ARTICLE Integrative Functional Genomics Implicates EPB41 Dysregulation in Hepatocellular Carcinoma Risk Xinyu Yang,1,2,7 Dianke Yu,3,7 Yanli Ren,2 Jinyu Wei,2 Wenting Pan,2 Changchun Zhou,4 Liqing Zhou,5 Yu Liu,6 and Ming Yang1,* Genome-wide association studies (GWASs) have provided many insights into cancer genetics. However, the molecular mechanisms of many susceptibility SNPs defined by GWASs in cancer heritability and in promoting cancer risk remain elusive. New research strategies, including functional evaluations, are warranted to systematically explore truly causal genetic variants. In this study, we developed an integrative functional genomics methodology to identify cancer susceptibility SNPs in transcription factor-binding sites across the whole genome. Employing integration of functional genomic data from c-Myc cistromics, 1000 Genomes, and the TRANSFAC matrix, we successfully annotated 12 SNPs present in the c-Myc cistrome with properties consistent with modulating c-Myc binding affinity in hepatocellular carcinoma (HCC). After genotyping these 12 SNPs in 1,806 HBV-related HCC case subjects and 1,708 control subjects, we identified a HCC susceptibility SNP, rs157224G>T, in Chinese populations (T allele: odds ratio ¼ 1.64, 95% confidence interval ¼ 1.32– 2.02; p ¼ 5.2 3 106). This polymorphism leads to HCC predisposition through modifying c-Myc-mediated transcriptional regulation of EPB41, with the risk rs157224T allele showing significantly decreased gene expression. Based on cell proliferation, wound healing, and transwell assays as well as the mouse xenograft model, we identify EPB41 as a HCC susceptibility gene in vitro and in vivo. Consistent with this notion, we note that EPB41 expression is significantly decreased in HCC tissue specimens, especially in portal vein metastasis or intrahepatic metastasis, compared to normal tissues. Our results highlight the involvement of regulatory genetic variants in HCC and provide pathogenic insights of this malignancy via a genome-wide approach.

Introduction Hepatocellular carcinoma (HCC [MIM: 114550]) is a lethal malignancy generally refractory to clinical treatments and shows the highest morbidity in Asia and sub-Saharan Africa.1,2 Remarkably, China alone accounts for about 50% of all HCC case subjects.1 Chronic hepatitis B or C virus (HBV or HCV) infection, excessive drinking, and exposure to dietary aflatoxin B have been identified as main epidemiological risk factors for HCC. Among them, chronic HBV infection is predominantly vital and showed a coherent distribution with HCC in China.1,2 However, only a segment of chronic HBV carriers eventually developed HCC, indicating that genetic makeup also plays an important role in HBV-related HCC etiology.3–6 As a pleiotropic transcription factor, the proto-oncoprotein c-Myc plays an essential role in carcinogenesis.7–9 Through controlling a wide variety of downstream effecter genes, c-Myc participates in the regulation of cell growth, cell cycle progression, apoptosis, and differentiation.7–9 To perform these functions, c-Myc binds to the consensus E-box DNA sequence (50 -CACGTG-30 ) by forming a heterodimer with MAX, a member of the basic helixloop-helix leucine zipper (bHLH) transcription factor

family.7–9 Forced overexpression of both c-Myc and transforming growth factor alpha in the mouse liver resulted in tremendously accelerated spontaneous HCC development.10 In contrast, inactivation of the c-Myc oncogene is sufficient to induce regression of invasive HCC, such as differentiation of tumor cells into hepatocytes.11 Recently, Qu et al. showed that hepatocyte-specific temporal disruption of c-Myc by the Cre-ERT2 system significantly suppressed hepatocellular proliferation and liver tumorigenesis,12 which provided direct evidence for the importance of endogenous c-Myc in HCC development. Genome-wide association studies (GWASs) provided a powerful tool for examining the genetic architecture of HCC.3–6 Multiple SNPs in different genomic regions have been identified for their potential importance in the HCC susceptibility, such as SNPs in chromosome regions 1p36.22, 8p12, 6p21.32, and 21q21.3 as well as the STAT4 (MIM: 600558) and HLA-DQ.3–6 These GWASs provided important insights into the genetic complexities of HCC. However, thus far most identified SNPs could explain only a small proportion of genetic susceptibility of this lethal disease, raising the question of how to disclose the remaining ‘‘missing’’ heritability.13,14 In this study, we developed an approach to investigate functional

1 Shandong Provincial Key Laboratory of Radiation Oncology, Cancer Research Center, Shandong Cancer Hospital affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, Shandong Province 250117, China; 2College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China; 3National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA; 4Clinical Laboratory, Shandong Cancer Hospital, Shandong Academy of Medical Sciences, Jinan, Shandong Province 250117, China; 5Department of Radiation Oncology, Huaian No. 2 Hospital, Huaian, Jiangsu Province 223002, China; 6Department of Etiology and Carcinogenesis, Cancer Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China 7 These authors contributed equally to this work *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ajhg.2016.05.029. Ó 2016 American Society of Human Genetics.

