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Supplementary Figure 4 - PDF file 111K, Reduction of Wnt signaling by β-catenin knockdown or GSK3B overexpression reduces tumor growth in xenograft models

Supplementary Figure S4. Serum metabolite profiles of experimental animals.

Supplementary Table 2 - PDF 69K, QPCR primer sequences

Supplementary Figure 2 - PDF file 109K, Overexpression of activated β-catenin and knockdown of AXIN1 or GSK3B results in increased Wnt signaling and an induction of oncogenic properties in immortalized human Schwann cells

Chronic hepatitis B viral (HBV) infection remains a high underlying cause for hepatocellular carcinoma (HCC) worldwide, while the genetic mechanisms behind this remain unclear. This study elucidated the mechanisms contributing to tumor development induced by the HBV X (HBx) gene of predominantly Asian genotype B HBV and its common HBx variants. To compare the potential tumorigenic effects of K130M/V131I (Mut) and wild-type (WT) HBx on HCC, the Sleeping Beauty (SB) transposon system was used to deliver HBx Mut and WT into the livers of fumarylacetoacetate hydrolase (Fah)-deficient mice and in the context of transformation related protein 53 (Trp53) deficiency. From our results, HBx Mut had a stronger tumorigenic effect than its WT variant. Also, inflammation, necrosis, and fibrosis were evident in HBx experimental animals. Reduction of forkhead box O1 (FOXO1) with increased phosphorylation of upstream serine/threonine kinase (AKT) was detected under HBx Mut overexpression. Thus, it is proposed that HBx Mut enhances disease progression by reducing FOXO1 via phosphorylation of AKT. At the metabolomic level, HBx altered the expression of genes that participated in arachidonic acid (AA) metabolism, as a result of inflammation via accumulation of proinflammatory factors such as prostaglandins and leukotriene in liver. Taken together, the increased rate of HCC observed in chronic hepatitis B patients with K130M/V131I-mutated X protein, may be due to changes in AA metabolism and AKT/FOXO1 signaling.Implications:Our findings suggested that HBx-K130M/V131I–mutant variant promoted HCC progression by activating AKT/FOXO1 pathway and inducing stronger inflammation in liver via AA metabolism.

Supplementary Table and Figure Legends-PDF file 123K, Supplementary Table and Figure Legends

Supplementary materials and methods for PCR and serum preparation.

Supplementary Table S3. Up-stream regulator of IGF2.

Supplementary Figure 1 - PDF file 141K, Human tissue microarray shows that C-MYC staining intensity increases with tumor progression

Supplementary Table 1 - PDF 95K, Genes listed were identified in a murine SB screen for drivers of Schwann cell tumorigenesis, and all have known roles in canonical Wnt/β-catenin signaling. Listed are the percentage of neurofibromas or MPNSTs with an SB insertion within or near the gene listed, as well as the total number of tumors within a given gene.

Supplementary Information

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

The survival benefit derived from sorafenib treatment for patients with hepatocellular carcinoma (HCC) is modest due to acquired resistance. Targeting cancer stem cells (CSC) is a possible way to reverse drug resistance, however, inhibitors that specifically target liver CSCs are limited. In this study, we established two sorafenib-resistant, patient-derived tumor xenografts (PDX) that mimicked development of acquired resistance to sorafenib in patients with HCC. RNA-sequencing analysis of sorafenib-resistant PDXs and their corresponding mock controls identified EPH receptor B2 (EPHB2) as the most significantly upregulated kinase. EPHB2 expression increased stepwise from normal liver tissue to fibrotic liver tissue to HCC tissue and correlated with poor prognosis. Endogenous EPHB2 knockout showed attenuation of tumor development in mice. EPHB2 regulated the traits of liver CSCs; similarly, sorted EPHB2High HCC cells were endowed with enhanced CSC properties when compared with their EPHB2-Low counterparts. Mechanistically, EPHB2 regulated cancer stemness and drug resistance by driving the SRC/AKT/GSK3β/β-catenin signaling cascade, and EPHB2 expression was regulated by TCF1 via promoter activation, forming a positive Wnt/β-catenin feedback loop. Intravenous administration of rAAV-8-shEPHB2 suppressed HCC tumor growth and significantly sensitized HCC cells to sorafenib in an NRAS/AKT-driven HCC immunocompetent mouse model. Targeting a positive feedback loop involving the EPHB2/β-catenin axis may be a possible therapeutic strategy to combat acquired drug resistance in HCC.Significance:This study identifies a EPHB2/β-catenin/TCF1 positive feedback loop that augments cancer stemness and sorafenib resistance in HCC, revealing a targetable axis to combat acquired drug resistance in HCC.

