAV1 and MCF2 identified that the active form of two Rho family members, RAC1 and CDC42, increases following treatment with vemurafenib, suggesting a PAK-mediated pathway of resistance. In addition, all candidates that were tested elevated ERK signaling in the presence of vemurafenib. Many of the extracellular signaling pathways known to drive increased vemurafenib resistance activate Rho signaling. Our results suggest that Dbl family members may play an important role in this process. Understanding how Rho activation occurs and its consequences for drug resistance in melanoma will provide critical insights into the design and validation of future targeted therapies. Citation Format: Jacob L. Schillo, Charlotte R. Feddersen, Afshin Varzavand, Hayley R. Vaughn, Lexy S. Wadsworth, Andrew P. Voigt, Eliot Y. Zhu, Jesse D. Riordan, Christopher S. Stipp, Adam J. Dupuy. Identification and characterization of Rho family GTPases as drivers of drug resistance in BRAFV600 mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B08.

Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in vitro RNA editing, A1cf–/– mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression. There were minimal changes in hepatic and intestinal apoB RNA editing in A1cf–/– mice and no changes in either liver- or intestine-specific A1CF transgenic mice. Rbm47 liver-specific knockout (Rbm47LKO) mice demonstrated reduced editing in a subset (11 of 20) of RNA targets, including apoB. By contrast, apoB RNA editing was virtually eliminated (Rbm47IKO) mice with only five of 53 targets exhibiting C-to-U RNA editing. Double knockout of A1cf and Rbm47 in liver (ARLKO) eliminated apoB RNA editing and reduced editing in the majority of other targets, with no changes following adenoviral APOBEC1 administration. Intestinal double knockout mice (ARIKO) demonstrated further reduced editing (ARIKO mice. These data suggest that A1CF and RBM47 each function independently, yet interact in a tissue-specific manner, to regulate the activity and site selection of APOBEC1 dependent C-to-U RNA editing.

A high-fat diet induces obesity and is associated with heart disease characterized by cardiac hypertrophy, diastolic dysfunction, fibrosis, and mitochondrial dysfunction. However, the molecular mechanisms involved in the pathogenesis of cardiac disease are not known. Therefore, the objective of this study was to investigate the molecular changes in the heart associated with a high-fat diet at early time points in the progression of cardiac disease. To do this, male C57BL/6 mice were fed either a chow diet or a high-fat (58 kcal%), low-sucrose (HFLS) diet starting at 5 weeks of age for 14 days and 100 days. At each time point, hearts were extracted and morphometric measurements were recorded. HFLS diet had no effect on weight gain at 14 days but did significantly increase body weight by two-fold at 100 days. At 14 days, there was no significant difference in cardiac size as measured by heart weight-to-tibia length ratio (HW/TL) between mice on HFLS diet and chow diet. However, there was a 24% increase in HW/TL following 100 days on the HFLS diet. Total RNA was extracted from left ventricular tissue from each group and gene expression was determined by RNA-Seq transcriptome analysis. DAVID functional pathway analysis of the top 50 genes with differential expression between diets at 14 days and 100 days identified significant enrichment for mitochondrial proteins and lipid metabolism at both time points on the HFLS diet. Moreover, there was enrichment for hypoxia response genes following 100 days on the HFLS diet. Analysis of genes with differential expression between 14 days and 100 days on HFLS diet identified enrichment for actin binding proteins/Z disc, glutathione transferase activity, and oxidation/reduction activity. These findings demonstrate significant differences in gene regulation in the hearts of mice fed a high fat diet at early time points. It is possible that these early molecular changes play a role in the progression of cardiac disease.

