Transposon mutagenesis identifies and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

Takahiro Kodamaa, Justin Y. Newberga, Michiko Kodamaa, Roberto Rangela, Kosuke Yoshiharab, Jean C. Tienc, Pamela H. Parsonsd, Hao Wud, Milton J. Finegoldd, Neal G. Copelanda, and Nancy A. Jenkinsa,1

aCancer Research Program, Houston Methodist Research Institute, Houston, TX 77030; bDepartment of Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 9518510, Japan; cMichigan Center for Translational Pathology, Department of Pathology, University of Michigan, Ann Arbor, MI 48109; and dDepartment of Pathology and Immunology, Baylor College of Mecidine and Texas Children’s Hospital, Houston, TX 77030

Contributed by Nancy A. Jenkins, April 30, 2016 (sent for review March 23, 2016; reviewed by Zoltán Ivics and Roland Rad)

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis (10, 11). Two recent publications have challenged this metastasis and chemoresistance in patients with hepatocellular dogma for breast and pancreatic cancers and have suggested carcinoma (HCC), leading to their poor prognosis. The genes driving instead that EMT confers chemoresistance rather than meta- EMT in HCC are not yet fully understood, however. Here, we show static potential to tumor cells (12, 13). In the case of HCC, that mobilization of Sleeping Beauty (SB) transposons in immortal- growing evidence suggests that EMT contributes to metastasis – ized mouse hepatoblasts induces mesenchymal liver tumors on and poorer patient prognosis (14 17). EMT is also positively correlated with resistance to sorafenib, cisplatin, and doxorubicin transplantation to nude mice. These tumors show significant – down-regulation of epithelial markers, along with up-regulation of (18 20). Interestingly, sorafenib has been shown to have an in- hibitory effect on the migration of HCC cells through inactiva- mesenchymal markers and EMT-related transcription factors (EMT- tion of the EMT program (21), which is one of the potential TFs). Sequencing of transposon insertion sites from tumors identi- mechanisms of its antineoplastic action in HCC. Collectively, the fied 233 candidate cancer genes (CCGs) that were enriched for genes foregoing studies show that EMT has a significant negative im- and cellular processes driving EMT. Subsequent trunk driver analysis pact on survival in patients with HCC through the induction of identified 23 CCGs that are predicted to function early in tumorigen- metastasis and drug resistance. To understand the mechanism(s) esis and whose mutation or alteration in patients with HCC is cor- regulating EMT in HCC, it will be important to discover all EMT related with poor patient survival. Validation of the top trunk genes to identify therapeutic targets and improve the high mor- drivers identified in the screen, including MET (MET proto-oncogene, tality of patients with HCC. receptor tyrosine kinase), GRB2-associated binding 1 (GAB1), Insertional mutagenesis is a powerful in vivo screening tool for HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific cancer discovery. Our laboratory has successfully modeled demethylase 6A (KDM6A), and protein-tyrosine phosphatase, non- more than 20 different types of human cancers in mice using receptor-type 12 (PTPN12), showed that deregulation of these genes Sleeping Beauty (SB) transposon mutagenesis and has identified activates an EMT program in human HCC cells that enhances tumor hundreds of candidate cancer genes (CCGs), which show striking cell migration. Finally, deregulation of these genes in human HCC overlaps with genes mutated or deregulated in human cancers (22– was found to confer sorafenib resistance through apoptotic toler- 24). We also have developed in vitro cell-based SB mutagenesis ance and reduced proliferation, consistent with recent studies show- screens that have made it possible to identify genes involved in the ing that EMT contributes to the chemoresistance of tumor cells. Our malignant transformation of neural stem cells into glioma-initiating unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human Significance HCC and potentially for identifying new drug targets. Epithelial-mesenchymal transition (EMT) contributes to metas- transposon | EMT | HCC | Met | -mediated proteolysis tasis and chemoresistance in patients with hepatocellular car- cinoma (HCC), but the genes driving EMT are poorly understood. CC is the sixth most common cancer and the third-leading Here, we describe a transposon mutagenesis screen that made it Hcause of cancer-related deaths worldwide (1). In the United possible to identify 233 candidate cancer genes (CCGs) that are States, the incidence of HCC is increasing, and overall 5-y sur- enriched for genes driving EMT in HCC. Twenty-three CCGs are vival is now <12%, despite recent progress in diagnostic and predicted to function early in tumorigenesis, and alterations in therapeutic modalities (2). This high mortality rate is related these genes are associated with poor HCC patient survival. Vali- mainly to a high recurrence rate and associated intrahepatic or dation studies showed that deregulation of the most highly mu- extrahepatic metastases (1). Patients with HCC who develop tated CCGs activates an EMT program that enhances HCC cell metastasis are no longer eligible for liver transplantation therapy migration and also confers sorafenib resistance. Thus, transposon and have very limited therapeutic options. Currently, sorafenib is mutagenesis appears to provide an excellent resource for identi- the sole systemic antineoplastic agent for HCC described in the fying genes regulating EMT in human HCC and for potentially National Comprehensive Cancer Network (NCCN) guideline identifying new drug targets for HCC. (3); however, its clinical benefit is limited (4), and alternative Author contributions: T.K., N.G.C., and N.A.J. designed research; T.K., M.K., R.R., J.C.T., effective treatments are needed for these patients. P.H.P., H.W., and M.J.F. performed research; T.K., J.Y.N., and K.Y. analyzed data; and T.K., Epithelial-mesenchymal transition (EMT) is a complex dif- N.G.C., and N.A.J. wrote the paper. ferentiation process whereby epithelial cells lose their identity Reviewers: Z.I., Paul Ehrlich Institute; and R.R., Klinikum Rechts der Isar, Technische and acquire mesenchymal characteristics (5). EMT is observed Universität München. routinely in developmental processes, but is also believed to play The authors declare no conflict of interest. an important role in the migration, invasion, and metastasis of 1To whom correspondence should be addressed. Email: [email protected]. – various cancers (6 9). It also promotes the generation of can- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cer stem cells, thereby contributing to tumor recurrence and 1073/pnas.1606876113/-/DCSupplemental.

E3384–E3393 | PNAS | Published online May 31, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1606876113 Downloaded by guest on September 23, 2021 cells (25). This versatility and high relevance to human cancers (referred to as IHBC/WT lines). The immortalization rates for the PNAS PLUS makes SB an invaluable tool for cancer gene identification in mice. IHBC/WT and IHBC/SB lines were not significantly different Here, we report that mobilization of SB transposons in im- (33.3% vs. 58.3%; P = 0.18, χ2 test), suggesting that SB muta- mortalized mouse hepatoblasts induces mesenchymal liver tu- genesisplayedlittleornoroleinimmortalizationoftheIHBC/ mors on transplantation into nude mice. Cloning and sequencing SB lines (Fig. S1). We subsequently confirmed the mobilization of the transposon insertions sites from these tumors enabled us of SB transposons in IHBC/SB lines by excision PCR (Fig. 1A), to identify 233 CCGs that are enriched for genes driving EMT in providing additional evidence that these lines originate from human HCC. Twenty-three of these CCGs are predicted to func- hepatoblasts, because SB transposase activity is restricted to tion early in tumorigenesis, and alterations in these genes are as- cells expressing Cre under the albumin promoter. sociated with poor survival in patients with HCC. Validation studies To further demonstrate that these cell lines are derived from showed that deregulation of the most highly mutated CCGs ac- hepatoblasts, we measured the mRNA levels of hepatoblast tivates an EMT program that enhances HCC cell migration and markers Afp, Dlk1, and Epcam in three IHBC/SB cell lines. Each marker was expressed at high levels in IHBC/SB cells also confers sorafenib resistance. Thus, this unique cell-based – transposon mutagenesis screen appears to be an excellent re- compared with control adult mouse liver (Fig. 1 B D), further source for discovering genes involved in EMT in human HCC confirming these cells are derived from hepatoblasts. A unique and for potentially identifying new drug targets for HCC. property of hepatoblasts is their ability to differentiate into mature hepatocytes. To determine whether IHBC/SB cells can Results differentiate into hepatocytes, we cultured these cells in hepa- tocyte differentiation medium containing hepatocyte growth SB Transposition in Immortalized Hepatoblasts Induces Mesenchymal factor (HGF), oncostatin M (OSM), and dexamethazone (Dex) Tumors Following Transplantation. Immortalized hepatoblast cell (27). After 14 d in culture, the expression of mature hepatocyte lines were generated from WT (C57BL6/J) and SB transposon- markers Alb, Apob, Aldob, and Glul was greatly increased (Fig. expressing (Alb-Cre/+;T2Onc2/+;Rosa26-lsl-SB11/+) mice (22) 1 E–H), whereas the expression of hepatoblast markers Dlk1 and using the method described by Strick-Marchand et al. (26). In Epcam was decreased (Fig. 1 I and J). Thus, IHBC/SB cells re- brief, dissociated E13.5 fetal liver cells were plated in culture, tain their ability to differentiate into hepatocytes. and the cultures were incubated until colonies appeared. Seven To determine whether these cell lines are tumorigenic in nude of 12 plates from Alb-Cre/+;T2Onc2/+;Rosa26-lsl-SB11/+ mice mice, we injected cells from seven IHBC/SB lines and six IHBC/ developed colonies that subsequently gave rise to immortalized WT lines into the flanks of nude mice and then monitored the hepatoblast lines (referred to as IHBC/SB lines), whereas six mice for tumor formation. Although none of the IHBC/WT lines lines from 18 plates were established from C57BL6/J mice generated tumors following transplantation, visible tumors (>5mm