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causal genetic variants in HCC that are of potential impact on c-Myc function. In brief, we hypothesized that SNPs locating in the E-box consensus 50 -CACGTG-30 DNA sequence may influence c-Myc binding, expression of adjacent oncogenes or tumor suppressors, and, thus, HCC susceptibility. To test this hypothesis, we conducted a genome-wide screening of the genetic polymorphisms by integrated analyses of c-Myc chromatin immunoprecipitation (ChIP)-chip data of HCC HepG2 cells, the dbSNP and 1000 Genomes databases, as well as the TRANSFAC matrix. Among 12 candidate SNPs identified, only Erythrocyte Membrane Protein Band 4.1 (EPB41) (MIM: 130500) rs157224 SNP was significantly associated with HCC risk in a two-stage case-control study of HBV-related HCC from different regions of China. Interestingly, the rs157224 genetic variant can interrupt c-Myc binding and expression regulation of EPB41 in vivo and in vitro.

Material and Methods Study Case-Control Sets This study consisted of two case-control sets (Table S1). The Shandong set (discovery set) consisted of 1,186 individuals with HBV-related HCC, sex- and age-matched (55 years); 508 chronic HBV carriers were recruited at Shandong Cancer Hospital (Jinan, Shandong Province, China). The Jiangsu set (validation set) consisted of 620 HBV-related HCC individuals from Huaian No. 2 Hospital (Huaian, Jiangsu Province, China) and sex- and age-matched 1,200 chronic HBV carriers as control subjects. Case and control subjects were recruited at Huaian No. 2 Hospital. Part of these case-control sets has been reported previously.15–17 A total of 48 pairs of HCC tissue specimens were collected from 48 HCCaffected individuals recruited in this study. All HCC individuals received curative resection in Huaian No. 2 Hospital. Prior to the surgery, no HCC-affected individuals received any local or systemic anticancer treatments. All subjects were ethnic Han Chinese. At recruitment, written informed consent was obtained from each subject. This study was approved by the institutional Review Boards of Shandong Cancer Hospital and Huaian No. 2 Hospital.

Candidate SNPs Selection and Genotyping The c-Myc-binding DNA segments were identified using genomewide HepG2 ChIP-chip data from hmChIP (Table S2). SNPs in the c-Myc-binding DNA segments were identified from the dbSNP database. Match 1.0 software based on the TRANSFAC matrix was used to identify SNPs whose one allele shows c-Myc binding but another allele does not. Information from the 1000 Genomes project database was used to exclude SNPs with minor allele frequency (MAF) less than 0.01 in Han Chinese populations. Further genotyping was performed by the MassArray system (Sequenom) in the discovery case-control set. The rs157224 polymorphism was genotyped in the validation case-control set using the same method. A 15% blind, random sample of study subjects was genotyped in duplicate and the reproducibility was 100%. A haplotype tag SNP (htSNP) approach was utilized to analyze the EPB41 genetic polymorphisms globally. Genotyped HapMap SNPs among Han Chinese with a MAF > 5% were included in the selection. EPB41 htSNPs were genotyped through the

MassArray system (Sequenom). A 5% blind, random DNA samples was analyzed in duplicates and the reproducibility was 99%. Sequences of primers and probes for each SNP are available on request.

Electrophoretic Mobility-Shift Assays Synthetic double-stranded and 30 biotin-labeled oligonucleotides corresponding to the c-Myc consensus binding sequence, rs157224G or rs157224T sequences (Table S3), and SMMC7721, HepG2, or Huh7 cell nuclear extracts were incubated at 25 C for 20 min using the Light Shift Chemiluminescent EMSA Kit (Pierce). The reaction mixture was separated on 6% PAGE, and the products were detected by Stabilized Streptavidin-Horseradish Peroxidase Conjugate (Pierce). Unlabeled probes at 100-fold molar excess were added to the reaction mixture before the addition of biotin-labeled probes in competition assays.

Chromatin Immunoprecipitation SMMC7721 cells were cross linked in 1% formaldehyde. Genomic DNA was extracted from the fixed-chromatin cells and then subjected to IP using a ChIP assay kit (Upstate) and antibodies against c-Myc (N-262; Santa Cruz cat# sc-764, RRID: AB_631276) or nonspecific rabbit IgG (Santa Cruz). Purified DNA was analyzed by PCR with the primers shown in Table S3.