Much research has gone into the development of hybrid gene delivery systems that combine the broad tropism and efficient transduction of adenoviral vectors with the ability to achieve stable expression of cargo genes. In addition to gene therapy applications, such a system has considerable advantages for studies of gene function in vivo, permitting fine-tuned genetic manipulation with higher throughput than can be achieved using standard transgenic and DNA targeting techniques. Existing strategies are limited, however, by low integration efficiencies, small cargo capacity, and/or a dependence on target cell division. The utility of this approach could be enhanced by a system that provides all of the following: (1) efficient delivery, (2) stable expression in a high percentage of target cells (whether mitotic or not), (3) large cargo capacity, (4) flexibility to use with a wide range of additional experimental conditions, and (5) simple experimental technique. Here we report the initial characterization of a hybrid system that meets these criteria by utilizing piggyBac (PB) transposition to achieve genomic integration from adenoviral vectors. We demonstrate stable expression of an adenovirus (Ad)-PB-delivered reporter gene in ∼20–40% of hepatocytes following standard tail vein injection. Its high efficiency and flexibility relative to existing hybrid adenoviral gene delivery approaches indicate a considerable potential utility of the Ad-PB system for therapeutic gene delivery and in vivo studies of gene function.

Most hepatocellular carcinomas (HCCs) develop in a chronically injured liver, yet the extent to which this microenvironment promotes neoplastic transformation or influences selective pressures for genetic drivers of HCC remains unclear. We sought to determine the impact of hepatic injury in an established mouse model of HCC induced by Sleeping Beauty transposon mutagenesis. Chemically-induced chronic liver injury dramatically increased tumor penetrance and significantly altered driver mutation profiles, likely reflecting distinct selective pressures. In addition to established human HCC genes and pathways, we identified several novel injury-associated candidates that represent promising loci for further study. Among them, we found that FIGN is overexpressed in human HCC and promotes hepatocyte invasion. We also validated Gli2's oncogenic potential in vivo, providing direct evidence that Hedgehog signaling can drive liver tumorigenesis in the context of chronic injury. Finally, we show that a subset of injury-associated candidate genes identifies two distinct classes of human HCCs. Further analysis of these two subclasses revealed significant trends among common molecular classification schemes of HCC. The genes and mechanisms identified here provide functional insights into the origin of HCC in a chronic liver damage environment. Conclusion: A chronically damaged liver microenvironment influences the genetic mechanisms that drive hepatocarcinogenesis. This article is protected by copyright. All rights reserved.

Mutations of RAS are believed to be early events in thyroid tumorigenesis but are insufficient to induce transformation. A forward genetic screen with transposons engineered to integrate randomly into the mouse Ras-mutant thyroid cell genome and to disrupt genes at their insertion sites resulted in tumors that phenocopied human RAS-driven, poorly differentiated thyroid cancers. Analysis of the transposon-integration sites revealed recurrent mutations of chromatin modifiers and PI3K pathway genes, consistent with mutations seen in human advanced thyroid cancers. These human cancers have a high mutation burden, which confounds distinctions between driver and passenger mutations. This unbiased screen for genes selected during tumorigenesis provides strong functional support for genetic disruptions in these pathways in RAS-induced thyroid tumor progression.

Oncogenic RAS mutations are present in 15-30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with HrasG12V in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in Tpo-Cre/homozygous FR-HrasG12V mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway. ATXN7, a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with RAS or NF1 mutations. Expression of ATXN7 mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to ATXN7 as a previously unrecognized cancer gene.

Hepatocellular carcinoma (HCC) is associated with poor survival for patients and few effective treatment options, raising the need for novel therapeutic strategies. MicroRNAs (miRNAs) play important roles in tumor development and show deregulated patterns of expression in HCC. Because of the liver’s unique affinity for small nucleic acids, miRNA-based therapy has been proposed in the treatment of liver disease. Thus, there is an urgent need to identify and characterize aberrantly expressed miRNAs in HCC. In our study, we profiled miRNA expression changes in de novo liver tumors driven by MYC and/or RAS, two canonical oncogenes activated in a majority of human HCCs. We identified an up-regulated miRNA megacluster comprised of 53 miRNAs on mouse chromosome 12qF1 (human homolog 14q32). This miRNA megacluster is up-regulated in all three transgenic liver models and in a subset of human HCCs. An unbiased functional analysis of all miRNAs within this cluster was performed. We found that miR-494 is overexpressed in human HCC and aids in transformation by regulating the G1/S cell cycle transition through targeting of the Mutated in Colorectal Cancer tumor suppressor. miR-494 inhibition in human HCC cell lines decreases cellular transformation, and anti-miR-494 treatment of primary MYC-driven liver tumor formation significantly diminishes tumor size. Conclusion: Our findings identify a new therapeutic target (miR-494) for the treatment of HCC. (Hepatology 2014;58:202–215)

