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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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)

Hepatocellular carcinoma (HCC) is the sixth most common cancer and third leading cause of cancer-related death.1 It is an aggressive tumor with a dismal prognosis given that less than 30% of patients will be eligible for potential curative treatment at the time of diagnosis.2 HCC is prevalent worldwide, but differences in disease incidence rates reflect regional diversity mostly related to geographic distribution of viral hepatitis.2 Sex also influences risk, with males showing a higher increase in prevalence over females, with limited existing preliminary molecular data that explain this sex discrepancy.3

In order to screen for cancer-associated genes in different types of cancer using Sleeping Beauty (SB) transposons, conditional SB transposition systems have been successfully used to generate various solid tumors.4-6 As described, we used a hepatocyte-specific albumin (Alb) promoter driving Cre recombinase transgene to activate transposase expression and initiate insertional mutagenesis specifically in the liver.5 Because mutations in tumor protein p53 are the most frequently described mutations in HCC, a conditional dominant negative transformation-related protein 53 (Trp53) transgene was also included in the original screen.5, 7 Triple transgenic (transposition in a wild-type genetic background) and quadruple-transgenic (transposition in a Trp53-deficient genetic background) mice from both sexes were generated and aged for liver tumorigenesis.

In the present study, liver cancer-associated genes were identified from liver nodules isolated from different sexes and genetic backgrounds using a conditional SB transposon forward insertional mutagenesis screen combined with the Roche 454 FLX high-throughput sequencing platform. Common insertion sites (CISs), representing regions of the genome with increased frequency of transposon integration than would be expected by random chance, indicate cancer-associated genes that confer selective growth advantages to a cell. A revised and fully automated method for calculating common insertion sites based on Poisson distribution was used in this study.8 A higher incidence of HCC was observed in male animals, which corresponded with increased insertions in the Egfr locus in SB-induced HCCs from male mice (10/10, 100%) but was infrequent in female animals (2/9, 22%). Interestingly, when comparing the homologues of our CIS gene lists with 235 human HCC patients to determine sex bias differences, the most striking results were for EGFR with significant differences in copy number changes, representing the Poly7 molecular subclass of HCC that has been described.9 Poly7 was more frequent in males than females, and patients with EGFR copy number gains also had significantly higher EGFR messenger RNA (mRNA) levels compared with patients without gains. It is our hypothesis that EGFR is associated and/or involved with sex bias occurrences in HCC. Using SB in a reverse genetic manner, the sex bias of EGFR was confirmed using our in vivo mouse model as described.5, 10, 11 Furthermore, genetic information from human HCC data with in vivo validation confirms the role of EGFR in sex disparities involved with human HCC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Generation of Transgenic Animals and Polymerase Chain Reaction Genotyping.

Generation of transgenic animals and polymerase chain reaction (PCR) genotyping for the identification of the various genotypes was performed as described.5

Liver Tumor Analysis.

Isolation of liver tumors for DNA and RNA extraction, processing for histological analysis were performed as described.5, 10

Pyrosequencing.

The protocol for amplicon sequencing using the GS20 Flex pyrosequencing machine was followed as described.5

Egfr PCR Genotyping.

PCR genotyping was used to confirm the presence of the T2/Onc transposon insertion in the Egfr gene as described.5

Details regarding selection criteria for CISs, reverse-transcription polymerase chain reaction (RT-PCR), gene expression and copy number changes in human HCC samples, vectors used for hydrodynamic injection, and oPOSSUM analyses are described in the Supporting Information.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Hepatocyte-Specific Transposition and Tumorigenesis.