EPB41 Reporter Gene or Expression Constructs Specific primer pairs (Table S2) with XhoI and HindIII restriction sites were used to amplify multiple deletion fragments spanning 50 -region of EPB41 (from 769 bp to 1 bp, relative to the transcription start site) from human genomic DNA using Pyrobest DNA Polymerase (TaKaRa). The PCR products were then digested with XhoI and HindIII (New England Biolabs) and ligated into an appropriately digested pGL3-Basic vector (Promega) containing the firefly luciferase gene as a reporter. The resultant plasmid, designated p-769, was sequenced to confirm that it contained exclusively G allele at rs157224 SNP position. After the p-769 plasmid was digested with XhoI and NdeI, XhoI and PstI, or NdeI and HindIII (New England Biolabs), the long restricted DNA products were recovered, blunted with Mung Bean Nuclease (TaKaRa), and ligated. The resultant constructs were named p-654, p-127, and p-116. Similarly, the p-654 plasmid was digested with PstI and HindIII (New England Biolabs). The long restricted DNA products were then recovered, blunted with Mung Bean Nuclease, and ligated. The resultant plasmid was designated p-G. The p-G construct was then site-specifically mutated at the rs157224G position to create the construct p-T, which contains rs157224T, with the mutagenesis primers shown in Table S2. Both p-G and p-T constructs were identical, except for the different allele at the rs157224 polymorphic site. Restriction analysis and complete DNA sequencing confirmed the orientation and integrity of these constructs. The EPB41 CDS sequence was directly synthesized (Genewiz Co.) and cloned after the CMV promoter into pcDNA3.1 vector. The plasmid was named pcDNA3.1-EPB41.

Dual Luciferase Reporter Assays SMMC7721 and Huh7 HCC cells (4 3 104) were placed in 24-well plates and transfected with 50 ng of reporter constructs (pGL3Basic, p-769, p-654, p-116, p-G, p-T, and p-127) using Lipofectamine 2000 (Invitrogen) when grown to 50% confluence. pRL-SV40 (1 ng) (Luciferase Assay System; Promega) containing renilla reniformis luciferase was cotransfected to standardize

276 The American Journal of Human Genetics 99, 275–286, August 4, 2016

der microscopy at 24 hr, 48 hr, and 72 hr after transfection. In apoptosis detection, cells were transfected with pcDNA3.1-EPB41 or pcDNA3.1 as well as 20 nmol/L siEPB41-1, siEPB41-2, or NC RNA. After nonadherent and adherent cells were collected at 48 hr after transfection, apoptosis was determined using the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen) with FACSCalibur flow cytometer (FCM) (BD Biosciences). For cell cycle analyses, transfected cells were dyed with PI and detected with the FACSCalibur FCM.

Colony Formation Assays SMMC7721, HepG2, or Huh7 cells were transfected with pcDNA3.1 (vector), pcDNA3.1-EPB41 (EPB41), 20 nM EPB41 siRNAs (siEPB41-1 and siEPB41-2) or NC RNA. A total of 1,500 SMMC7721, HepG2 or Huh7 cells were seeded into a 6-well cell culture plate. After 10 days, cells were washed with cold PBS twice and fixed with 3.7% formaldehyde. After cells were dyed with crystal violet, the colony number in each well was counted.

HCC Xenograft

Figure 1. Flowchart of an Integrative Functional Genomics Methodology to Identify Cancer Susceptibility Genetic Variants in c-Myc-Binding Sites across the Whole Genome

transfection efficiency. Dual luciferase activities were determined at 48 hr after transfection using a luciferase assay system (Promega) as previously described.18,19 For each plasmid construct, three independent transfection experiments were performed, and each was done in triplicate. Fold increase was calculated by defining the activity of the empty pGL3-Basic vector as 1. In brief, the relative luciferase value for each pGL3-Basic derived plasmid equals the ratio of its firefly luciferase activity (standardized by the renilla luciferase activity of the same sample) and pGL3-Basic’s firefly luciferase activity (standardized by the renilla luciferase activity of the same sample). That is, the relative luciferase value of pGL3-Basic transfected cells was 1, and that value for each pGL3-Basic derived plasmid equals to folds of 1.

Quantitative Reverse Transcription PCR After isolation from culture cells or clinical tissues with Trizol reagent (Invitrogen), each RNA sample was treated with RNaseFree DNase to remove genomic DNA (Invitrogen). These RNA samples were then reverse transcribed into cDNAs using Revert Ace kit (TOYOBO). EPB41 and b-actin (MIM: 102630) mRNAs were measured through the SYBR-Green qRT-PCR. The EPB41 expression was calculated relative to the b-actin expression.18–20

Cell Proliferation, Apoptosis, and Cell Cycle Analyses SMMC7721, HepG2, and Huh7 cells (8 3 104) were seeded in 12well plates and transfected with pcDNA3.1-EPB41 or pcDNA3.1 as well as 20 nmol/L each EPB41 siRNA duplexes (siEPB41-1, siEPB41-2, and siEPB41-3) (Table S2) or NC RNA (Genepharma), respectively. Cells were then harvested by trypsin digestion, washed by cold PBS twice, dyed with trypan blue, and counted un-

To evaluate the tumor suppressor role of EPB41 in vivo, we first established a stable HepG2 cell clone with high expression of EPB41 after pcDNA3.1-EPB41 transfection and G418 (Geneticin) selection. 5-week-old female nude BALB/c mice were purchased from Vital River Laboratory. 5 3 106 HepG2 cells with stable transfection of pcDNA3.1-EPB41 or normal HepG2 cells were inoculated subcutaneously into fossa axillaris of 8 nude mice (n ¼ 4 per group). Tumor volumes were measured every day after tumor volumes equaled to or were greater than 80 mm3. All procedures involving mice were approved by the institutional Review Board of Huaian No. 2 Hospital.