Forward genetic screens using Sleeping Beauty (SB)-mobilized T2/Onc transposons have been used to identify common insertion sites (CISs) associated with tumor formation. Recurrent sites of transposon insertion are commonly identified using ligation-mediated PCR (LM-PCR). Here, we use RNA sequencing (RNA-seq) data to directly identify transcriptional events mediated by T2/Onc. Surprisingly, the majority (∼80%) of LM-PCR identified junction fragments do not lead to observable changes in RNA transcripts. However, in CIS regions, direct transcriptional effects of transposon insertions are observed. We developed an automated method to systematically identify T2/Onc-genome RNA fusion sequences in RNA-seq data. RNA fusion-based CISs were identified corresponding to both DNA-based CISs (Cdkn2a, Mycl1, Nf2, Pten, Sema6d, and Rere) and additional regions strongly associated with cancer that were not observed by LM-PCR (Myc, Akt1, Pth, Csf1r, Fgfr2, Wisp1, Map3k5, and Map4k3). In addition to calculating recurrent CISs, we also present complementary methods to identify potential driver events via determination of strongly supported fusions and fusions with large transcript level changes in the absence of multitumor recurrence. These methods independently identify CIS regions and also point to cancer-associated genes like Braf. We anticipate RNA-seq analyses of tumors from forward genetic screens will become an efficient tool to identify causal events.

Both genetic and dietary factors contribute to the metabolic syndrome (MetS) in humans and animal models. Characterizing their individual roles as well as relationships among these factors is critical for understanding MetS pathogenesis and developing effective therapies. By studying phenotypic responsiveness to high-risk versus control diet in two inbred mouse strains and their derivatives, we estimated the relative contributions of diet and genetic background to MetS, characterized strain-specific combinations of MetS conditions, and tested genetic and phenotypic complexity on a single substituted chromosome.Ten measures of metabolic health were assessed in susceptible C57BL/6 J and resistant A/J male mice fed either a control or a high-fat, high-sucrose (HFHS) diet, permitting estimates of the relative influences of strain, diet and strain-diet interactions for each trait. The same traits were measured in a panel of C57BL/6 J (B6)-Chr(A/J) chromosome substitution strains (CSSs) fed the HFHS diet, followed by characterization of interstrain relationships, covariation among metabolic traits and quantitative trait loci (QTLs) on Chromosome 10.We identified significant genetic contributions to nine of ten metabolic traits and significant dietary influence on eight. Significant strain-diet interaction effects were detected for four traits. Although a range of HFHS-induced phenotypes were observed among the CSSs, significant associations were detected among all traits but one. Strains were grouped into three clusters based on overall phenotype and specific CSSs were identified with distinct and reproducible trait combinations. Finally, several Chr10 regions were shown to control the severity of MetS conditions.Generally strong genetic and dietary effects validate these CSSs as a multifactorial model of MetS. Although traits tended to segregate together, considerable phenotypic heterogeneity suggests that underlying genetic factors influence their co-occurrence and severity. Identification of multiple QTLs within and among strains highlights both the complexity of genetically regulated, diet-induced MetS and the ability of CSSs to prioritize candidate loci for mechanistic studies.