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)

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)

PDF file - 137K, Effect of Nf1 dosage with Pten inactivation in peripheral nerve tumorigenesis

PDF file - 80K, Transgenes used to establish a novel PNST mouse model

PDF file - 181K, Histological and immunohistochemical (IHC) analyses of peripheral nervous system phenotype

PDF file - 93K, Semi-quantitative analysis of proliferative peripheral nerve cells in various control and experimental cohorts

The survival benefit derived from sorafenib treatment for patients with hepatocellular carcinoma (HCC) is modest due to acquired resistance. Targeting cancer stem cells (CSC) is a possible way to reverse drug resistance, however, inhibitors that specifically target liver CSCs are limited. In this study, we established two sorafenib-resistant, patient-derived tumor xenografts (PDX) that mimicked development of acquired resistance to sorafenib in patients with HCC. RNA-sequencing analysis of sorafenib-resistant PDXs and their corresponding mock controls identified EPH receptor B2 (EPHB2) as the most significantly upregulated kinase. EPHB2 expression increased stepwise from normal liver tissue to fibrotic liver tissue to HCC tissue and correlated with poor prognosis. Endogenous EPHB2 knockout showed attenuation of tumor development in mice. EPHB2 regulated the traits of liver CSCs; similarly, sorted EPHB2High HCC cells were endowed with enhanced CSC properties when compared with their EPHB2-Low counterparts. Mechanistically, EPHB2 regulated cancer stemness and drug resistance by driving the SRC/AKT/GSK3β/β-catenin signaling cascade, and EPHB2 expression was regulated by TCF1 via promoter activation, forming a positive Wnt/β-catenin feedback loop. Intravenous administration of rAAV-8-shEPHB2 suppressed HCC tumor growth and significantly sensitized HCC cells to sorafenib in an NRAS/AKT-driven HCC immunocompetent mouse model. Targeting a positive feedback loop involving the EPHB2/β-catenin axis may be a possible therapeutic strategy to combat acquired drug resistance in HCC. Significance: This study identifies a EPHB2/β-catenin/TCF1 positive feedback loop that augments cancer stemness and sorafenib resistance in HCC, revealing a targetable axis to combat acquired drug resistance in HCC.

A forward genetic Sleeping Beauty (SB) insertional mutagenesis screen, followed by high-throughput transcriptome sequencing, was used to identify driver genes responsible for hepatocellular carcinoma (HCC)-associated metastasis. Using RNA-sequencing (RNA-seq) to identify transposon-endogenous transcriptome fusion genes, the phylogenetic lineage between the parental liver tumor and secondary metastasis can be determined to provide mechanistic insight to genetic changes involved in the metastatic evolution process. In the current study, two novel candidate genes were identified to be potentially involved in HCC-associated metastatic progression, canopy FGF signaling regulator 2 (Cnpy2) and actinin alpha 2 (Actn2). Transposon-Cnpy2 fusion transcripts were identified in both primary liver tumors and lung metastases. Its significant association with clinicopathological characteristics and correlated gene enrichment in metastasis-related mechanisms suggest its potential role in modulating local invasion and angiogenesis. Other known driver genes for human HCC that can also promote metastatic progression include epidermal growth factor receptor (Egfr) and RNA imprinted and accumulated in nucleus (Rian). Metabolic pathway related gene carbamoyl phosphate synthetase (Cps1) was identified to play an important role in early HCC development, while cell junction-related pathway gene Rac family small GTPase 1 (Rac1) was identified to take part in both HCC and pro-metastatic progression. Importantly, actinin alpha 2 (Actn2) was identified exclusively in the secondary metastasis site and its role in HCC-related metastatic process was elucidated using in vitro approaches. ACTN2-overexpression in human liver cancer cells displayed enhanced cellular motility and invasion abilities, indicating its possible function in later stage of metastasis, such as extravasation and lung colonization.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that occur spontaneously, or from benign plexiform neurofibromas, in the context of the genetic disorder Neurofibromatosis Type 1 (NF1). The current standard treatment includes surgical resection, high-dose chemotherapy, and/or radiation. To date, most targeted therapies have failed to demonstrate effectiveness against plexiform neurofibromas and MPNSTs. Recently, several studies suggested that the mTOR and MAPK pathways are involved in the formation and progression of MPNSTs. Everolimus (RAD001) inhibits the mTOR and is currently FDA approved for several types of solid tumors. PD-0325901 (PD-901) inhibits MEK, a component of the MAPK pathway, and is currently in clinical trials. Here, we show in vitro than MPNST cell lines are more sensitive to inhibition of cellular growth by Everolimus and PD-901 than immortalized human Schwann cells. In combination, these drugs synergistically inhibit cell growth and induce apoptosis. In two genetically engineered mouse models of MPNST formation, modeling both sporadic and NF1-associated MPNSTs, Everolimus, or PD-901 treatment alone each transiently reduced tumor burden and size, and extended lifespan. However, prolonged treatment of each single agent resulted in the development of resistance and reactivation of target pathways. Combination therapy using Everolimus and PD-901 had synergistic effects on reducing tumor burden and size, and increased lifespan. Combination therapy allowed persistent and prolonged reduction in signaling through both pathways. These data suggest that co-targeting mTOR and MEK may be effective in patients with sporadic or NF1-associated MPNSTs.