As shown previously, liver nodules were first detected at around 160 days in both triple-transgenic male (transposition in a wild-type genetic background) and quadruple-transgenic (transposition in a Trp53-deficient genetic background) animals.5 However, insertion data analyzed in the original study were mainly obtained from quadruple transgenic mice.5 In the present study, 61 liver nodules were isolated from triple-transgenic male mice (n = 5; range, 160-528 days), and genomic DNA was isolated for genetic interrogation to identify SB transposon insertions (Fig. 1A and Supporting Table 1). Triple transgenic male mice (n = 3) also developed HCC between 330 and 528 days (Supporting Table 1). Genetic insertion data of 68 liver nodules isolated from quadruple-transgenic male mice (n = 5) described previously5 were reanalyzed separately from the wild-type male liver nodules (Fig. 1A, Supporting Table 1). Quadruple transgenic male mice (n = 4) also developed HCC between 156 and 432 days (Supporting Table 1). Interestingly, the majority of liver nodules isolated from male mice were histopathologically classified as HCCs (Supporting Table 1).

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Figure 1. Sex disparities seen in mouse model experimental animals with mutagenic SB-induced liver tumors. (A) HCC nodules in a 528-day-old triple-transgenic male mouse (top left) and 432-day-old quadruple-transgenic male mouse (top right). Adenoma/HCC nodules in a 588-day-old triple-transgenic female mouse (bottom left) and 588-day-old quadruple-transgenic female mouse (bottom right) are also shown. Scale bars = 0.5 cm. (B) A significantly higher number of liver nodules were isolated from male mice (n = 11) compared with female mice (n = 9). The P value was determined using an unpaired t test. (C) Top 17 candidate CIS genes from the combined analyses (P < 0.001) in Supporting Table 7, shown as a heat map indicating putative sex bias genes responsible for HCC. Insertion within a CIS for a given tumor is indicated by the presence of a red bar. The sex and genotype are described in the bar above the heat map. Genes found in the neighborhood of the CISs are listed at the right of the heat map. To determine whether sex bias occurrences of these CIS exist, a Fisher exact test was performed to assess the relationship between insertion sites within CIS and phenotypes. The results are shown as a heat map (right), where increase in intensity indicates higher statistical significance. P values for associations are provided in Supporting Table 7. (D) Diagram of transposon insertions into the Egfr gene. A schematic representation of the mutagenic transposon (T2/Onc) is shown. Red triangles indicate inverted repeat/direct repeat transposon flanking sequences (MSCV, long terminal repeat of the murine stem cell virus; polyA, polyadenalytion signal; SA, splice acceptor; SD, splice donor). Top: White arrowheads indicate sense-orientated insertion of the T2/Onc relative to the Egfr gene; black arrowheads indicate anti-sense–orientated insertion of the T2/Onc relative to the Egfr gene; gray arrows indicate endogenous and vector primers used for PCR genotyping. Bottom: Numbers in parentheses indicate the frequency of transposon insertions at each particular site from different liver tumor nodules of experimental animals; black and red arrowheads indicate transposon insertions in either male or female animals, respectively.

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Triple- and quadruple-transgenic female animals sacrificed between 178 and 342 days and 178 and 344 days, respectively, did not display any macroscopic liver lesions.5 In the current study, female triple- and quadruple-transgenic animals aged further presented liver nodules that were histopathologically classified as dysplastic lesions, adenomas, or HCCs (Supporting Table 1). The low frequency and late latency of liver nodules in female animals mirrors the strong sex bias in tumor incidence seen in human HCC patients. In the present study, 20 liver nodules were isolated from triple-transgenic female mice (n = 6; range, 512-621 days) (Fig. 1A, Supporting Table 1) and 14 liver nodules were isolated from quadruple-transgenic female experimental mice (n = 3; range, 432-624 days) (Fig. 1A, Supporting Table 1) for genetic interrogation to identify SB transposon insertions. There was a statistical significantly higher number of liver nodules isolated from male experimental mice (P = 0.0044), indicating sex bias occurrence of HCC was also observed in our mouse model (Fig. 1B, Supporting Table 1).

Sequencing for CISs From Tumor Samples.