Background Animal models of cancer are useful to generate complementary datasets for comparison to human tumor data. Insertional mutagenesis screens, such as those utilizing the Sleeping Beauty (SB) transposon system, provide a model that recapitulates the spontaneous development and progression of human disease. This approach has been widely used to model a variety of cancers in mice. Comprehensive mutation profiles are generated for individual tumors through amplification of transposon insertion sites followed by high-throughput sequencing. Subsequent statistical analyses identify common insertion sites (CISs), which are predicted to be functionally involved in tumorigenesis. Current methods utilized for SB insertion site analysis have some significant limitations. For one, they do not account for transposon footprints – a class of mutation generated following transposon remobilization. Existing methods also discard quantitative sequence data due to uncertainty regarding the extent to which it accurately reflects mutation abundance within a heterogeneous tumor. Additionally, computational analyses generally assume that all potential insertion sites have an equal probability of being detected under non-selective conditions, an assumption without sufficient relevant data. The goal of our study was to address these potential confounding factors in order to enhance functional interpretation of insertion site data from tumors. Results We describe here a novel method to detect footprints generated by transposon remobilization, which revealed minimal evidence of positive selection in tumors. We also present extensive characterization data demonstrating an ability to reproducibly assign semi-quantitative information to individual insertion sites within a tumor sample. Finally, we identify apparent biases for detection of inserted transposons in several genomic regions that may lead to the identification of false positive CISs. Conclusion The information we provide can be used to refine analyses of data from insertional mutagenesis screens, improving functional interpretation of results and facilitating the identification of genes important in cancer development and progression. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1150) contains supplementary material, which is available to authorized users.

Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC). Although these conditions also lead to integrated stress response (ISR) or unfolded protein response (UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases., Author Summary Liver cancer is the third most common cause of cancer death worldwide. It is most commonly caused by viral hepatitis, alcoholism, or obesity, all of which activate cellular stress responses in the liver. However, the contribution of these responses to disease pathogenesis was unknown. We found that expression of the stress-regulated transcription factor CHOP—widely thought to be anti-oncogenic because of its cell death-promoting properties—was associated with both human liver cancer and two mouse models thereof. In response to challenge with a tumor-causing agent, mice lacking CHOP developed fewer tumors, exhibited less cell death, compensatory cellular proliferation, and liver scarring (fibrosis), and showed lower expression of immune and inflammatory genes. These findings establish CHOP as a biomarker for liver cancer and demonstrate its importance in promoting liver tumor formation. They raise the possibility that promotion of tumorigenesis by CHOP is a common feature of liver cancer caused by viral infection, alcoholism, and obesity.

The adaptation of transposable elements inserted within the genome to serve novel functions in a host cell, a process known as molecular domestication, is a widespread phenomenon in nature. Around fifty protein-coding genes in humans have arisen through this mechanism. Functional characterization of these domesticated genes has revealed involvement in a multitude of diverse cellular processes. Some of these functions are related to cellular activities and pathways known to be involved in cancer development. In this mini-review we discuss such roles of domesticated genes that may be aberrantly regulated in human cancer, as well as studies that have identified disrupted expression in tumors. We also describe studies that have provided definitive experimental evidence for transposable element-derived gene products in promoting tumorigenesis.

TCL1 oncogene is overexpressed in aggressive form of human chronic lymphocytic leukemia (CLL) and its dysregulation in mouse B cells causes a CD5-positive leukemia similar to the aggressive form of human CLLs. To identify oncogenes that cooperate with Tcl1, we performed genetic screen in Eμ−TCL1 mice using Sleeping Beauty transposon-mediated mutagenesis. Analysis of transposon common insertion sites identified 7 genes activated by transposon insertions. Overexpression of these genes in mouse CLL was confirmed by real time reverse transcription-polymerase chain reaction. Interestingly, the main known function of 4 of 7 genes (Nfkb1, Tab2, Map3K14, and Nfkbid) is participation in or activation of the nuclear factor-kB (NF-kB) pathway. In addition, activation of the NF-kB is 1 of main functions of Akt2, also identified in the screen. These findings demonstrate cooperation of Tcl1 and the NF-kB pathway in the pathogenesis of aggressive CLL. Identification cooperating cancer genes will result in the development of combinatorial therapies to treat CLL.