AB C D GENETICS

Tail/ IHBC/ IHBC/ IHBC/ SB SB-1 SB-2 SB-3 EF G K Fig. 1. Sleeping Beauty transposition in immortal- ized hepatoblasts results in the formation of mesenchymal tumors following transplantation. (A) Excision PCR analysis of tail DNA from a T2Onc2/+; Rosa26-lsl-SB11/+ mouse (Tail/SB) and three immortal- HI J * ized hepatoblast cell lines derived from Alb-Cre/+; T2Onc2/+;Rosa26-lsl-SB11/+ mice (IHBC/SB). The 2,200-bp band in tail DNA denotes unmobilized transposons, whereas the 225-bp band in the three hepatoblast lines represents mobilized transposons. (B–D) Afp (B), Dlk1 (C), and Epcam (D) mRNA expression in three IHBC/SB lines and one C57BL6/J adult mouse liver was quantitated by qPCR. (E–J) To induce hepatocyte LMN differentiation, an IHBC/SB line was cultured with 20 ng/mL HGF, 20 ng/mL oncostatin M, and 10−6 M dexamethazone for 14 d. mRNA expression levels of mature hepatocyte markers Alb (E), Apob (F), Aldob (G), and Glul (H), and two hepatoblast markers, Dlk1 (I) and Epcam (J), were then quantitated by qPCR. (K) The rate of tumor formation after s.c. injection of OP Q seven IHBC/SB and six IHBC/WT lines into the flanks of nude mice. Each line was injected into four flanks each. *P < 0.05, log-rank test. (L–Q) Histological analysis of tumors derived from IHBC/SB lines: H&E

(L), Ki-67 (M), Vimentin (N), Pan-CK (O), Epcam (P), and SB transposase (Q). (Original magnification, 200×; scale bar: 100 μm.)

Kodama et al. PNAS | Published online May 31, 2016 | E3385 Downloaded by guest on September 23, 2021 diameter) were obtained from five of the seven IHBC/SB lines chymal tumors (Fig. 1 L–N). However, immunohistochemical between 47 and 80 d after transplantation (Fig. 1K). Thus, trans- analysis also showed that these tumors also expressed the epithelial poson mutagenesis appears to be required for the malignant markers pan-cytokeratin and Epcam, as well as SB transposase (Fig. transformation of IHBC cells. Histological analysis showed that 1 O–Q). These findings support the hypothesis that these tumors tumors were composed of highly proliferative, spindle-shaped originate from transposase-active hepatoblasts, but acquire mesen- cells with vimentin (Vim) immunopositivity, typical of mesen- chymal characteristics during their growth in vivo. To confirm these

A IHBC/SB-3 IHBC/SB-1 IHBC/SB-2 IHBC/WT AML12 IHBC/SB-4 IHBC/SB-5 IHBC/SB-2 tumor IHBC/SB-1 tumor IHBC/SB-3 tumor IHBC/SB-4 tumor Ilk Mitf Vim Fn1 Dsp Gsc Tcf4 Egfr Krt7 Msn Il1rn F11r Ptk2 Sip1 Akt1 Ocln Esr1 Itgav Jag1 Tfpi2 Fzd7 Itgb1 Itga5 Zeb1 Zeb2 Vcan Dsc2 Rac1 Stat3 Rgs2 Spp1 Cav2 Krt14 Krt19 Cdh1 Cdh2 Mst1r Plek2 Tgfb2 Tgfb3 Tgfb1 Snai1 Snai2 Snai3 Tcf7l1 Sparc Bmp1 Bmp7 Nodal Cald1 Erbb3 Foxc2 Timp1 Mmp9 Mmp2 Mmp3 Igfbp4 Wnt11 Twist1 Sox10 Ahnak Wnt5b Wnt5a Gsk3b Gng11 Pdgfrb Tmeff1 Fgfbp1 Steap1 Smad2 Ptp4a1 Notch1 Col1a2 Col5a2 Col3a1 Ctnnb1 Nudt13 Mtap1b Vps13a Pppde2 Tspan13 Serpine1 Camk2n1 Tmem132a B CD * * * * *

EF G * * * * * *

HI * * * *

Fig. 2. An EMT program is active in IHBC/SB-derived tumors. (A) Heat map and hierarchical clustering depicting the expression of 84 EMT-related genes in five IHBC/SB lines, one IHBC/WT line, the mouse immortalized hepatocyte cell line (AML12), and four IHBC/SB-derived tumors. (B–I) mRNA expression levels of EMT markers and EMT-TFs quantitated by qPCR in five IHBC/SB lines, 45 IHBC/SB-derived tumors, and five well-differentiated HCCs derived from SB mice: Col1a2 (B), Vim (C), Fn1 (D), Cdh1 (E), Snai1 (F), Twist1 (G), Zeb1 (H), and Zeb2 (I). *P < 0.05, Kruskal–Wallis test.

E3386 | www.pnas.org/cgi/doi/10.1073/pnas.1606876113 Kodama et al. Downloaded by guest on September 23, 2021 results, we reinjected the five tumorigenic IHBC/SB lines into nude ABA PNAS PLUS mice and generated a total of 52 tumors. Once again, all of these tumors were spindle-shaped mesenchymal tumors. IHBC/SB-tumor Cancer Gene 7 IHBC/SB 52 IHBC/SB- CISs Census To determine whether the subcutaneous microenvironment 28 might have provided a selective pressure that led to the gener- lines 1 derived tumors 199 542 ation of mesenchymal tumors, we orthotopically injected IHBC/ 60 232 SB cells into the livers of nude mice. All 10 tumors that de- veloped in the liver were histologically identical to those that p < 0.0001 developed in the flank (Fig. S2). These results indicate that the C subcutaneous microenvironment did not provide a selective 3000 pressure that resulted in the formation of mesenchymal tumors. Gab1 2500 Ptpn12 IHBC/SB Tumors Have an Activated EMT Program. The mesenchymal Fto nature of these tumors prompted us to ask whether an EMT program had been activated in tumors. We did this by assaying the 2000 mRNA expression of 84 EMT-related genes in five IMBC/SB