From triple-transgenic male mice, 4,111 nonredundant insertions were subsequently cloned from 61 liver nodules identified as having 25 CIS loci harboring 27 annotated genes and one ENSMUST gene (Table 1). From quadruple-transgenic male mice, 9,323 nonredundant insertions cloned from 68 liver nodules identified 18 CIS loci harboring 18 annotated genes and two GENSCAN predicted genes (Supporting Table 2). When all male samples were combined, 47 CIS loci were identified harboring 70 annotated genes and one GENSCAN gene (Supporting Table 3). From triple-transgenic female mice, 1,819 nonredundant insertions cloned from 20 liver nodules identified 17 CIS loci harboring 22 annotated and 1 GENSCAN genes (Supporting Table 4). Subsequently, from quadruple-transgenic female mice, 1,750 nonredundant insertions cloned from 14 liver nodules identified 12 CIS loci harboring 16 annotated genes (Supporting Table 5). When all female samples were combined, 42 CIS loci were identified harboring 50 annotated genes, one RIKEN gene, and one GENSCAN predicted gene (Supporting Table 6). In order to obtain an overall genetic landscape profiling, nonredundant insertions from both sexes and genetic backgrounds were pooled for analysis. This identified 83 CIS loci harboring 114 annotated genes and two RIKEN genes that can provide useful genetic mechanisms associated with the strong sex bias of this deadly disease (Supporting Table 7). Out of the 83 CIS loci identified in this screen, 11 CISs were previously identified in the original screen using predominantly male quadruple-transgenic mice5 (Supporting Table 7).

Table 1. Common Insertion Sites for HCC-Associated Genes in Wild-Type Males
GeneChromosomePosition of Transposon InsertionsP ValueFrequency*n
StartEndRange (bp)
  • Positions were based on the Ensembl NCBI m37 April 2007 mouse assembly.

  • *

    Number of liver nodules from which the CIS was determined.

  • Number of mice from which the nodules were isolated.

Egfr11168093001683330024,0003.91E−06285
Crebbp164126000420400078,0002.89E−0552
Ppp1r12a1010764740010767140024,0000.00047443143
Kif1b414860400014862800024,0000.00047443141
BC0313539748207007484470024,0000.00047443131
Rbm10X202013002022530024,0000.00047443152
Rabgap1l1162423600162709600286,0000.00344250574
Ctnna118353204003534440024,0000.0460413932
Ppp2ca11519150005193900024,0000.0460413932
Lsamp16400050004002900024,0000.0460413932
ENSMUST0000007137417170877001711170024,0000.0460413932
Usp3411233460002337000024,0000.0460413932
Gm976610530775005310150024,0000.0460413933
Nbeal11602618006028580024,0000.0460413932
Tsc22d23582421005826610024,0000.0460413932
Rere414988390014990790024,0000.0460413931
Palld8640268006405080024,0000.0460413932
Arih19592451005926910024,0000.0460413932
Arih2910852630010855030024,0000.0460413942
Pard3812975760012983560078,0000.04824249243
Btbd71210406900010414700078,0000.04824249243
Smap11238646002394260078,0000.04824249254
Dpyd3118304800118590800286,0000.04906067963
Phf21a, Pex1629205780092224800167,0000.04990628152
Atn1, Usp5, Lpcat36124624400124791400167,0000.04990628152

Combined Insertion Profiles.

Transposon insertion data from both sexes and all genotypes (16,977 nonredundant) were combined in order to obtain an overall relationship between sex/genotype and insertion sites (Supporting Table 7). Relationships between the insertions and the tumor libraries for the most statistically significant CISs (P < 0.001) are shown (Fig. 1C). The insertion profiles for all 163 samples were sorted by sex and genotype (Fig. 1C, left heat map). Fisher exact test was performed on all insertion sites to determine whether sex bias occurrences of these CISs exist. The result of this analysis is shown as a heat map (Fig. 1C, right heat map). From the statistical analytical heat map, there are clusters of genes that are clearly enriched or absent in liver nodules from both sexes and genotypes. These clusters indicate the putative sex bias genes responsible for HCC.

Frequent Transposon Insertions in Egfr for Male Liver Nodules.