Cutaneous squamous cell carcinoma (SCC) is the second most common form of cancer in the human population, yet the underlying genetic mechanisms contributing to the disease are not well understood. We recently identified Zmiz1 as a candidate oncogene in nonmelanoma skin cancer through a transposon mutagenesis screen. Here we show that transposon-induced mutations in Zmiz1 drive expression of a truncated transcript that is similar to an alternative endogenous ZMIZ1 transcript found to be overexpressed in human SCCs relative to normal skin. We also describe an original mouse model of invasive keratoacanthoma driven by skin-specific expression of the truncated Zmiz1 transcript. Unlike most mouse models, Zmiz1-induced skin tumors develop rapidly and in the absence of promoting agents such as phorbol esters. In addition, we found that the alternative Zmiz1 isoform has greater protein stability than its full-length counterpart. Finally, we provide evidence that ZMIZ1 is overexpressed in a significant percentage of human breast, ovarian, and colon cancers in addition to human SCCs, suggesting that ZMIZ1 may play a broader role in epithelial cancers.

Hepatocellular carcinoma (HCC) is one of the deadliest solid cancers and is the third leading cause of cancer-related death. There is a universal estimated male/female ratio of 2.5, but the reason for this is not well understood. The Sleeping Beauty (SB) transposon system was used to elucidate candidate oncogenic drivers of HCC in a forward genetics screening approach. Sex bias occurrence was conserved in our model, with male experimental mice developing liver tumors at reduced latency and higher tumor penetrance. In parallel, we explored sex differences regarding genomic aberrations in 235 HCC patients. Liver cancer candidate genes were identified from both sexes and genotypes. Interestingly, transposon insertions in the epidermal growth factor receptor (Egfr) gene were common in SB-induced liver tumors from male mice (10/10, 100%) but infrequent in female mice (2/9, 22%). Human single-nucleotide polymorphism data confirmed that polysomy of chromosome 7, locus of EGFR, was more frequent in males (26/62, 41%) than females (2/27, 7%) (P = 0.001). Gene expression–based Poly7 subclass patients were predominantly male (9/9) compared with 67% males (55/82) in other HCC subclasses (P = 0.02), and this subclass was accompanied by EGFR overexpression (P < 0.001). Conclusion: Sex bias occurrence of HCC associated with EGFR was confirmed in experimental animals using the SB transposon system in a reverse genetic approach. This study provides evidence for the role of EGFR in sex bias occurrences of liver cancer and as the driver mutational gene in the Poly7 molecular subclass of human HCC. (HEPATOLOGY 2013)

The many advantages of retroviral insertional mutagenesis have been discussed in the previous chapters, and this strategy has played a significant role in furthering our current understanding of the genetic basis of cancer. However, retroviral insertional mutagenesis has two main limitations that have prevented this approach from being applied to many forms of cancer. First, naturally-occurring slow transforming retroviruses have a restricted cellular tropism within the infected host, thus limiting the types of cells that can be mutagenized by these viruses [29]. Second, retroviruses require the host cell to undergo mitosis in order to gain access to the nuclear genome and integrate as a provirus. The combined effects of these drawbacks have limited the application of retroviral insertional mutagenesis to the study of hematopoietic malignancies and mammary cancer in mice.