Western Blotting After total cellular proteins were separated with SDS-PAGE gel, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The PVDF membrane was then incubated with EPB41 primary antibody (P-20, Santa Cruz cat# sc-25968; RRID: AB_2246481) or b-actin primary antibody (C4, Santa Cruz cat# sc-47778; RRID: AB_626632) overnight at 4 C. Target proteins were visualized with enhanced chemiluminescence (ECL) reagents (Millipore).

Wound-Healing Assays and Transwell Assays When reaching ~90% confluence, the SMMC7721 or HepG2 cell layer was scratched. HCC cells were then continued cultured at 37 C. The average extent of wound closure was quantified. For transwell assays, the transwell chambers were coated with 100 mL BD Matrigel overnight in cell incubator. SMMC7721 or HepG2 cells (1 3 104 cells in 200 mL medium with 0.2% BSA) transfected with pcDNA3.1 (vector), pcDNA3.1-EPB41 (EPB41), 20 nM EPB41 siRNAs (siEPB41-1 and siEPB41-2), or NC RNA were added to upper transwell chambers (pore 8 mm, Corning). A medium containing 10% FBS (650 mL) was added to the lower wells. After 48 hr incubation, cells were fixed and stained, and the nonmigratory cells were scraped from the upper part of the filter. Cells migrated to the lower wells through pores were stained with 0.2% crystal violet solution and counted.

Immunohistochemical Analysis Formalin-fixed, paraffin-embedded HCC or normal liver tissue samples were used for IHC. The goat polyclonal antibody to

The American Journal of Human Genetics 99, 275–286, August 4, 2016 277

Table 1.

Associations between Candidate c-Myc Binding Site SNPs and HBV-Related HCC Risk in Shandong Case-Control Set

#

Identity

Chr. Positiona

Case

ORb (95% CI) for p AA No. (%) AB No. (%) BB No. (%) AB Genotype

1

rs157224

1

28886822

HCC

975 (82.2)

rs11575516 7

50468172

G>T 2

control 450 (88.6)

C>A 3

rs6478804

9

4

rs7038077

rs1180015

72926683

14

89956320

rs61996919 14

90235459

T>C 8

A>C 9

rs4787645

16

30446029

T>C 10 rs8062766

16

9089009

20

62953331

G>A 11 rs2277770 G>A 12 rs5000466 C>A

121 (23.8)

7 (1.4)

566 (47.7)

501 (42.2)

119 (10.1)

control 239 (47.0)

219 (43.2)

50 (9.8)

465 (39.2)

542 (45.7)

179 (15.1)

control 175 (34.4) 121089600 HCC

rs12587742 14

rs942190

control 380 (74.8)

12

G>A 7

1 (0.2) 15 (1.2)

127952643 HCC

G>C 6

57 (11.2) 270 (22.8)

9

A>G 5

20

26208472

10 (0.8)

901 (76.0)

HCC

127952045 HCC

C>T

201 (17.0)

253 (49.8)

80 (15.7)

470 (39.6)

564 (47.6)

151 (12.7)

control 219 (43.1)

219 (43.1)

70 (13.8)

HCC

492 (41.5)

544 (45.9)

150 (12.6)

control 214 (42.1)

235 (46.3)

59 (11.6)

HCC

776 (65.4)

370 (31.2)

40 (3.4)

control 309 (60.9)

176 (34.6)

23 (4.5)

HCC

315 (26.6)

614 (51.8)

257 (21.7)

control 130 (25.6)

261 (51.4)

117 (23.0)

HCC

931 (78.5)

233 (19.6)

22 (1.9)

control 404 (79.5)

96 (18.9)

8 (1.6)

704 (59.4)

405 (34.1)

77 (6.5)

control 305 (60.0)

176 (34.6)

27 (5.3)

HCC

602 (50.8)

477 (40.2)

107 (9.0)

control 267 (52.6)

192 (37.8)

49 (9.6)

HCC

644 (54.3)

476 (40.1)

66 (5.6)

control 278 (54.7)

192 (37.8)

38 (7.5)

HCC

ORb (95% CI) for BB Genotype p

1.62 (1.18–2.22)

2.7 3 103 NC

NC

1.08 (0.86–1.29)

0.537

1.12 (0.53–2.76)

0.816

1.10 (0.75–1.27)

0.726

0.01 (0.74–1.37)

0.954

1.29 (0.91–1.54)

0.079

1.24 (0.87–1.54)

0.271

0.81 (0.62–1.09)

0.114

0.92 (0.68–1.35)

0.914

0.97 (0.74–1.31)

0.915

0.92 (0.65–1.34)

0.528

1.24 (0.84–1.62)

0.129

1.52 (0.74–2.31)

0.187

1.01 (0.84–1.47)

0.842

0.14 (0.83–1.68)

0.583

0.91 (0.64–1.34)

0.684

0.91 (0.41–1.83)

0.725

0.98 (0.84–1.27)

0.854

0.88 (0.54–1.41)

0.411

0.85 (0.64–1.24)

0.374

0.86 (0.64–1.37)

0.841

0.95 (0.70–1.24)

0.481

0.37 (0.80–1.86)

0.196

Abbreviations are as follows: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; AA, common genotype; AB, heterozygous genotype; BB, rare genotype; OR, odds ratio; CI, confidence interval; NC, not calculated. Position in NCBI build 38. b Data were calculated by unconditional logistic regression, adjusted for age, sex, drinking, and smoking. a

EPB41 (P-20, Santa Cruz cat# sc-25968; RRID: AB_2246481) was incubated with the tissue sections at 4 C overnight and was then detected with the ABC kit (Pierce). Stained slides were read independently by two pathologists.