lines and one IMBC/WT line, four IMBC/SB tumors, and an 1500 immortalized mouse hepatocyte cell line (AML12). Hierarchical Mllt10 Huwe1 clustering showed distinct expression differences in the IHBC/SB Lpp Kdm6a line-derived and IHBC/SB-derived tumors (Fig. 2A), whereas read counts Average 1000 similar expression patterns were observed in the IHBC/SB lines, Stag2 Ltbp1 Epc2 Met IHBC/WT lines and AML12 cells. We also individually checked Rasa1 500 Arhgef28 Epb4.1l2 the expression levels of EMT-related genes in the IHBC/SB lines, Chd9 Nf1 Pbx1 Dnajc1 Ttc3 Psen1 IHBC/SB-derived tumors, and HCC tumors obtained from SB Nedd4l Trio Fam120a transposon expressing mice (SB/HCCs) by quantitative PCR 0 1 10 100 1000 (qPCR). SB/HCC tumors are well-differentiated HCCs and thus - log P maintain their epithelial characteristics. Thus, they are different from poorly differentiated HCCs, which often exhibit mesen- Fig. 3. CCGs and trunk drivers identified in IHBC/WT lines and IHBC/SB- chymal properties (28). Mesenchymal markers Col1a2, Vim, and derived tumors. (A) Venn diagram showing the overlap between 61 CCGs fibronectin 1 (Fn1) were markedly elevated in IHBC/SB-derived identified from seven IHBC/SB lines and 233 CCGs identified from 52 IHBC/SB- tumors but not in IHBC/SB lines and well-differentiated SB/ derived tumors. (B) Venn diagram showing the overlap of 227 CCGs with HCC tumors (Fig. 2 B–D). We also observed a significant de- human orthologs identified from 52 IHBC/SB-derived tumors and 570 human cancer genes listed in the Cancer Gene Census database. P values were cal- crease in expression of epithelial markers Cdh1 (cadherin 1, type ’ 1, E-cadherin) in IHBC/SB-derived tumors (Fig. 2E). Finally, we culated using Fisher s exact test. (C) Transposon insertion sites identified in 52 IHBC/SB-derived tumors that were represented by >100 sequence read found significant up-regulation of EMT-TFs, snail family zinc counts were selected for gCIS analysis, which identified 23 trunk drivers that finger 1 (Snai1), twist family BHLH 1 (Twist1),

were mutated in six or more tumors. Shown are the 23 trunk drivers, including GENETICS E-box binding 1 (Zeb1), and Zeb2 (6) in – – their average sequence read counts and log P values after Bonferroni cor- IHBC/SB-derived tumors (Fig. 2 F I). These data indicate rection of the gCIS results. The seven trunk drivers listed in the Cancer Gene that an EMT program has been activated in IHBC/SB-derived Census are shown in red. tumors.

Identification of CCGs Driving the Development of Mesenchymal (24). To identify the subset of CCGs that function early in tumor Tumors. To identify CCGs driving mesenchymal tumor develop- development, we selected transposon insertion sites represented ment, we PCR-amplified and sequenced the transposon insertion by ≥100 sequencing reads, arguing that these insertions would be sites from seven IHBC/SB lines and all 52 IHBC/SB-derived present in the largest number of tumor cells. We then performed tumors. To obtain the maximum number of sequencing reads gCIS analysis on these insertions and identified 23 CCGs that and avoid PCR bias (29), we used Illumina sequencing and were mutated in six or more tumors. We refer to these CCGs as acoustic shearing of tumor DNA (Materials and Methods). Using trunk drivers (Fig. 3C). Strikingly, Met (MET proto-oncogene, this modified method, we identified 871,231 and 6,541,525 receptor tyrosine kinase) and its binding partner GRB2-associ- mapped reads, corresponding to 50,215 and 118,530 nonredundant ated binding protein 1 (Gab1) were the first and third most transposon insertion sites from IHBC/SB lines and IHBC/SB- significantly mutated trunk drivers. The Met pathway has various derived tumors, respectively (Datasets S1 and S2). Using the important roles in tumor development, including increased gene-centric common integration site (CIS) calling method proliferation, migration, invasion, and angiogenesis. It is also an (gCIS) (30), we identified 61 CCGs from IHBC/SB lines and 233 inducer of EMT (32). Seven of the 23 trunk drivers are known CCGs from IHBC/SB-derived tumors (P < 0.05, χ2 test followed human cancer genes, whereas five—Met, Lpp, Pbx1, lysine-spe- by Bonferroni correction) (Tables S1 and S2). CISs are genomic cific demethylase 6A (Kdm6a), and Nf1—are reportedly involved regions that contain more transposon insertions than predicted in regulating EMT (32–37). Another trunk driver, Rasa1, is a Ras by chance and thus are likely to mark the location of CCGs (22). GTPase-activating protein that suppresses oncogenic Ras sig- Only one CCG overlapped between these two datasets (Fig. 3A), naling, another EMT inducer (5). The trunk driver Ltbp1 con- indicating that tumors have acquired new insertional mutations trols the activity of TGF-β, which is also an EMT inducer (38). important for their development during growth in vivo. Although there could be some PCR bias in this trunk driver To determine whether CCGs identified in tumors are signifi- analysis, these analytical results are certainly in agreement with cantly enriched for human cancer genes, we compared our list of the hypothesis that transposon mutagenesis plays an important CCGs to the list of 570 known human cancer genes in the Cancer role in activating the EMT program and driving a mesenchymal Gene Census database (31), and found that 28 GCCs are human tumor phenotype in tumors generated from immortalized hep- cancer genes (Fig. 3B), which is a highly significant enrichment atoblasts. Importantly, examination of the cancer genome atlas (P < 0.0001). These results provide additional evidence that in- (TCGA), which contains the mutation and copy number data for sertional mutations in these CCGs are involved in driving the 193 HCC patients, shows that deregulation of the 23 trunk drivers in development of mesenchymal tumors. human HCC is significantly correlated with poor patient survival Previous studies have suggested that most CCGs identified by (Fig. S3), suggesting the important role of these trunk drivers in SB mutagenesis function during late stages of tumor progression human HCC.

Kodama et al. PNAS | Published online May 31, 2016 | E3387 Downloaded by guest on September 23, 2021 A Met/Gab1 pathway (40/52, 76.9%)

Gab1 61.5%

Met 38.5% B Fig. 4. Met/Gab1 pathway activation induces EMT and enhanced migration of human HCC cells. Gab1 (A) Oncoprints showing the number of transposon insertions in Gab1 and Met in 52 IHBC/SB-derived tu- 5′ 3′ C mors. Color density is determined by the sequence read counts for each insertion. The higher the read Met count, the darker the color. Each column represents a 5′ 3′ single tumor. (B and C) Insertion maps showing the D E F location of transposon insertions in Gab1 (B) and Met SNU-423 SNU-449 SNU-475 (C) in 52 IHBC/SB-derived tumors. Each bar represents * * p-Met a single transposon insertion event. (D) mRNA ex- pression levels of Gab1 determined by qPCR in five Met IHBC/SB lines and 20 IHBC/SB-derived tumors that had p-Gab1 transposon insertions in Gab1.*P < 0.05, Mann– Gab1 Whitney U test. (E) mRNA expression levels of Met determined by qPCR of five IHBC/SB lines and 13 IHBC/ β-actin SB-derived tumors that had transposon insertions in Met.*P < 0.05, Mann–Whitney U test. (F) Protein Vehicle Vehicle Vehicle expression levels of phosphor-Met (Tyr1234), Met, HGF 1h HGF 3h HGF 1h HGF 3h HGF 1h HGF 3h phosphor-Gab1 (Tyr307), Gab1, and β-actin deter- GHIJmined by Western blot analysis of three human HCC cell lines, including SNU-423, SNU-449, and SNU-475, * * after treatment with 10 ng/mL of recombinant human * HGF or DMSO. (G) Expression levels of CDH1 deter- mined by qPCR in SNU-423, SNU-449, and SNU-475 cells at 2 d after treatment with 10 ng/mL recombi- nant human HGF or DMSO. (H–J) The migration ca- pacity of SNU-423 (H), SNU-449 (I), and SNU-475 (J)cells measured using a scratch assay at 2 d after treatment with 10 ng/mL recombinant human HGF or DMSO. n = 4. *P < 0.05, Student’s t test.