As seen in quadruple-transgenic males,5 transposon insertions in the Egfr gene also represented the most frequently hit gene in triple-transgenic male animals. Although Egfr insertions were cloned from all triple-transgenic male animals (n = 5), these insertions were only detected in 46% of liver nodules (n = 61) (Table 1). These transposon insertions were most frequently detected in intron 24 of Egfr, and the majority of insertions were in the antisense orientation, suggesting they are Egfr-truncating insertions. Most strikingly, Egfr insertions in combined male mice were detected in all mice (n = 10) and at a frequency of 67% total liver nodules (n = 129) (Supporting Table 3). In contrast, Egfr insertions were detected in 33% of quadruple-transgenic female mice (n = 3) and 17% of triple-transgenic female mice (n = 6). Egfr insertions in combined female mice were only detected in 22% of mice (n = 9) and at a frequency of 24% total liver nodules (n = 34) (Supporting Table 6). Therefore, Egfr alterations are more commonly found in male mice tumors, suggesting a contribution to the sex bias of HCC (Fig. 1D).

Sex Disparities in Human HCC.

Among the 91 human HCCs analyzed (training set), there were 70% (n = 64) males and 30% females (n = 27). As an initial step, we analyzed the distribution of males and females among our reported human HCC molecular subclasses.9 Interestingly, Poly7 subclass patients were all males (9/9) compared with 67% males (55/82) in other HCC subclasses (P = 0.02) (Fig. 2A and Table 2). EGFR mRNA levels were higher in tumors belonging to this Poly7 subclass of HCC (P < 0.001), which is characterized by gains in chromosome 7 (locus of EGFR in humans)9 (Fig. 2A, Supporting Data 4). According to our observations, polysomy of chromosome 7 occurred more frequently in men than in women (26/63 males versus 2/27 females; P = 0.001) (Fig. 2A, Table 2). Sex distribution was analyzed in a second independent dataset of 144 formalin-fixed paraffin-embedded HCC (validation set) including 107 males (74%) and 37 females (26%). In this set, Poly7 subclass also showed male sex bias (96% [23/24] of Poly7 subclass were males versus 70% [84/120] in the others subclasses; P < 0.01) (Fig. 2B, Table 2). Overall in the combined human dataset (training plus validation), we observed a male/female ratio of 33:1 in the Poly7 subclass, as opposed to a ratio of 2:1 in the non-Poly7 samples. Because DNA copy number data were not available for the validation set, FISH analysis using a probe specific for the centromeric region of human chromosome 7 probe was applied to 51 samples. FISH analysis revealed that patients belonging to the Poly7 subclass showed significant chromosome 7 polysomy (7/9 in the Poly7 subclass versus 7/42 in other subclasses; P < 0.001). Furthermore, in this subset it was observed that polysomy of chromosome 7 occurred more frequently in men than in women (13/35 men versus 1/16 women; P = 0.04) (Fig. 2B, Table 2). Finally, EGFR overexpression in Poly7 subclass samples was confirmed in the validation set (P < 0.001) (Fig. 2B, Supporting Data 5).

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Figure 2. Poly7 subclass accounts for sex disparities seen in human hepatocarcinogenesis. (A) Heat map showing previously identified Poly7 molecular subclass in the HCC training set (n = 91). Poly7 was enriched in patients of this subgroup (P = 0.02), and EGFR mRNA levels were higher in these tumors (P < 0.001) (Supporting Data 4). (B) Heat map showing samples belonging to Poly7 subclass within the validation set (n = 144). EGFR mRNA overexpression and polysomy of chromosome 7 were significantly enriched in patients of the Poly7 subclass (Supporting Data 5). (A and B) Patients belonging to Poly7 subclass are indicated in black; other classes are indicated in gray. Presence or absence of chromosome 7 polysomy is indicated as follows: present, black box; absent, gray box; missing, white box. EGFR gene expression levels are represented as indicated in the color scale.

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Table 2. Chromosome 7 Polysomy and Poly7 Molecular Subclass Sex Bias in Human HCC
 Training Set, nValidation Set, nCombined,* nP
MaleFemaleMaleFemaleMaleFemale
  • Abbreviations: Chr7 poly, polysomy of chromosome 7; FISH, gene copy number determined via fluorescence in situ hydridization using a probe specific for the centromeric region of human chromosome 7; SNP, gene copy number determined via single-nucleotide polymorphism array.

  • *

    Training plus validation sets.