Statistics Pearson’s c2 test was used to examine the differences in demographic variables, smoking status, drinking status, and genotype distributions of all candidate polymorphisms between case and control subjects. The associations between genotypes of the genetic variants and HBV-related HCC risk were estimated by odds ratios (OR) and their 95% confidence intervals (CI) computed by logistic regression models. All ORs were adjusted for age, sex, smoking, or drinking status, where it was appropriate. Student’s t test was used to examine the differences in luciferase reporter gene expression, cell counts, apoptosis, colony numbers, and EPB41 expression. A p value of less than 0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided. All analyses were performed using SPSS 16.0 (SPSS).

Results Genome-wide Screening of Candidate SNPs in c-Myc Binding Sites As shown in Figure 1, we conducted a genome-wide screening of genetic polymorphisms that may interrupt c-Myc binding. First, a total of 12,256 c-Myc-binding DNA segments were identified using genome-wide HepG2 ChIP-chip data from hmChIP (Table S2). Next, 34,436 SNPs in the 12,256 DNA segments were identified from dbSNP database. Using Match 1.0 software based on the TRANSFAC matrix, we found that there were 20 SNPs that have one allele exhibiting c-Myc binding but another allele not. After screening the 1000 Genomes project database, we excluded 8 SNPs with MAF less than 0.01 in Han Chinese populations, finally resulting in a total of 12 c-Myc binding site SNPs (rs157224, rs11575516, rs6478804, rs7038077, rs1180015, rs12587742, rs942190,

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Table 2.

Genotype Frequencies of the rs157224 G>T Polymorphism among Case and Control Subjects and Their Association with HCC Risk

Studies

ORa (95% CI)

pa

Genotypes

Cases No. (%)

Controls No. (%)

n ¼ 1,186

n ¼ 508

GG

975 (82.2)

450 (88.6)

1.00 (reference)

GT

201 (17.0)

57 (11.2)

1.62 (1.18–2.22)

2.7 3 103

TT

10 (0.8)

1 (0.2)

NC

NC

211 (17.8)

58 (11.4)

1.68 (1.23–2.30)

1.1 3 103

Shandong set

GTþTT

n ¼ 620

n ¼ 1,200

GG

510 (82.3)

1,053 (87.8)

GT

107 (17.3)

143 (11.9)

TT

3 (0.4)

3 (0.3)

110 (17.7)

146 (12.2)

Jiangsu set

GTþTT

1.00 (reference) 1.77 (1.32–2.37)

1.2 3 104

NC

NC

1.77 (1.33–2.36)

1.1 3 104

Abbreviations are as follows: HCC, hepatocellular carcinoma; OR, odds ratio; CI, confidence interval; NC, not calculated. a Data were calculated by logistic regression with adjustment for age, sex, smoking, and drinking status.

rs61996919, rs4787645, rs8062766, rs2277770, and rs5000466). These 12 candidate SNPs were genotyped in the Shandong discovery case-control set (Table S4). rs157224, a c-Myc Binding Site SNP, Is Associated with HCC Risk The frequency matching of age and sex was adequate for both the Shandong and Jiangsu sets (Table S4). Unconditional logistic regression analysis was utilized to calculate associations between genotypes of the 12 candidate c-Myc binding site SNPs and HBV-related HCC risk in the Shandong discovery set (Table 1). Among these SNPs, only rs157224G>T located 269 bp telomeric to the EPB41 TSS (the transcription start site) was significantly associated with HCC risk. As shown in Table 2, the rs157224T allele was a risk allele; subjects with the GT genotype had an OR of 1.62 (95% CI ¼ 1.18–2.22, p ¼ 2.7 3 103) for developing HCC, compared with subjects with the GG genotype. Significantly elevated HCC risk was also observed among rs157224GT or TT carriers compared to the GG carriers (OR ¼ 1.68, 95% CI ¼ 1.23– 2.30, p ¼ 1.1 3 103). The association of HCC risk with the rs157224 SNP was further verified in an independent Jiangsu case-control set. Similarly, a significantly increased cancer risk was associated with rs157224 (GT: OR ¼ 1.77, 95% CI ¼ 1.32–2.37, p ¼ 1.2 3 104; GT or TT: OR ¼ 1.77, 95% CI ¼ 1.33–2.36, p ¼ 1.1 3 104). In the metaanalyses, we found that the rs157224 GT or TT carriers had an increased risk, 1.61 times, to develop HCC compared to the GG carriers (95% CI ¼ 1.31–1.97) (Figure S1). Considering that genetic interactions are complex, we explored whether other SNPs in the EPB41 locus might contribute to HCC susceptibility besides rs157224. We genotyped 11 htSNPs of the EPB41 locus in the discovery set and found that rs157224 is the only polymorphism significantly associated with HCC risk (Table S5). Additionally, we did not find significant gene-environment interac-