Oncogenic Activation of the Met/Gab1 Pathway Induces EMT and genesis plays an important role in driving EMT and inducing a Enhances the Migration of Human HCC Cells. We next sought to mesenchymal phenotype in IHBC/SB-derived tumors. confirm that the Met/Gab1 pathway plays an important role in Ubiquitin-mediated proteolysis was the most enriched path- regulating EMT in human HCC. We focused on this pathway way in tumors with 26 CCGs functioning in this process. Whereas because 40 of 52 IHBC/SB-derived tumors (76.9%) contain in- ubiquitin-mediated proteolysis is not commonly associated with sertions in either or both Met and Gab1 genes (Fig. 4A). EMT, all IHBC/SB-derived tumors had one or more mutations Transposon insertions are located in the 5′ end of these genes, be- in a CCG that functions in this process (Fig. 5B). Interestingly, these fore exon 3 or 2 (Fig. 4 B and C), respectively, suggesting that they 26 CCGs also appear to be clinically relevant in human HCC. are functioning as oncogenes. Consistent with this, the expression of TCGA data show that deregulation of these CCGs in human HCC Met and Gab1 was significantly higher in tumors with insertions in was significantly correlated with poor patient survival (Fig. S4). Met or Gab1 compared with IHBC/SB lines (Fig. 4 D and E). To determine whether the Met/Gab1 pathway plays an im- Ubiquitin-Mediated Proteolysis Is Linked to EMT in Human HCC Cells. portant role in regulating EMT in human HCC cells, we treated To determine whether ubiquitin-mediated proteolysis is linked three human HCC cell lines with hepatocyte growth factor to EMT in human HCC, we focused on the HECT, UBA, and WWE domain containing 1 (Huwe1) gene, the most frequently (HGF), which is the ligand for Met (32). HGF strongly increased mutated gene linked to ubiquitin-mediated proteolysis in tumors Met phosphorylation and slightly up-regulated Gab1 phosphor- (Fig. 5B). Transposon insertions in Huwe1 are scattered throughout ylation in human HCC cell lines (Fig. 4F). HGF treatment de- thegene(Fig.5C), and its expression is significantly down-regulated creased the expression of epithelial markers, such as E-cadherin, in tumors with insertions in Huwe1 (Fig. 5D), suggesting that Huwe1 in all three HCC cell lines (Fig. 4G). Finally, in vitro scratch assays is a tumor suppressor gene. Therefore, we investigated whether (39) showed that HGF treatment significantly enhanced the mi- HUWE1 knockdown effects EMT in human HCC cells. – gration of HCC cells in vitro (Fig. 4 H J). These results, along with Knockdown of HUWE1 using two different siRNAs increased the those reported by others (32), indicate that activation of the Met/ expression of mesenchymal markers CDH2 (cadherin 2, type 1, N- Gab1 pathway is an important driver of EMT in human HCC cells. cadherin), FN,andVIM (Fig. 5E) and the EMT-TFs SNAI1 and ZEB1 (Fig. 5E). HUWE1 knockdown also enhanced the migration CCGs Function in Multiple Pathways Important for EMT. We next of cells from three human HCC cell lines (Fig. 5 F–H), suggesting used KEGG pathway analysis to determine whether any signal- that HUWE1 is an EMT suppressor in human HCC. Importantly, we ing pathways or cellular processes are enriched among the CCGs also found a significant negative correlation between the expression identified in tumors. Strikingly, multiple pathways and cellular of HUWE1 and poor prognosis in patients with HCC, indicating processes known to be involved in EMT, including Wnt signal- that HUWE1 also has clinical relevance in human HCC (Fig. 5I). ing, adherens junctions, focal adhesion, Notch signaling, MAPK signaling, regulation of actin cytoskeleton, and TGFβ signaling Protein-Tyrosine Phosphatase, Nonreceptor-Type 12 and Kdm6a Also (5), were enriched among the CCGs identified in tumors (Fig. Regulate EMT in Human HCC Cells. We also asked whether two 5A), further supporting the hypothesis that transposon muta- other SB-identified trunk drivers, protein-tyrosine phosphatase,

E3388 | www.pnas.org/cgi/doi/10.1073/pnas.1606876113 Kodama et al. Downloaded by guest on September 23, 2021 PNAS PLUS A Ubiquitin mediated proteolysis Wnt signaling pathway Pathway in cancer Adherens junction Endocytosis Focal adhesion Axon guidance Notch signaling pathway Chronic myeloid leukemia MAPK signaling pathway Regulation of actin cytoskeleton TGF-beta signaling pathway 0 2 4 6 8 10 12 - log P B D Huwe1 71.2% Fbxw7 36.5% Rfwd2 32.7% * Cblb 26.9% Trip12 26.9% Ube3c 25.0% Anapc1 23.1% Ube3a 23.1% Ube2l3 23.1% Cul1 23.1% Itch 21.2% Cbl 21.2% Cul3 19.2% Fbxw11 19.2% Wwp2 19.2% E Ube2d3 19.2% Ube2k 17.3% Cul5 15.4% Rchy1 11.5% Cul4a 11.5% 9.6%

Uba2 GENETICS Birc2 9.6% Ppil2 9.6% Ube2g2 7.7% Prpf19 5.8% Ube2b 5.8%

C

Huwe1 5′ 3′ I FHG HUWE1 1.0 high low 0.8

* 0.6 * 0.4 * 0.2

Ratio of survived patients p 0.0 logrank = 0.03

0 2000 4000 (Days)

Fig. 5. Ubiquitin-mediated proteolysis plays a role in regulating EMT in human HCC cells. (A) KEGG pathway analysis of the CCGs identified from 52 IHBC/SB- derived tumors. P values for enrichment are represented on a –log scale. Pathways known to be involved in EMT are shown in red. (B) Oncoprints showing the number of IHBC/SB-derived tumors with transposon insertions in 26 CCGs that function in ubiquitin-mediated proteolysis. Color density is determined by the sequence read counts for each insertion. The higher the read count, the darker the color. Each column represents a single tumor. (C) Insertion maps showing the location of transposon insertions in Huwe1 in IHBC/SB-derived tumors. Each bar represents a single transposon insertion event. (D) mRNA expression levels of Huwe1 quantitated by qPCR in five IHBC/SB lines and 21 IHBC/SB-derived tumors that had transposon insertion in Huwe1.*P < 0.05, Mann–Whitney U test. (E) mRNA expression levels of CDH2, FN1, VIM, SNAI1, ZEB1, and HUWE1 quantitated by qPCR in SNU-387 cells at 3 d after transfection with negative control (NC) or HUWE1 siRNA. (F–H) Migration capacity of SNU-387 (F), SNU-423 (G), and SNU-475 (H) cells measured using a scratch assay at 3 d after transfection with negative control (NC) or HUWE1 siRNA. n = 4. P < 0.05 vs. all, one-way ANOVA. (I) Kaplan–Meier plot of patient survival based on mRNA abundance of HUWE1 in human HCC tumor samples (GSE10141). Statistical analysis was performed using the log-rank test.

Kodama et al. PNAS | Published online May 31, 2016 | E3389 Downloaded by guest on September 23, 2021 A Ptpn12 5′ 3′ B Kdm6a

5′ 3′ CD * *

Fig. 6. SB-identified trunk drivers Ptpn12 and Kdm6a regulate EMT in human HCC cells. (A and B) Insertion maps showing the location of transposon insertions in Ptpn12 (A)andKdm6a (B) in IHBC/SB-derived tumors. EF Each bar represents a single transposon insertion event. (C) mRNA expression levels of Ptpn12 quantitated by qPCR in five IHBC/SB lines and seven IHBC/SB-derived tumors that had transposon insertions in Ptpn12.*P < 0.05, Mann–Whitney U test. (D) mRNA expression levels of Kdm6a quantitated by qPCR in five IHBC/SB lines and nine IHBC/SB-derived tumors that had transposon in- sertion in Kdm6a.*P < 0.05, Mann–Whitney U test. (E and F) mRNA expression levels of CDH2, FN1, VIM, TWIST1, ZEB,andPTPN12 (E)orKDM6A (F) quantitated by qPCR in human SNU-387 HCC cells at 3 d after transfection with negative control (NC) or PTPN12 GH I J siRNA (E)orKDM6A siRNA (F). (G and H) Migration capacity of human HCC cells in SNU-387 (G) and SNU- 475 (H) cells at 3 d after transfection with negative control (NC) or PTPN12 siRNA, assessed using a scratch * * * assay. *P < 0.05 vs. all, one-way ANOVA. (I and J) * Migration capacity of human HCC cells in SNU-387 (I) and SNU-475 (J) cells at 3 d after transfection with negative control (NC) or KDM6A siRNA, as- sessed using a scratch assay. n = 4. *P < 0.05 vs. all, one-way ANOVA.