The neuromuscular junction (NMJ) is formed by motor nerve terminals, post-junctional muscle membranes, and terminal Schwann cells (SCs). The formation of NMJ requires complex and dynamic molecular interactions. Nerve- and muscle-derived molecules have been well characterized but the mechanistic involvement of SC in NMJ development remains poorly understood. SC-specific phosphatase and tensin homolog (Pten) inactivation and epidermal growth factor receptor (EGFR) overexpression (Dhh-Cre; Cnp-EGFR; Ptenflox/flox or DET) mice were used and NMJ malformation was observed in these mice. Acetylcholine receptors (AChRs) were distorted and varicose presynaptic nerve terminals appeared in the tibialis anterior (TA) muscle of DET mice. Agrin signaling related to NMJ development, was downregulated in TA muscle. Both RAS/MEK/ERK and PI3K/AKT/mTOR signaling pathways were activated in the sciatic nerves of DET mice. In addition, autophagy was downregulated in these sciatic nerves. Interestingly, the use of Torin 2, an mTOR inhibitor, rescued the phenotype. The downregulated-autophagy might account for Agrin signaling abnormity, which induced NMJ malformation. Taken together, our results indicate that SCs-specific Pten and EGFR cooperation are essential for NMJ development.

Non-alcoholic fatty liver disease (NAFLD) is becoming an epidemic disease in adults and children worldwide. Importantly, there are currently no approved treatments available for NAFLD. This study aims to investigate the potential applications of sodium tanshinone IIA sulfonate (STS) on improving the NAFLD condition using both in vitro and in vivo approaches. The results showed that STS markedly inhibited lipid accumulation in oleic acid (OA) and palmitic acid (PA) treated HepG2 and primary immortalized human hepatic (PIH) cells. STS suppressed lipogenesis by inhibiting expression of sterol regulatory element binding transcription factor 1 (SREBF1), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD). In addition, STS reduced inflammation in cells treated with OA-PA, shown by decreased transcriptional levels of tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFB1) and interleukin 1 beta (IL1B). Consistently, protective effects on hepatic steatosis in db/db mice were observed after STS administration, demonstrated by decreased lipid accumulation in mouse hepatocytes. This protective effect might be associated with STS induced activation of sirtuin 1 (SIRT1)/protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1) pathways. Our findings suggest a potential therapeutic role for STS in the treatment of NAFLD.