We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the Dlk1-Dio3 imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of Retrotransposon-like 1 (Rtl1) expression as the only consistent alteration detected in all SB–induced tumors with Dlk1-Dio3 integrations, suggesting that Rtl1 activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple Dlk1-Dio3 domain members and HCC, we show here that direct modulation of a single domain member, Rtl1, can promote hepatocarcinogenesis in vivo. Overexpression of Rtl1 in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of RTL1 in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The Rtl1 locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying Rtl1 as a novel driver of HCC, our study represents one of the first direct in vivo demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis., Author Summary HCC is the third deadliest cancer worldwide, largely due to a lack of effective treatment options. Therapeutic approaches targeted at the molecular mechanisms underlying tumor formation and progression have shown great efficacy for treating other tumor types. Unfortunately, however, much remains to be learned about the molecular pathogenesis of HCC. There is an urgent need to identify and characterize genetic alterations that drive HCC in order to facilitate the development of more effective targeted therapeutics for patients. Here, we present data showing that recurrent mutations identified in a mouse model of HCC result in overexpression of the Rtl1 gene. We have validated Rtl1 as a driver of HCC by demonstrating that its overexpression in mouse liver causes tumor formation. We also detected overexpression of this gene in a significant proportion of human HCC samples, suggesting that it may be a relevant therapeutic target for patients with this disease.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed and deadliest cancers worldwide. In the United States, age-adjusted incidence has tripled over the last three decades. While traditional cancer treatments, such as radiation and chemotherapy, are ineffective in treating HCC, therapeutic approaches targeted at the underlying molecular causes are proving to be more efficacious. However, compared to other common cancers, relatively little is known about the molecular pathogenesis of HCC. We have used a Sleeping Beauty transposon-based insertional mutagenesis screen to identify novel oncogenes and tumor suppressors involved in HCC development and progression. This forward genetics approach identified a link between disruption of gene expression within the Dlk1-Dio3 imprinted domain and development of HCC in mice; over half of the induced HCCs contained transposon integrations within a ten kilobase region of this domain. Analysis of gene expression within the domain identified two consistent and significant events associated with transposon integration: activation of Delta-like 1 homolog (Dlk1) and inhibition of microRNA-370 (miR-370). These results led us to hypothesize that Dlk1 and miR-370 possess oncogenic and tumor suppressive functions, respectively, in HCC pathogenesis. The Dlk1 gene encodes a Notch receptor ligand. Overexpression has been documented in a subset of human HCC cases, and the effects of modulating expression level in cultured hepatocytes are consistent with a tumor-promoting role. To test Dlk1's oncogenic potential in vivo, we drove overexpression in adult mouse livers using a hydrodynamic gene delivery technique. Hepatic nodule formation was observed in ∼50% of mice, supporting our hypothesis that Dlk1 functions as an oncogene in HCC. miR-370 is a relatively uncharacterized microRNA, although it has been demonstrated to suppress proliferation of cultured cholangiocytes. Similarly, manipulation of miR-370 activity in cultured hepatocytes has proliferative consequences consistent with a tumor suppressor role. Efforts to examine the effect of modulating miR-370 activity in vivo are currently underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 236.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world and its prevalence is rising. In the absence of disease progression, fatty liver poses minimal risk of detrimental health outcomes. However, advancement to non-alcoholic steatohepatitis (NASH) confers a markedly increased likelihood of developing severe liver pathologies, including fibrosis, cirrhosis, organ failure, and cancer. Although a substantial percentage of NAFLD patients develop NASH, the genetic and molecular mechanisms driving this progression are poorly understood, making it difficult to predict which patients will ultimately develop advanced liver disease. Deficiencies in mechanistic understanding preclude the identification of beneficial prognostic indicators and the development of effective therapies. Mouse models of progressive NAFLD serve as a complementary approach to the direct analysis of human patients. By providing an easily manipulated experimental system that can be rigorously controlled, they facilitate an improved understanding of disease development and progression. In this review, we discuss genetically- and chemically-induced models of NAFLD that progress to NASH, fibrosis, and liver cancer in the context of the major signaling pathways whose disruption has been implicated as a driving force for their development. Additionally, an overview of nutritional models of progressive NAFLD is provided.