nonreceptor-type 12 (Ptpn12)andKdm6a, regulate EMT in human Finally, to study the potential mechanism of EMT-induced HCC cells. We chose these genes because they had the highest av- sorafenib resistance, we assessed the proliferation and apoptosis erage read counts of all trunk driversidentifiedintumors(Fig.3C). of HCC cells on knockdown of these three EMT suppressors. Similar to Huwe1, transposon insertions in both genes were located Knockdown of these genes significantly reduced HCC cell pro- throughout the coding region (Fig. 6 A and B), and their expression liferation (Fig. 7 D–F), and HCC cells with siRNAs targeting was significantly down-regulated in tumors with SB insertions (Fig. 6 these genes significantly attenuated the apoptosis induced by C and D), suggesting they are tumor suppressor genes as well. sorafenib treatment (Fig. 7 G–I). These findings suggest that Knockdown of these genes using two independent siRNAs in EMT confers sorafenib resistance to human HCC cells through human HCC cells once again led to the up-regulation of mes- reduced cell proliferation and increased apoptotic tolerance. enchymal markers CDH2, FN, and VIM and the EMT-TFs TWIST1 and ZEB1 (Fig. 6 E and F). Down-regulation of these Discussion genes also significantly increased the migration of cells from two In this study, we have shown that mobilization of SB transposons human HCC cell lines, SNU-387 and SNU-475 (Fig. 6 G–J), in immortalized mouse hepatoblasts induces mesenchymal liver further confirming that these genes are suppressors of EMT in tumors following transplantation into nude mice. The tumors human HCC cells. Combined with our previous studies, all five have reduced of epithelial markers and in- of the top trunk drivers identified in IHBC/SB-derived tumors creased expression of mesenchymal markers and EMT-TFs such are confirmed drivers of EMT in human HCC cells. as Snail, Twist1, and Zeb1. Sequencing of transposon insertion sites from 52 tumors identified 233 CCGs that are enriched for SB-Identified EMT Regulators Promote Sorafenib Resistance in Human genes and cellular processes driving EMT, whereas trunk driver HCC Cells. Two recent reports have shown that EMT promotes analysis identified 23 CCGs that appear to function early in tu- chemoresistance of tumor cells (12, 13). To determine whether morigenesis and whose mutation or alteration in patients with EMT contributes to chemoresistance in human HCC cells, we HCC correlates with poor survival of these patients. Validation examined the ability of three SB-identified EMT suppressors— studies have shown that deregulation of the most highly mutated Kdm6a, Huwe1, and Ptpn12—to modify the chemosensitivity of CCGs in human HCC activates an EMT program that enhances HCC cells. We knocked down the expression of each of these cell migration and also confers sorafenib resistance to HCC cells. genes in human HCC cells using siRNA and then treated the Thus, transposon mutagenesis appears to provide an excellent cells with sorafenib, the sole potentially effective antineoplastic resource for identifying genes driving EMT in human HCC and agent against HCC identified to date (4). Intriguingly, on sorafenib for potentially identifying new drug targets for HCC. treatment, the viability of HCC cells containing siRNAs targeting Strikingly, Met and its binding partner Gab1 were the first and these genes was significantly higher than that of cells receiving third most significantly mutated trunk drivers identified in tumors. negative control siRNA (Fig. 7 A–C). Thus, EMT also seems Activating insertional mutations in Met and/or Gab1 were identi- to confer chemoresistance to human HCC cells. fied in >75% of tumors and showed very high sequencing read

E3390 | www.pnas.org/cgi/doi/10.1073/pnas.1606876113 Kodama et al. Downloaded by guest on September 23, 2021 counts, suggesting that activation of this signaling pathway is a ABC PNAS PLUS major driver of these tumors. This pathway has been extensively studied in various cancers and shown to be a potent oncogenic * pathway involved in cancer formation, progression, and metastasis * * (32, 40, 41). This pathway is activated in human HCC mainly through copy number alterations (42), and its overexpression has been correlated with an increased incidence of intrahepatic me- tastases and poor prognosis in patients with HCC (43). In HCC, HGF stimulation has been shown to activate an EMT program and to enhance the migration capacity of PLC/PRF/5 and HepG2 hu- DEF man liver cancer cells (21). In the present study, we have shown that * * * activation of the Met/Gab1 pathway by HGF treatment decreases the expression of epithelial markers and increases the migration capacity of SNU-423, SNU-449, and SNU-475 human liver cancer cells, confirming that this pathway is an important activator of EMT in HCC. These data suggest that the Met pathway could be a promising therapeutic target in HCC. Indeed, tivantinib, a selective Met inhibitor, has recently been shown to significantly improve the overall survival of Met-positive HCC patients who experienced GHI failure of or intolerance to previous systemic therapy (44). Phase III *** trials of several MET inhibitors in patients with HCC are currently underway, but the results of these trials have not yet been reported. Pathway analysis revealed that 26 CCGs identified in mesen- chymal tumors function in ubiquitin-mediated proteolysis. Huwe1 was the most frequently mutated CCG and was inactivated by SB insertions in more than 70% of tumors. Huwe1 is a proven tumor suppressor gene that acts via the degradation of c-/Miz1 in skin cancer (45) or Mcl-1 in HCC (46); however, a role of Huwe1 in Fig. 7. siRNA knockdown of KDM6A, HUWE1, and PTPN12 induces sorafenib EMT has not yet been described. In this study, we found that resistance in human HCC cells. (A–C) Cell viability following sorafenib siRNA-mediated knockdown of Huwe1 led to increased expression treatment of human HCC cells measured using the WST-1 assay. SNU-387 of a variety of mesenchymal markers as well as EMT-TFs SNAI1 human HCC cells were transfected with negative control (NC), KDM6A (A), and ZEB1, leading to enhanced migration and sorafenib resistance HUWE1 (B), or PTPN12 (C) siRNA. At 2 d after transfection, the cells were treated of multiple HCC cell lines, demonstrating a new role for Huwe1 with 4 μM sorafenib or vehicle for another 3 d. Relative cell viability was calcu- as an EMT suppressor. We also showed that down-regulation of lated as the ratio of the WST-1 value between sorafenib-treated and vehicle- Huwe1 is correlated with poor prognosis in patients with HCC, treated cells. n = 4. *P < 0.05 vs. all, one-way ANOVA. (D–F) Cell proliferation of human HCC cells assessed using the WST-1 assay. SNU-387 human HCC cells were suggesting that inactivation of HUWE1 may contribute to the GENETICS transfected with negative control (NC), KDM6A (D), HUWE1 (E), or PTPN12 (F) progression of HCC through activation of EMT. = < The second most frequently mutated CCG that functions in siRNA. At 5 d after transfection, the WST-1 assay was performed. n 4. *P 0.05 vs.all,one-wayANOVA.(G–I) Measurement of apoptosis using the caspase-3/7 ubiquitin-mediated proteolysis was Fbxw7, which was mutated in assay following sorafenib treatment of human HCC cells. SNU-387 human HCC 35% of tumors. Fbxw7 is highly mutated in various human can- cells were transfected with negative control (NC), KDM6A (G), HUWE1 (H), or cers and functions as a tumor suppressor through degradation of PTPN12 (I) siRNA. At 2 d after transfection, the cells were treated with 4 μM several growth promoters, including c-Myc, m-TOR, cyclin E, and sorafenib or vehicle for another 2 d. Relative caspase-3/7 activity was calculated c-Jun (47). FBXW7 is also a known EMT suppressor and contrib- as the ratio of the caspase-3/7 activity value between sorafenib-treated and utes to chemosensitivity in human HCC (48). In IHBC/SB-derived vehicle-treated cells. n = 4. *P < 0.05 vs. all, one-way ANOVA. tumors, insertions in Fbxw7 are scattered throughout the coding regions, where they are predicted to inactivate Fbxw7 expression Kdm6a is an H3K27me3 demethylase that can reverse histone and potentially activate EMT in IHBC/SB-derived tumors. lysine methylation induced by Ezh2, a member of the polycomb We also have shown that two other SB-identified trunk drivers, repressive complex 2 (58). Loss-of-function mutations of KDM6A Ptpn12 and Kdm6a, regulate EMT in human HCC cells. PTPN12 are frequently observed in various human cancers, including bladder tyrosine phosphatase was originally identified in a genetic screen for cancers, medulloblastomas, and renal cancers (59), suggesting tumor suppressors in triple-negative breast cancers, where it inhibits that KDM6A functions as a tumor suppressor. The role of Kdm6a multiple oncogenic tyrosine kinases, including HER2 and EGFR in cancer progression remains controversial, however. Choi et al. (49). In addition, studies of Ptpn12-deficient mice have shown that (36) reported that Kdm6a represses EMT genes and inhibits EMT- Ptpn12 regulates the migration of endothelial cells, dendritic cells, induced cancer stemness properties in breast cancer. Similarly, van and macrophages, probably through dephosphorylation of Prk2, – den Beucken et al. (37) showed that hypoxia inhibits KDM6A and FAK, Cas, and paxillin (50 52). These antimigratory functions of induces the hypermethylation of DICER, leading to the up-regulation PTPN12 also have been observed in several types of human can- of ZEB1 expression and promotion of EMT and cancer stemness. In – cers, including breast, ovary, and prostate cancers (53 55). Con- contrast, Thieme et al. (58) showed that Kdm6a positively regulates sistent with this, Ptpn12 was inactivated in our IHBC/SB-derived the migration of hematopoietic stem cells. In addition, Kim et al. (60) tumors by transposon insertion. We also showed that knockdown of reported that knockdown of KDM6A decreased the migration and PTPN12 significantly increased the migration capacity and expres- invasion capacity of breast cancer cell lines. sion of mesenchymal markers and EMT-TFs in multiple human In the present study, Kdm6a was inactivated in IHBC/SB- HCC cell lines, indicating that PTPN12 is a universal EMT sup- derived tumors by transposon insertions. Knockdown of Kdm6a pressor in cancer cells as well as in normal tissues. Decreased ex- accelerated the migration of cells from two human HCC cell pression of PTPN12 is also negatively correlated with tumor lines and increased ZEB1 expression. Because histone lysine recurrence and survival of HCC patients (56), further supporting demethylase can potentially regulate the activation of hundreds the importance of this tumor suppressor in HCC. Interestingly, of genes, its function may vary and may depend on cell context, Ptpn12-deficient mouse embryos exhibited a failure of liver devel- which could possibly generate contradictory findings. opment, leading to lethality (57), suggesting that Ptpn12 mayplaya Why IHBC/SB lines preferentially generate mesenchymal tu- fundamental role in liver development as well. mors following transplantation into nude mice remains unclear.