  • Fisher exact test versus sex in the combined training plus validation dataset.

Entire cohort64271073717164 
Poly7 subclass90231321 
Non-Poly7 subclass5527843613963<0.001
Chr7 poly (SNP or FISH)262131393 
No Chr7 poly (SNP or FISH)372522155940<0.001

Validation of EGFR in Sex Disparities Involved With Liver Tumorigenesis.

To test whether EGFR could contribute to neoplastic growth in vivo specifically in a sex bias manner, a fumarylacetoacetate hydrolase (Fah)-deficient mouse model was used as described.10, 11 SB transposon-based expression vector for the truncated EGFR (pT2/PGK-Trunc. EGFR), full-length EGFR (pT2/PGK-EGFR), source of Fah (pT2/PGK-FAHIL) and a short hairpin RNA vector directed against the Trp53 gene (pT2/shp53) were coadministered to both sexes of Fah-deficient mice that express the SB11 transposase knocked into the Rosa26 locus (Fah/SB11) by tail vein hydrodynamic injection5, 12 (Fig. 3A, Supporting Fig. 1A). Upon withdrawal of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), the mice underwent liver repopulation, as evidenced by stable weight gain and increasing luciferase (Luc) expression (data not shown). At around 130 days post-hydrodynamic injection (PHI), Fah/SB11 mice of both sexes coadministered with either truncated or full-length EGFR, source of Fah and a short hairpin RNA vector directed against the Trp53 gene were sacrificed and observed for liver nodules. Interestingly, for truncated EGFR, pT2/PGK-FAHIL and pT2/shp53 coinjected mice, male Fah/SB11 mice (n = 9) had a significantly increased number of liver nodules compared with female mice (n = 4) (P = 0.0382) (data not shown). Striking differences in sex bias occurrences of HCC associated with EGFR can be seen in Fah/SB11 mice at 300 days PHI (Fig. 3B, Supporting Fig. 1B). Male Fah/SB11 mice (n = 4) coadministered with pT2/PGK-Trunc. EGFR and pT2/shp53 had significantly increased number of liver tumor nodules compared with female mice (n = 4) (P = 0.0086) (Fig. 3C). Interestingly, liver tumors from Fah/SB11 mice of both sexes were classified as HCCs according to the histopathological analysis (data not shown). Semiquantitative RT-PCR analyses for several markers indicated that both short hairpin against Trp53 (livers were green fluorescent protein–positive due to the presence of the reporter gene in the plasmid construct [data not shown]) and truncated EGFR vectors were being stably integrated into the mouse genome (Fig. 3D). Analysis of two known liver tumor markers, alpha-fetoprotein (Afp) (P = 0.0640) (Fig. 3D) and secreted phosphoprotein 1 (Spp1) (P = 0.0355) (Fig. 3E), also indicated higher expression levels in male mice compared with female mice. Interestingly, this male sex bias was also observed in Fah/SB11 animals coadministered with pT2/PGK-EGFR and pT2/shp53 (P = 0.0388) (Supporting Fig. 1C). Taken together, the genetic information from human HCC data with in vivo studies in Fah/SB11 mice validates the role of EGFR in sex disparities involved with human HCC.