tions between rs157224 polymorphism and age, sex, smoking, or drinking history. SNP rs157224 Mediated Allele-Specific c-Myc Binding in HCC Cells Because rs157224 SNP is located in the c-Myc E-box consensus binding sequence and 269 bp telomeric to the EPB41 TSS, we then conducted EMSA to distinguish the differences in binding capacity between the rs157224G and T alleles to c-Myc (Figures 2A–2C). As shown in Figure 2A, we found that c-Myc-containing SMMC7721 nuclear extracts bound only to the biotin-labeled oligonucleotide probe with the G allele or the c-Myc consensus sequence but not the T allele probe. A 100-fold excess of unlabeled G allele oligonucleotides or unlabeled c-Myc consensus oligonucleotides efficiently competed for the binding activity of the G allele. However, unlabeled T allele oligonucleotides did not affect the binding activity of the G allele. Similar results were observed when using Huh7 or HepG2 nuclear extracts (Figures 2B and 2C). We also did a competition assay using c-Myc antibody in SMMC7721 and HepG2 cells (Figure 2D). Interestingly, although we did not find super-shift bands, we did observe gradually attenuated c-Myc binding bands with increased amount of c-Myc antibody used. We further confirmed that the binding of c-Myc to the region telomeric to EPB41 occurred in vivo in HCC cells using ChIP assays in SMMC7721 cells with the rs157224GG genotype. The region telomeric to EPB41 was able to be specifically precipitated with the c-Myc antibody but not with IgG (Figure 2E). We speculated the possibility that the c-Myc binding SNP rs157224 may be located in a potential proximal promoter of EPB41. Therefore, we examined the promoter activity of this region using a set of luciferase reporter constructs in SMMC7721 and HepG2 cells (Figure 2F). Intriguingly, we observed a significantly higher level of promoter

The American Journal of Human Genetics 99, 275–286, August 4, 2016 279

Figure 2. Abolishment of a c-Myc Binding Site in the EPB41 Promoter by the rs157224G>T Genetic Polymorphism Influences Promoter Activities (A) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled c-Myc consensus, rs157224G, or rs157224T probes and SMMC7721 nuclear extract. Left: EMSA with c-Myc consensus or rs157224G probes. Lanes numbered from left to right. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5, and 7, probe and nuclear extracts plus 1003 unlabeled rs157224G (lanes 1 and 5) or c-Myc consensus probes (lanes 3 and 7). Right: EMSA with rs157224G or rs157224T probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3–5 and 8–10, probe and nuclear extracts plus 1003 unlabeled rs157224G (lanes 5 and 8), rs157224T (legend continued on next page)

280 The American Journal of Human Genetics 99, 275–286, August 4, 2016

activities derived from the p-769 construct of a 769 bp region telomeric to EPB41 TSS (769 through 1 bp from TSS), the p-654 construct of a 654 bp region (654 through 1 bp), and the p-G construct of a 527 bp region (654 through 127 bp), compared with other constructs. Among them, p-G showed the highest level of activity, indicating that the EPB41 proximal promoter might exist between 654 bp and 127 bp. We next examined whether the rs157224 SNP has an allele-specific effect on the promoter activity. Either SMMC7721 or HepG2 cells transfected with rs157224G allelic reporter construct (p-G) showed significantly higher luciferase activities than cells expressing rs157224T allelic reporter construct (p-T) (both p < 0.01) (Figure 2F). As shown in Figure S2, this was validated in the Raji cell line with a high c-Myc protein level and silencing c-Myc protein significantly suppressed reporter gene activities. Our results indicate that c-Myc could bind the rs157224G allelic EPB41 promoter and prompt elevated EPB41 expression. Identification of EPB41 as a Susceptibility Gene of HCC Because the role of EPB41 in HCC development is largely unknown, we next investigated the impact of EPB41 on biological behaviors of HCC cells. We first studied whether overexpression or silencing of EPB41 could modulate cell proliferation. Elevated EPB41 significantly suppressed cell growth in different HCC cells (all p < 0.05) (Figure 3A). In contrast, EPB41 downregulation by siRNAs significantly accelerated proliferation of HCC cells compared to cells transfected with NC RNA (all p < 0.01) (Figure 3B). To gain insight into the functional relevance of EPB41, we examined the impact of EPB41 on apoptosis and cell cycle progression of HCC cells (Figures 3C and 3D). EPB41 overexpression could induce obvious apoptosis in three HCC cell lines (p < 0.05) (Figure 3C). Compared with NC RNA-transfected HCC cells, both siEPB41-1 and siEPB41-2 resulted in a significant reduction of apoptotic population in all HCC cell lines (p < 0.05) (Figure 3D). However, EPB41 showed no significant impacts on cell cycle progression in all HCC cell lines. Colony formation assays also supported its tumor suppressor role (Figure 3E). We then evaluated the anti-cancer ability of EPB41 in vivo. We found that the growth of tumors of the EPB41-upregulated HepG2 xenografts in mice was significantly inhibited