Kodama et al. PNAS | Published online May 31, 2016 | E3391 Downloaded by guest on September 23, 2021 One possibility is that IHBC/SB lines acquire mesenchymal prop- In Vitro Hepatocyte Differentiation of Hepatoblasts. To induce hepatocyte erties during growth in culture, and the tumors simply reflect this differentiation in vitro, IHBC/SB lines were cultured for 14 d in medium supplemented with 20 ng/mL recombinant mouse HGF (Peprotech), 20 ng/mL preselection. Our qPCR EMT array results suggest that this is not −6 the case, however, and instead indicate that the mesenchymal recombinant mouse oncostatin M (R&D Systems), 10 M dexamethazone properties of tumors are acquired during growth in vivo. Consistent (Sigma-Aldrich), and insulin-transferrin-selenium (Thermo Fisher Scientific) (27). with this, there was almost no overlap between the CCGs detected Allograft Tumor Formation Assay. Female athymic nude mice [Crl:Nu(NCr)- in IHBC/SB lines and those detected in IHBC/SB-derived tumors, × 6 indicating that these EMT-related cancer genes were selectively Foxn1nu] were purchased from Charles River Laboratories. A total of 1.0 10 IHBC/SB or IHBC/WT cells were injected s.c. into the flanks of nude mice mutated during growth in vivo. Another possible explanation is that within 2 wk after their resuscitation from cryopreservation. The mice were the subcutaneous microenvironment provided selective pressure for monitored for tumor formation until 90 d of age. These experiments were the generation of mesenchymal tumors. This also does not appear repeated several times. Tumors >5 mm in diameter were collected for se- to be the case, however, given that IHBC/SB-derived tumors gen- quence and biochemical analysis. erated after orthotopic injection into the liver were identical to the tumors generated after s.c. injection. EMT is linked to various Sequencing of Transposon Insertion Sites. Transposon insertion sites were fundamental processes in cancer development and progression, sequenced using splinkerette PCR to produce barcoded PCR products, which including migration, invasion, cancer stemness, and DNA repair (5, were then pooled and sequenced (22–24). To obtain the maximum amount 6, 8, 61), and it is possible that transposon-induced activation of an of sequencing reads and avoid PCR bias (29), we used Illumina sequencing EMT program was selected during growth of IHBC/SB cells in vivo and acoustic shearing of genomic DNA (gDNA). gDNA (3 μg per sample) was becauseitgavethecellsastrong survival advantage. acoustically sheared to 300-bp fragments using a Covaris S220 Focused- Although these tumors originated from epithelial cells and ultrasonicator and end-repaired using a Fast DNA End Repair Kit (Thermo acquired many mesenchymal properties, some of our findings Fisher Scientific). Adapters (linker+:5′-GTA ATA CGA CTC ACT ATA GGG CTC were not consistent with typical EMT. For instance, whereas the CGC TTA AGG GAC-3′; linker−:5′-phos-GTC CCT TAA GCG GAG-C3spacer-3′) were then annealed and ligated to repaired DNA. Purified DNA was PCR- expressions of many known EMT markers and EMT regulators ′ ′ ′ were up-regulated in tumors, including extracellular matrix amplified using linker (5 -GTA ATA CGA CTC ACT ATA GGG C-3 ), IRR (5 -GGA β TTA AAT GTC AGG AAT TGT GAA AA-3′), and IRL (5′-AAA TTT GTG GAG TAG , TGF- family proteins, and EMT-TFs, the expression ′ β TTG AAA AAC GA-3 ) primers. Nested secondary PCR was performed using levels of -catenin and N-cadherin were low in these tumors. indexed SB-2ndR primer [5′-CAA GCA GAA GAC GGC ATA CGA GAT (8-bp Moreover, these tumors still maintained immunopositivity for index) GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TTA GGG CTC epithelial markers as well as mesenchymal markers. Therefore, CGC TTA AGG GAC-3′], SB-2ndRF primer [AAT GAT ACG GCG ACC ACC GAG the phenotype that these tumors acquired was not complete EMT ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T (1- to 4-bp stagger) in a strict sense, which requires a complete transition, but rather (8-bp index) GGC TAA GGT GTA TGT AAA CTT CCG ACT TCA ACT G-3′], and incomplete EMT caused by epithelial plasticity of cancer cells, SB-2ndLF primer [AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC which is often a transient and reversible process. This important CTA CAC GAC GCT CTT CCG ATC T (1- to 4-bp stagger) (8-bp index) AAC TTA feature of cancer cells may contribute to metastasis, cancer stem- AGT GTA TGT AAA CTT CCG ACT TCA ACT G-3′]. All oligos were purchased ness, and drug resistance; thus, our transposon screen provides from Integrated DNA Technologies with standard desalting. important global insights into the genes regulating this process. DNA fragments of 300–400 bp from amplicon libraries were excised from In summary, our unique cell-based SB transposon screen pro- the agarose gel and quantified using a Qubit fluorometer (Thermo Fisher vides, to our knowledge, the first high-throughput approach to Scientific), an Agilent 2100 bioanalyzer, and real-time qPCR. Excised libraries identifying and validating genes driving EMT in human HCC. were then pooled, loaded onto two flow cell lanes, and sequenced using an CCGs identified in this screen provide an important new resource Illumina Hi Seq2500 sequencer at Eurofins Genomics. Sequencing reads were for further studying the genes and cellular processes driving EMT converted to FASTQ format and demultiplexed using bcl2fastq-1.8.4 (Illu- in human HCC, as well as new potential therapeutic targets. mina) at Eurofins Genomics. To reduce bleed-through across samples in the same flow cell, a custom Materials and Methods dual-indexing routine was incorporated. This checked that a dual index se- quence near the beginning of a read matched the barcode associated with Mice. Rosa26-loxP-STOP-loxP-SB11 transposase knock-in mice [Gt(ROSA) the read, and then stripped away the index and the preceding sequence. 26Sortm2(sb11)Njen] (62); T2/Onc2 (6070) transposon transgenic mice [TgTn After these preprocessing steps, reads to the mouse genome were mapped (sb-T2/Onc2)6070Njen] (62), which have 214 copies of the T2/Onc2 trans- using a modified form of our SBCapSeq pipeline. The likelihood of local poson inserted into a single site on 4; B6.Cg-Tg(Alb-cre)21Mgn/J hopping of the SB transposon is increased on the chromosome where the transgenic mice (Jackson Laboratory strain 003574); and C.129S4-Ptentm1Hwu/J transposon concatamer is located (62); thus, all insertions on chromosome 4, mice (Jackson Laboratory strain 004597) were used in this study. Mice were the site of the T2/Onc2 donor concatamer, were removed from the dataset housed in a specific pathogen-free facility with a 12-h-light/12-h-dark cycle. The before subsequent analysis, unless indicated otherwise. Sfi1 is listed as a single- Institutional Animal Care and Use Committee of Houston Methodist Research copy gene in the reference mouse genome; however, it is estimated that the Institute approved all mouse procedures. mouse genome has 20–30 copies of Sfi1 (63). Insertions in multiple different Sfi1 Mice heterozygous for the Alb-Cre transgene were crossed to mice loci are erroneously annotated to a single Sfi1 gene located on chromosome 11; homozygous for Rosa26-lsl-SB11 and T2/Onc2 (6070) to generate thus, we removed Sfi1 from our lists of CIS genes, because this is a known arti- Alb-Cre/+;T2Onc2/+;Rosa26-lsl-SB11/+ embryos used for hepatoblast isola- fact. We reported the resulting TA sites and their read depths in BED format. A tion. For qPCR analysis, well-differentiated HCCs were collected from total of 871,231 and 6,541,525 mapped reads, corresponding to 50,215 and flox/flox Alb-Cre/+;T2Onc2/+;Rosa26-lsl-SB11/+;Pten mice and confirmed 118,530 nonredundant insertion sites, were identified from seven IHBC/SB lines histologically by a pathologist. and 52 IHBC/SB-derived tumors, respectively (Datasets S1 and S2). CCGs were identified using gCIS (30), which looks for a higher density of Isolation and Immortalization of Embryonic Hepatoblasts. Immortalized transposon insertions within the coding regions of all RefSeq genes than hepatoblasts were obtained using the method reported by Strick-Marchand H would be predicted by chance. gCIS analysis was performed using insertions et al. (26). In brief, Alb-Cre/+;T2Onc2/+;Rosa26-lsl-SB11/+ embryos were dissected with >10 sequence read counts. Genes with a P value <0.05, as calculated by at E13.5, and dissociated cells from embryonic livers were plated into type I the χ2 test followed by Bonferroni correction, were defined as CCGs. For collagen-treated dishes. Cells were cultured in vitro with DMEM/F12 medium trunk driver analysis, gCIS analysis was performed using insertions with >100 containing 10% FCS, penicillin-streptomycin, 50 ng/mL EGF (Gibco), 30 ng/mL sequence read counts. Genes with a P value < 0.05, as calculated by the χ2 IGF-II (Peprotech), and 10 μg/mL insulin (Thermo Fisher Scientific). Spontane- test followed by Bonferroni correction, and detected in more than six tu- ously immortalized hepatoblast colonies were inoculated and further cultured mors were defined as candidate trunk drivers. For pathway analysis, gCIS to obtain immortalized IHBC/SB lines. Soon after the immortalized lines were analysis was performed using insertions with >10 sequence read counts, and established, they were cryopreserved for transplantation. Transposons in these genes with a P value < 0.05 calculated by the χ2 test followed by false dis- lines are continuously transposing during in vitro culture as well as after covery rate correction were used. For all other information on Materials and transplantation, because of constitutive activity of the SB11 transposase. Methods, please see SI Materials and Methods. A list of siRNA oligonucleo- Embryos from C57BL6/J mice were used to establish IHBC/WT lines as a control. tides and TaqMan probes is also provided in Table S3.