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Figure 3. Validating the oncogenic sex bias potential of EGFR using the Fah-deficient/Rosa26-SB11 (Fah/SB11) mouse model. (A) Vectors used for hydrodynamic injections into the livers of Fah/SB11 mice. Truncated EGFR complementary DNA (exon 1 to exon 24) placed under the control of the phosphoglycerate kinase (PGK) promoter (pT2/PGK-Trunc. EGFR), Fah complementary DNA placed under the control of the PGK promoter fused with the IRES-luciferase (Luc) reporter gene (pT2/PGK-FAHIL) and short-hairpin directed against Trp53 (pT2/shp53), all flanked by SB inverted repeat/direct repeat recognition sequences essential for transposition (red triangles). (B) Representative images of whole livers taken from male (left) and female (right) Fah/SB11 mice 300 days PHI with pT2/PGK-Trunc. EGFR and pT2/shp53. Scale bars = 0.5 cm. (C) Significant increase in the number of liver tumor nodules found in male versus female Fah/SB11mice hydrodynamically injected with pT2/PGK-Trunc. EGFR and pT2/shp53 via the tail vein. Mice were sacrificed at 300 days PHI and liver tumor nodules were counted. P value was determined using an unpaired t test. (D) Semiquantitative RT-PCR analyses of various markers in livers taken from 130 days PHI male and female Fah/SB11 mice injected with transgenes described in (A). Actb, β-actin; Total RNA, total RNA loading showing intact 28S, 18S, and 5S ribosomal bands; WT, normal liver taken from a wild-type FVB/N mouse. (E) Arbitrary expression level of the liver tumor marker Afp relative to Actb was obtained using ImageJ software (National Institutes of Health) shows a nonsignificant trend toward higher expression in male mice compared with female mice. P value was determined using an unpaired t test. (F) Arbitrary expression level of the liver tumor marker Spp1 relative to Actb shows significantly higher expression in male mice compared with female mice. P value was determined using an unpaired t test.

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Sex Has no Effect on CTNNB1-Driven Liver Tumorigenesis.

As a control, a SB transposon-based expression vector for constitutively active catenin beta 1 (CTNNB1S33Y) transgene and pT2/shp53 were coadministered to both sexes of Fah/SB11 mice (Fig. 4A). Upon withdrawal of NTBC, the mice underwent liver repopulation, as evidenced by stable weight gain and increasing Luc expression (data not shown). At around 80 and 83days PHI, Fah/SB11 mice of both sexes coadministered with activated CTNNB1S33Y and a short hairpin RNA vector directed against the Trp53 gene were sacrificed and observed for liver nodules (Fig. 4B). Interestingly, both male (n = 8) and female (n = 9) Fah/SB11 mice had comparable numbers of macroscopic liver tumors (P = 0.4016) (Fig. 4C). Liver tumors from Fah/SB11 mice of both sexes coadministered with activated CTNNB1S33Y and short hairpin RNA vectors directed against the Trp53 gene were classified histologically as HCCs (data not shown). Semiquantitative RT-PCR analyses for several markers indicated that both short hairpin against Trp53 and CTNNB1S33Y vectors were being stably integrated into the mouse genome (Fig. 4D). Analysis of liver tumor markers Afp (P = 0.3669) (Fig. 4E) and Spp1 (P = 0.6509) (Fig. 4F) indicated comparable expression levels in both male and female mice. Taken together, overexpression of constitutively active CTNNB1 in the context of Trp53 knockdown did not induce strong sex bias occurrences of HCC.

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Figure 4. Validating the non–sex bias oncogenic potential of CTNNB1 in an Fah/SB11 mouse model. (A) Vectors carrying the constitutively active CTNNB1S33Y gene (pT2/GD-IRES-Gfp-CTNNB1) and pT2/shp53 delivered into the livers of Fah/SB11 mice via hydrodynamic injection. (B) Representative images of whole livers taken from male (left) and female (right) Fah/SB11 mice injected at 83 days PHI with pT2/GD-IRES-Gfp-CTNNB1 and pT2/shp53. Scale bars = 0.5 cm. (C) Comparable number of liver tumor nodules found in both male and female Fah/SB11mice hydrodynamically injected with the vectors described in (A) via the tail vein. Mice were sacrificed at 80 and 83 days PHI, and liver tumor nodules were counted. P value was determined using an unpaired t test. (D) Semiquantitative RT-PCR analyses of various markers in livers taken from both male and female Fah/SB11 mice injected with transgenes described in (A). Actb, β-actin; CTNNB1-S33Y, constitutively active CTNNB1S33Y; Total RNA, total RNA loading showing intact 28S, 18S, and 5S ribosomal bands; WT, normal liver taken from a wild-type FVB/N mouse. (E) Arbitrary expression level of the liver tumor marker Afp relative to Actb showed similar expression levels in both male and female mice. P value was determined using an unpaired t test. (F) Arbitrary expression level of the liver tumor marker Spp1 relative to Actb showed similar expression levels in both male and female mice. P value was determined using an unpaired t test.