compared with that of control xenografts after 15 days (Figures 3F and 3G). EPB41 protein levels in xenografts stably transfected with pcDNA3.1-EPB41 were maintained and higher than those in control xenografts (Figure 3H). There were no significant differences of mice weight between controls and the EPB41-overexpressed group (Figure S3). EPB41 Inhibits Migration and Invasion of HCC Cells We then examined whether EPB41 could influence migration and invasion of HCC cells. The wound-healing assays demonstrated that EPB41 impaired the motility of the SMMC7721 and HepG2 cells (Figure 4A). In contrast, EPB41 knockdown stimulated migration of both cell lines (Figure 4B). Next, the impact of EPB41 on invasiveness of SMMC7721 and HepG2 cells was determined via the Matrigel invasion assays. Reduced invasion ability of HCC cells was observed after elevated expression of EPB41 (Figure 4C). In line with this observation, EPB41 siRNAs can enhance invasion of these HCC cells (Figure 4C). Decreased EPB41 Expression in Human HCC Tissue Specimens We further examined EPB41 expression in 48 pairs of HCC tissue specimens and adjacent normal tissues. A significant EPB41 mRNA and protein downregulation in HCC tissues was observed compared to those in normal tissues (p < 0.01) (Figures 5A–5C). Interestingly, we found an obvious allele-differential expression between rs157224Gallele carriers and T-allele carriers in either HCC or normal specimens (both p < 0.05) (Figure 5D). We also examined whether the differential EPB41 expression exists in public HCC profiling databases. In a cohort of Taiwanese HCC individuals (GEO: GSE45267), a much lower EPB41 expression in HCC tissues was observed compared to that in normal tissues (Figure 5E), consistent with our findings. Interestingly, Ye et al. analyzed the gene expression profiles of HCC samples with or without metastases and found that subjects without metastasis showed much higher EPB41 expression than ones with portal vein metastasis or intrahepatic metastasis (GEO: GSE364) (Figure 5F).21 These data further supported the tumor suppressor nature of EPB41 in HCC development.

(lanes 3 and 10), or c-Myc consensus probes (lanes 4 and 9). Probes: EPB41-rs157224G, 50 -GAAGCAATTTGACACGTGGTACTGCTCC TAA-30 ; EPB41-rs157224T, 50 -GAAGCAATTTGACACTTGGTACTGCTCCTAA-30 ; c-Myc-consensus, 50 -CAGGAAGCAGACCACGTGGT CAGGCTATA-30 . (B and C) EMSA with biotin-labeled rs157224G or rs157224T probes and Huh7 (B) or HepG2 (C) nuclear extracts. (D) EMSA competition assay using c-Myc antibody in SMMC7721 and HepG2 cells. (E) ChIP assays using SMMC7721 cells carrying the rs157224GG genotype. The presence of c-Myc-binding EPB41 or P53 promoter was verified by PCR, with GAPDH as the negative control. (F) Transient luciferase reporter gene expression assays with constructs containing different lengths or different rs157224 allele of the region telomeric to EPB41 in SMMC7721 or HepG2 cells. pRL-SV40 were cotransfected with these constructs to standardize transfection efficiency. Fold-changes were detected by defining the luciferase activity of cells co-transfected with pGL3-basic as 1. All experiments were performed in triplicate in three independent transfection experiments and each value represents mean 5 SD. Compared with pGL3-Basic transfected cells, *p < 0.05; **p < 0.01.

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Figure 3. Impacts of EPB41 on HCC Cell Proliferation In Vitro and In Vivo (A) Overexpression of EPB41 significantly suppressed cell growth of SMMC7721, HepG2, and Huh7 cells. Cell number was counted at 24 hr, 48 hr, and 72 hr after transfection. (B) Silencing EPB41 with three siRNAs (siEPB41-1, siEPB41-2, and siEPB41-3) accelerates cell proliferation of SMMC7721, HepG2, and Huh7 cells. (legend continued on next page)

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Figure 4. EPB41 Reduces Migration and Invasion Ability of HCC Cells (A) Enforced EPB41 expression inhibits wound healing in SMMC7721 and HepG2 cells. Wound fields were observed directly after removal of inserts (0 hr) and cell migration was followed for 24 hr and 48 hr. Wound-healing area in HCC cells was presented by histogram. (B) Silencing EPB41 accelerates wound healing in SMMC7721 and HepG2 cells. Wound fields were observed directly after removal of inserts (0 hr) and cell migration was followed for 18 hr and 36 hr. Woundhealing area in HCC cells was presented by histogram. (C) EPB41 inhibits invasion abilities of SMMC7721 and HepG2 cells. Cells on the lower surface of the chamber were stained by crystal violet at 48 hr after transfection. *p < 0.05, **p < 0.01.