E3392 | www.pnas.org/cgi/doi/10.1073/pnas.1606876113 Kodama et al. Downloaded by guest on September 23, 2021 ACKNOWLEDGMENTS. We thank E. Freiter and H. Lee for monitoring mice (CPRIT) and Public Health Service Grant DK56338, which funds the Texas PNAS PLUS and providing technical assistance with the animals. This research was Medical Center Digestive Diseases Center. N.G.C. and N.A.J. are CPRIT supported in part by the Cancer Prevention Research Institute of Texas Scholars in Cancer Research.

1. Forner A, Llovet JM, Bruix J (2012) Hepatocellular carcinoma. Lancet 379(9822): 34. Risolino M, et al. (2014) Transcription factor PREP1 induces EMT and metastasis by 1245–1255. controlling the TGF-β-SMAD3 pathway in non-small cell lung adenocarcinoma. Proc 2. Mittal S, El-Serag HB (2013) Epidemiology of hepatocellular carcinoma: Consider the Natl Acad Sci USA 111(36):E3775–E3784. population. J Clin Gastroenterol 47(Suppl):S2–S6. 35. Arima Y, et al. (2010) Decreased expression of neurofibromin contributes to epithe- 3. National Comprehensive Cancer Network (2016) NCCN Clinical Practice Guidelines in lial-mesenchymal transition in neurofibromatosis type 1. Exp Dermatol 19(8): Oncology: Hepatobiliary Cancers (National Comprehensive Cancer Network, Fort e136–e141. Washington, PA). 36. Choi HJ, et al. (2015) UTX inhibits EMT-induced breast CSC properties by epigenetic 4. Llovet JM, et al.; SHARP Investigators Study Group (2008) Sorafenib in advanced he- repression of EMT genes in cooperation with LSD1 and HDAC1. EMBO Rep 16(10): patocellular carcinoma. N Engl J Med 359(4):378–390. 1288–1298. 5. Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial-mesenchymal 37. van den Beucken T, et al. (2014) Hypoxia promotes stem cell phenotypes and poor transition. Nat Rev Mol Cell Biol 15(3):178–196. prognosis through epigenetic regulation of DICER. Nat Commun 5:5203–5215. 6. De Craene B, Berx G (2013) Regulatory networks defining EMT during cancer initia- 38. Chandramouli A, Simundza J, Pinderhughes A, Cowin P (2011) Choreographing me- tion and progression. Nat Rev Cancer 13(2):97–110. tastasis to the tune of LTBP. J Mammary Gland Biol Neoplasia 16(2):67–80. 7. Kalluri R, Weinberg RA (2009) The basics of epithelial-mesenchymal transition. J Clin 39. Liang CC, Park AY, Guan JL (2007) In vitro scratch assay: A convenient and inexpensive Invest 119(6):1420–1428. – 8. Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions method for analysis of cell migration in vitro. Nat Protoc 2(2):329 333. in development and disease. Cell 139(5):871–890. 40. Mazzone M, Comoglio PM (2006) The Met pathway: Master switch and drug target in – 9. Tsai JH, Donaher JL, Murphy DA, Chau S, Yang J (2012) Spatiotemporal regulation of cancer progression. FASEB J 20(10):1611 1621. epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. 41. Cecchi F, Rabe DC, Bottaro DP (2012) Targeting the HGF/Met signaling pathway in – Cancer Cell 22(6):725–736. cancer therapy. Expert Opin Ther Targets 16(6):553 572. 10. Mani V, et al. (2008) Serial in vivo positive contrast MRI of iron oxide-labeled em- 42. Giordano S, Columbano A (2014) Met as a therapeutic target in HCC: Facts and hopes. bryonic stem cell-derived cardiac precursor cells in a mouse model of myocardial in- J Hepatol 60(2):442–452. farction. Magn Reson Med 60(1):73–81. 43. Ueki T, Fujimoto J, Suzuki T, Yamamoto H, Okamoto E (1997) Expression of hepato- 11. Sampieri K, Fodde R (2012) Cancer stem cells and metastasis. Semin Cancer Biol 22(3): cyte growth factor and its receptor c-met proto-oncogene in hepatocellular carci- 187–193. noma. Hepatology 25(4):862–866. 12. Fischer KR, et al. (2015) Epithelial-to-mesenchymal transition is not required for lung 44. Qi XS, Guo XZ, Han GH, Li HY, Chen J (2015) MET inhibitors for treatment of advanced metastasis but contributes to chemoresistance. Nature 527(7579):472–476. hepatocellular carcinoma: A review. World J Gastroenterol 21(18):5445–5453. 13. Zheng X, et al. (2015) Epithelial-to-mesenchymal transition is dispensable for metas- 45. Inoue S, et al. (2013) Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by tasis but induces chemoresistance in pancreatic cancer. Nature 527(7579):525–530. preventing c-Myc/Miz1-mediated down-regulation of p21 and p15. Genes Dev 27(10): 14. Yang MH, et al. (2009) Comprehensive analysis of the independent effect of twist 1101–1114. and snail in promoting metastasis of hepatocellular carcinoma. Hepatology 50(5): 46. Gruber S, et al. (2013) Obesity promotes liver carcinogenesis via Mcl-1 stabilization – 1464 1474. independent of IL-6Rα signaling. Cell Reports 4(4):669–680. 15. Jou J, Diehl AM (2010) Epithelial-mesenchymal transitions and hepatocarcinogenesis. 47. Yumimoto K, et al. (2015) F-box protein FBXW7 inhibits cancer metastasis in a non– – J Clin Invest 120(4):1031 1034. cell-autonomous manner. J Clin Invest 125(2):621–635. 16. Xia L, et al. (2013) Overexpression of promotes tumor metastasis and 48. Yu J, et al. (2014) FBW7 increases chemosensitivity in hepatocellular carcinoma cells indicates poor prognosis in hepatocellular carcinoma. Hepatology 57(2):610–624. through suppression of epithelial-mesenchymal transition. Hepatobiliary Pancreat Dis 17. Ding W, et al. (2010) Epithelial-to-mesenchymal transition of murine liver tumor cells Int 13(2):184–191. promotes invasion. Hepatology 52(3):945–953.