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Other Sex Bias Genes Identified in the SB Mutagenesis Screen.

Male sex bias genes identified in our forward genetic screen included the following genes implicated in liver tumorigenesis from Trp53-deficient tumors: solute carrier family 25 member 13 (Slc25a13), nuclear factor I/B (Nfib), partitioning-defective protein 3 (Pard3), and zinc finger and BTB domain containing 20 (Zbtb20) (Supporting Table 3, Supporting Fig. 2A). Transposon insertion profiles in Slc25a13, Nfib, and Pard3 indicate loss-of-function activities, whereas gain-of-function activity is predicted for Zbtb20 (Supporting Fig. 2A). Semiquantitative RT-PCR for Nfib and Pard3 expression in tumors with transposon insertions showed reduced transcript levels, consistent with the expected consequence of the transposon insertion pattern (Supporting Fig. 2B,C). Transposon insertions in Zbtb20 were enriched upstream of the transcription start site, resulting in a predicted gain-of-function activity.

Female sex bias genes identified from combined genotypes include NHL repeat containing 2 (Nhlrc2), Rho GTPase activating protein 42 (Arhgap42), and adenosine kinase (Adk). Transposon insertion patterns in Nhlrc2 and Arhgap42 indicate gain-of-function activities, whereas loss-of-function activity was predicted for Adk (Supporting Table 6, Supporting Fig. 3).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In the current study, we analyzed liver nodules isolated from both sexes and different genetic backgrounds to elucidate the sex bias of HCC. Egfr insertions were enriched in male mice but were infrequent in female mice. Human data also confirmed that polysomy of chromosome 7 (locus of EGFR gene) occurred more frequently in males than females and was associated with EGFR overexpression. This sex bias occurrence of HCC associated with EGFR was confirmed in experimental animals using the SB transposon system in a reverse genetic manner.

Interestingly, transposon insertions in the Egfr gene that truncate the carboxy-terminus were common in SB-induced liver tumors from male mice but infrequent in female animals (Fig. 1D). Activation of EGFR triggers signaling processes that promote cell proliferation, migration, adhesion, angiogenesis, and inhibition of apoptosis.13 Carboxy-terminal domain deletions of EGFR (966-1006) result in higher autokinase activity and transforming ability.14 These naturally occurring EGFR deletion mutants display tumorigenic properties, probably resulting in constitutively active forms due to the destabilization of the inactive EGFR monomeric complex.14-17 EGFR is overexpressed in 15%-40% of HCCs, although there are data suggesting activation of EGF signaling is actually higher.18, 19 Extra copies of the EGFR gene were seen in 45% of HCC tumors, but increased expression did not correlate with the increase in EGFR copy number.20 Interestingly, analyses of human HCC indicate that males are highly enriched in a molecular subclass (Poly7) that show chromosome 7 polysomy and overexpression of EGFR.9 Mechanisms that could explain the differential EGFR tumorigenic activity in males and females have been suggested.21 Genetic information from human HCC data with in vivo studies in Fah/SB11 mice validates the role of EGFR in sex disparities involved with human HCC. Interestingly, overexpression of full-length EGFR in Fah/SB11 mice also resulted in male sex bias occurrence of HCC (Supporting Fig. 1). The CTNNB1 molecular subclass of human HCC has been shown to occur at similar frequency in both male and female patients.22, 23 The non–sex bias occurrence of HCC associated with the CTNNB1 molecular subclass of HCC was confirmed using our Fah/SB11 mouse model (Fig. 4). Li et al.24 demonstrated recently that Foxa1 and Foxa2 are essential for sexual dimorphism in liver cancer. Using hepatocyte-specific gene ablation, they were able to demonstrate that sexually dimorphic HCC was dependent on these Foxa genes that modulate the responses of sex hormones in HCC tumorigenesis. Using the web-based system oPOSSUM (version 3.0)25, 26 for the detection of overrepresented transcription factor binding sites in the promoters of our CIS genes (Supporting Table 7), 39 transcription factors (including Foxa1 and Foxa2) were identified (using selection criteria of z score ≥10 and Fisher score ≥7) in 116 genes (Supporting Data 1). Conserved Foxa1 and Foxa2 binding sites were identified near the transcription start site of the Egfr gene (Supporting Data 2 and 3 for Foxa1 and Foxa2, respectively). From this in silico analysis, the results suggest that our screen is indeed identifying genes and perhaps other transcription factors that may be involved in the sex bias occurrences of HCC.