The neuromuscular junction (NMJ) consists of three components, namely presynaptic motor neurons, postsynaptic muscle fibers and perisynaptic Schwann cells (PSCs). The role of Schwann cells (SCs) in regulating NMJ structural and functional development remains unclear. In this study, mice with conditional inactivation of neurofibromin 1 (Nf1) and phosphatase and tensin homolog (Pten), specifically in SCs, resulted in delayed NMJ maturation that led to delayed muscle growth, recapitulating the muscular dystrophy condition observed in human neurofibromatosis type I syndrome (NF1) patients. Expression levels of NMJ development related molecules such as cholinergic receptor, nicotinic, alpha polypeptide 1 (Chrna1), agrin (Agrn), dystrophin, muscular dystrophy (Dmd), laminin, beta 2 (Lamb2) and dystroglycan 1 (Dag1) were also downregulated. To further explore the molecular alterations in these SCs, NF1- and PTEN-related pathways were analyzed in mutant sciatic nerves. As expected, hyperactive RAS/PI3K/AKT/mTOR signaling pathways were identified, suggesting the importance of these pathways for NMJ development, and subsequent muscle maturation.

There is increasing evidence that the sympathetic nervous system (SNS) plays an important role in adipose tissue development. However, the underlying molecular mechanism(s) associated with this remains unclear. SNS innervation of white adipose tissue (WAT) is believed to be necessary and sufficient to elicit WAT lipolysis. In this current study, mice with Schwann cell (SC)-specific inactivation of phosphatase and tensin homolog (Pten) displayed enlarged inguinal white adipose tissue (iWAT). This serendipitous observation implicates the role of SCs in mediating SNS activity associated with mouse adipose tissue development. Mice with SC-specific Pten inactivation displayed enlarged iWAT. Interestingly, the SNS activity in iWAT of SC-specific Pten-deficient mice was reduced as demonstrated by decreased tyrosine hydroxylase (TH) expression level and neurotransmitters, such as norepinephrine (NE) and histamine (H). The lipolysis related protein, phosphorylated hormone sensitive lipase (pHSL), was also decreased. As expected, AKT-associated signaling pathway was hyperactivated and hypothesized to induce enlarged iWAT in SC-specific Pten-deficient mice. Moreover, preliminary experiments using AKT inhibitor AZD5363 treatment ameliorated the enlarged iWAT condition in SC-specific Pten-deficient mice. Taken together, SCs play an essential role in the regulation of SNS activity in iWAT development via the AKT signaling pathway. This novel role of SCs in SNS function allows for better understanding into the genetic mechanisms of peripheral neuropathy associated obesity.

Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive form of soft tissue neoplasm with extremely poor prognosis and no effective medical options currently available. MPNSTs can occur either sporadically or in association with the neurofibromatosis type 1 (NF1) syndrome. Importantly, activation of RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, and WNT/CTNNB1 signaling pathways has been reported in both NF1-related and late-stage sporadic MPNSTs. In this study, we found that DAW22, a natural sesquiterpene coumarin compound isolated from Ferula ferulaeoides (Steud.) Korov., could inhibit cell proliferation and colony formation in five established human MPNST cancer cell lines. Further molecular mechanism exploration indicated that DAW22 could target the main components in the MPNST tumorigenic pathways: namely suppress phosphorylation of AKT and ERK, and reduce levels of non-phospho (active) CTNNB1. Using the xenograft mouse model transplanted with human MPNST cancer cell line, daily treatment with DAW22 for 25 days was effective in reducing tumor growth. These results support DAW22 as an alternative therapeutic compound for MPNST treatment by affecting multiple signaling transduction pathways in its disease progression.

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.