49. Sun T, et al. (2011) Activation of multiple proto-oncogenic tyrosine kinases in breast GENETICS 18. Zhai B, Sun XY (2013) Mechanisms of resistance to sorafenib and the corresponding cancer via loss of the PTPN12 phosphatase. Cell 144(5):703–718. strategies in hepatocellular carcinoma. World J Hepatol 5(7):345–352. 50. Rhee I, Zhong MC, Reizis B, Cheong C, Veillette A (2014) Control of dendritic cell 19. Chow AK, et al. (2013) The enhanced metastatic potential of hepatocellular carci- noma (HCC) cells with sorafenib resistance. PLoS One 8(11):e78675. migration, T cell-dependent immunity, and autoimmunity by protein tyrosine phos- – 20. Chen X, et al. (2011) Epithelial mesenchymal transition and hedgehog signaling ac- phatase PTPN12 expressed in dendritic cells. Mol Cell Biol 34(5):888 899. tivation are associated with chemoresistance and invasion of hepatoma subpopula- 51. Souza CM, et al. (2012) The phosphatase PTP-PEST/PTPN12 regulates endothelial cell tions. J Hepatol 55(4):838–845. migration and adhesion, but not permeability, and controls vascular development 21. Nagai T, et al. (2011) Sorafenib inhibits the hepatocyte growth factor-mediated ep- and embryonic viability. J Biol Chem 287(51):43180–43190. ithelial mesenchymal transition in hepatocellular carcinoma. Mol Cancer Ther 10(1): 52. Rhee I, Davidson D, Souza CM, Vacher J, Veillette A (2013) Macrophage fusion is 169–177. controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12. Mol 22. Bard-Chapeau EA, et al. (2014) Transposon mutagenesis identifies genes driving he- Cell Biol 33(12):2458–2469. patocellular carcinoma in a chronic hepatitis B mouse model. Nat Genet 46(1):24–32. 53. Sahu SN, Nunez S, Bai G, Gupta A (2007) Interaction of Pyk2 and PTP-PEST with 23. Mann MB, et al. (2015) Transposon mutagenesis identifies genetic drivers of Braf leupaxin in prostate cancer cells. Am J Physiol Cell Physiol 292(6):C2288–C2296. (V600E) melanoma. Nat Genet 47(5):486–495. 54. Villa-Moruzzi E (2013) PTPN12 controls PTEN and the AKT signalling to FAK and HER2 24. Takeda H, et al. (2015) Transposon mutagenesis identifies genes and evolutionary in migrating ovarian cancer cells. Mol Cell Biochem 375(1-2):151–157. forces driving gastrointestinal tract tumor progression. Nat Genet 47(2):142–150. 55. Li J, et al. (2015) Loss of PTPN12 stimulates progression of ErbB2-dependent breast 25. Koso H, et al. (2012) Transposon mutagenesis identifies genes that transform neural cancer by enhancing cell survival, migration, and epithelial-to-mesenchymal transi- – stem cells into glioma-initiating cells. Proc Natl Acad Sci USA 109(44):E2998 E3007. tion. Mol Cell Biol 35(23):4069–4082. 26. Strick-Marchand H, Weiss MC (2002) Inducible differentiation and morphogenesis of 56. Luo RZ, et al. (2014) Decreased expression of PTPN12 correlates with tumor recurrence bipotential liver cell lines from wild-type mouse embryos. Hepatology 36(4 Pt 1): and poor survival of patients with hepatocellular carcinoma. PLoS One 9(1):e85592. – 794 804. 57. Sirois J, et al. (2006) Essential function of PTP-PEST during mouse embryonic vascu- 27. Zhang W, et al. (2012) Efficient generation of functional hepatocyte-like cells from larization, mesenchyme formation, neurogenesis and early liver development. Mech human fetal hepatic progenitor cells in vitro. J Cell Physiol 227(5):2051–2058. Dev 123(12):869–880. 28. van Zijl F, et al. (2009) Epithelial-mesenchymal transition in hepatocellular carcinoma. 58. Thieme S, et al. (2013) The histone demethylase UTX regulates stem cell migration Future Oncol 5(8):1169–1179. and hematopoiesis. Blood 121(13):2462–2473. 29. March HN, et al. (2011) Insertional mutagenesis identifies multiple networks of co- 59. Suvà ML, Riggi N, Bernstein BE (2013) Epigenetic reprogramming in cancer. Science operating genes driving intestinal tumorigenesis. Nat Genet 43(12):1202–1209. – 30. Brett BT, et al. (2011) Novel molecular and computational methods improve the ac- 339(6127):1567 1570. curacy of insertion site analysis in Sleeping Beauty-induced tumors. PLoS One 6(9): 60. Kim JH, et al. (2014) UTX and MLL4 coordinately regulate transcriptional programs for – e24668. cell proliferation and invasiveness in breast cancer cells. Cancer Res 74(6):1705 1717. 31. Futreal PA, et al. (2004) A census of human cancer genes. Nat Rev Cancer 4(3): 61. Zhang P, et al. (2014) ATM-mediated stabilization of ZEB1 promotes DNA damage – 177–183. response and radioresistance through CHK1. Nat Cell Biol 16(9):864 875. 32. Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G (2012) Targeting MET in 62. Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA (2005) Mammalian cancer: Rationale and progress. Nat Rev Cancer 12(2):89–103. mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. 33. Ngan E, Northey JJ, Brown CM, Ursini-Siegel J, Siegel PM (2013) A complex containing Nature 436(7048):221–226. LPP and α-actinin mediates TGFβ-induced migration and invasion of ErbB2-expressing 63. Quinlan AR, et al. (2010) Genome-wide mapping and assembly of structural variant breast cancer cells. J Cell Sci 126(Pt 9):1981–1991. breakpoints in the mouse genome. Genome Res 20(5):623–635.

Kodama et al. PNAS | Published online May 31, 2016 | E3393 Downloaded by guest on September 23, 2021