In addition to EGFR, we have identified many other candidate genes involved with sex bias occurrences for this deadly disease. As shown previously, Zbtb20 and Slc25a13 or citrin remain strong male sex bias cancer candidate genes.5 Interestingly, both Foxa1 and Foxa2 transcription binding sites were found to be overrepresented in Zbtb20 and Slc25a13 by oPOSSUM analysis (Supporting Data 2 and 3). Further examination of the transposon insertion profiles in Zbtb20 and Slc25a13 suggest a gain-of-function and loss-of-function activity, respectively (Supporting Fig. 2A). Zbtb20 is a key regulator of Afp and is developmentally upregulated in postnatal liver.27 In addition, ZBTB20 expression is increased in human HCC and associated with poor prognosis.28 SLC25A13 gene mutations result in type II citrullinemia, and mutations have been identified in male patients with nonviral HCC.29 Nfib has been shown to be a direct functional target of miR-372/373, and its knockdown promotes hepatitis B viral expression.30 Pard3 is an adapter protein involved in asymmetrical cell division and cell polarization and plays a role in the formation of epithelial tight junctions. Our preliminary validation seems to indicate that both Nfib and Pard3 are novel candidate tumor suppressor genes for liver tumorigenesis (Supporting Fig. 2A-C). Candidate genes identified in our screen to be female sex bias include Nhlrc2, Arhgap42, and Adk (Supporting Fig. 3). Interestingly, both Foxa1 and Foxa2 transcription binding sites were found to be overrepresented in all three genes via oPOSSUM analysis (Supporting Data 2 and 3). Transposon insertion pattern into Nhlrc2 and Arhgap42 predict gain-of-function activity, suggesting that these two genes are putative oncogenes. In contrast, transposon insertion pattern for Adk predicts a loss-of-function activity and a putative tumor suppressor gene. Interestingly, disruption of Adk results in neonatal hepatic steatosis31 and perturbs the methionine cycle, resulting in hypermethioninemia, encephalopathy, and abnormal liver function.32 Taken together, we are only in the beginning of unraveling the complex molecular mechanisms behind sex disparities in HCC. Although some evidence has been implicated between the role of sex hormones and modulation of gene expression through FOXA transcription factors, further studies are needed to define the implication of sexual hormones in the molecular pathogenesis of HCC.

This study provides convincing evidence for the role of EGFR in sex disparities and as the driver mutational gene in the Poly7 molecular subclass of human HCC. In addition, we have established two mouse models that recapitulate two of the molecular subclasses of human HCC, namely Poly7 and CTNNB1. This study also highlights other candidates through the use of the SB forward insertional mutagenesis system that could account for the sex disparities found between male and female HCC patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors are grateful to the Minnesota Supercomputing Institute for providing extensive computational resources (hardware and systems administration support) used to perform the sequence analysis.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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HEP_26004_sm_SuppFig1.pdf121KSupporting Information Figure 1
HEP_26004_sm_SuppFig2.pdf121KSupporting Information Figure 2
HEP_26004_sm_SuppFig3.pdf91KSupporting Information Figure 3
HEP_26004_sm_SuppInfo1.doc153KSupporting Information
HEP_26004_sm_SuppInfo2.doc5036KSupporting Information
HEP_26004_sm_SuppInfo3.doc3528KSupporting Information
HEP_26004_sm_SuppInfo4.doc66KSupporting Information
HEP_26004_sm_SuppInfo5.doc91KSupporting Information
HEP_26004_sm_SuppInfo6.doc82KSupporting Information
HEP_26004_sm_SuppTables.doc367KSupporting Information Tables

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