CCCTC-binding factor (CTCF) is a DNA-binding protein that interacts with a large number of highly divergent target sequences throughout the genome. It is implicated in a variety of functions, including chromatin organization and transcriptional control. The functional role of CTCF in tumour pathogenesis remains elusive. We showed that CTCF is frequently upregulated in a subset of primary hepatocellular carcinomas (HCCs) as compared with non-tumoural liver. Overexpression of CTCF was associated with shorter disease-free survival of patients. Short hairpin RNA (shRNA)-mediated suppression of CTCF inhibited cell proliferation, motility and invasiveness in HCC cell lines; these effects were correlated with prominent reductions in the expression of telomerase reverse transcriptase (TERT), the shelterin complex member telomerase repeat-binding factor 1, and forkhead box protein M1 (FOXM1). In contrast, upregulation of CTCF was positively correlated with FOXM1 and TERT expression in clinical HCC biopsies. Depletion of CTCF resulted in reduced motility and invasiveness in HCC cells that could be reversed by ectopic expression of FOXM1, suggesting that FOXM1 is one of the important downstream effectors of CTCF in HCC. Reporter gene analysis suggested that depletion of CTCF is associated with reduced FOXM1 and TERT promoter activity. Chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) analysis further revealed occupancy of the FOXM1 promoter by CTCF in vivo. Importantly, depletion of CTCF by shRNA significantly inhibited tumour progression and metastasis in HCC mouse models. Our work uncovered a novel functional role of CTCF in HCC pathogenesis, which suggests that targeting CTCF could be further explored as a potential therapeutic strategy for HCC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Each year, more than 25,000 people succumb to liver cancer in the United States, and this neoplasm represents the second cause of cancer‐related death globally. R‐spondins (RSPOs) are secreted regulators of Wnt signaling that function in development and promote tissue stem cell renewal. In cancer, RSPOs 2 and 3 are oncogenes first identified by insertional mutagenesis screens in tumors induced by mouse mammary tumor virus and by transposon mutagenesis in the colonic epithelium of rodents. RSPO2 has been reported to be activated by chromosomal rearrangements in colorectal cancer and overexpressed in a subset of hepatocellular carcinoma. Using human liver tumor gene expression data, we first discovered that a subset of liver cancers were characterized by high levels of RSPO2 in contrast to low levels in adjacent nontumor tissue. To determine if RSPOs are capable of inducing liver tumors, we used an in vivo model from which we found that overexpression of RSPO2 in the liver promoted Wnt signaling, hepatomegaly, and enhanced liver tumor formation when combined with loss of transformation‐related protein 53 (Trp53). Moreover, the Hippo/yes‐associated protein (Yap) pathway has been implicated in many human cancers, influencing cell survival. Histologic and gene expression studies showed activation of Wnt/β‐catenin and Hippo/Yap pathways following RSPO2 overexpression. We demonstrate that knockdown of Yap1 leads to reduced tumor penetrance following RSPO2 overexpression in the context of loss of Trp53. Conclusion: RSPO2 overexpression leads to tumor formation in the mouse liver in a Hippo/Yap‐dependent manner. Overall, our results suggest a role for Yap in the initiation and progression of liver tumors and uncover a novel pathway activated in RSPO2‐induced malignancies. We show that RSPO2 promotes liver tumor formation in vivo and in vitro and that RSPO2's oncogenic activity requires Hippo/Yap activation in hepatocytes. Both RSPO2 and YAP1 are suggested to represent novel druggable targets in Wnt‐driven tumors of the liver.

Understanding the complex genetic background of cancers is key in developing effective targeted therapies. The Sleeping Beauty (SB) transposon system is a powerful and unbiased genetic editing tool that can be used for rapid screening of candidate liver cancer driver genes. Manipulating their expression level using a reverse genetic mouse model involving hydrodynamic tail-vein injection delivery can rapidly elucidate the role of these candidate genes in liver cancer tumorigenesis.

Understanding the complex genetic background of cancers is key in developing effective targeted therapies. The Sleeping Beauty (SB) transposon system is a powerful and unbiased genetic editing tool that can be used for rapid screening of candidate liver cancer driver genes. Manipulating their expression level using a reverse genetic mouse model involving hydrodynamic tail-vein injection delivery can rapidly elucidate the role of these candidate genes in liver cancer tumorigenesis.

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.

The genetic complexity and heterogeneity of cancer has posed a problem in designing rationally targeted therapies effective in a large proportion of human cancer. Genomic characterization of many cancer types has provided a staggering amount of data that needs to be interpreted to further our understanding of this disease. Forward genetic screening in mice using Sleeping Beauty (SB) based insertional mutagenesis is an effective method for candidate cancer gene discovery that can aid in distinguishing driver from passenger mutations in human cancer. This system has been adapted for unbiased screens to identify drivers of multiple cancer types. These screens have already identified hundreds of candidate cancer-promoting mutations. These can be used to develop new mouse models for further study, which may prove useful for therapeutic testing. SB technology may also hold the key for rapid generation of reverse genetic mouse models of cancer, and has already been used to model glioblastoma and liver cancer.