Discussion Although GWASs have provided important insights into cancer genetics, the identified susceptibility genetic variants could not account for all heritability of cancers and their mechanisms promoting cancer risk are elusive. New research strategies would facilitate the systematically defining real causal variants with thoroughly functional evaluations. In this study, we combined cistromics, 1000 Genomes data, and the TRANSFAC matrix to develop an

integrative functional genomics strategy to identify genome-wide c-Mycbinding site SNPs. We successfully annotated 12 SNPs that are located in the c-Myc cistrome (the in vivo genome-wide location of trans-factor binding sites) and may modulate the c-Myc binding affinity in HCC cells. After genotyping 1,806 HBV-related HCC case subjects and 1,708 control subjects, we identified a HCC susceptibility SNP rs157224 in Chinese populations (p ¼ 5.2 3 106). The risk rs157224T allele showed no c-Myc-binding, thereby resulting in allele-specific decreased EPB41 expression in both HCC cell lines and tissue specimens. Moreover, we determined the tumor suppressor function of EPB41. c-Myc is moderately expressed in normal liver compared to other tissue types (Figure S4). We found that silencing c-Myc can significantly decrease EPB41 expression, but forced c-Myc expression did not stimulate EPB41 expression in HCC cells (Figure S5). As a result, we speculated that c-Myc at a physiological level is enough to drive EPB41 expression in the normal liver. However, there might be multiple other factors involved in EPB41 regulation with or without c-Myc. At transcriptional regulation level, several other potential trans-factors that may bind

(C) Enforced EPB41 expression induces HCC cell apoptosis. Apoptosis was determined with FACSCalibur flow cytometer. (D) Silencing EPB41 with siRNAs inhibits HCC cell apoptosis. (E) Colony formation assays. pcDNA3.1-EPB41 or pcDNA3.1 as well as 20 nmol/L NC RNA or EPB41 siRNAs was transfected into HCC cells, respectively. (F and G) EPB41 significantly inhibits growth of HepG2 xenografts compared with control xenografts after 15 days. (H) EPB41 and b-actin protein levels in xenografts. **p < 0.01.

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Figure 5. EPB41 Expression in HCC Tissue Specimens and Public Gene Profiling Databases (A–C) EPB41 mRNA and protein were quantified using qRT-PCR, immunohistochemical analyses, and western blot in 48 tumor-normal pairs. All data of EPB41 expression were normalized to b-actin expression levels. (D) EPB41 mRNA expression in HCC and normal tissues grouped by rs157224G>T genotypes. (E) EPB41 expression in GEO: GSE45267. (F) EPB41 expression in GEO: GSE364, the gene expression profiles of HCC samples with or without metastases. Subjects without metastasis showed much higher EPB41 expression than ones with portal vein metastasis or intrahepatic metastasis. *p < 0.05, **p < 0.01.

to the promoter region of EPB41 were predicted by Match 1.0 software (Figure S6). Nevertheless, further studies on identification of other EBP41 promoter-binding trans-factors would be beneficial to declare detailed molecular mechanisms of expression regulation on the EBP41 locus. The c-Myc transcription factor acts as a key proto-oncogene through controlling hepatocyte proliferation, liver regeneration, and malignant transformation after hepatic injury and carcinogen exposure. Therefore, it is plausible that genetic variants, which are located in c-Myc binding sites and modulate its binding affinity, may dysregulate expression of adjacent genes and contribute to HCC susceptibility. Several lines of evidence point to the involvement of a proportion of such cancer-risk SNPs in transcriptional regulation, including modulation of cisregulatory elements (promoters and enhancers).22–30 For instance, a common colorectal cancer (MIM: 114500) predisposition SNP, rs6983267, affects a transcription factor

TCF4 binding site, with the risk G allele showing stronger binding in vitro and in vivo. This provided evidence that the common 8q24 colorectal cancer predisposition arises from enhanced responsiveness to the Wnt signaling pathway.22 Additionally, multiple breast cancer (MIM: 114480) risk-associated SNPs are able to alter the affinity of chromatin for FOXA1 at distal regulatory elements and lead to allele-specific gene expression, which is exemplified by the role of the 16q12.1 rs4784227 SNP on TOX3 (MIM: 611416).23 EPB41, a component of cell membrane cytoskeleton, plays a part in erythrocyte shape and deformability. Though attenuated expression of EPB41 was proved in human nonsmall cell lung cancer (MIM: 211980),31 inconsistent results on its expression in meningioma (MIM: 606190) were reported.32,33 EPB41 belongs to the same erythrocyte membrane protein family as a well-known tumor suppressor EPB41L3 (also known as DAL1) (MIM: 605331).34–38 Our and others’ tissue profiling data support its decreased expression in HCC tissues compared to adjacent normal tissues. In vitro cell proliferation, colonic formation, scratch and transwell assays after gain- or loss-gene expression as well as in vivo mice xenograft data provide evidence that EPB41 could suppress HCC growth and progression significantly, demonstrating its tumor-suppressor nature in HCC. Interesting, aberrant splicing of EPB41 has previously been associated with solid tumors,39 supporting its role as a tumor-suppressor gene. Considering EPB41 is a component of cell membrane cytoskeleton together with spectrins and actins, we speculate that it may be involved in the p21-activated kinase (PAK)

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pathway. PAKs are serine/threonine protein kinases involved in both cytoskeletal rearrangements40 and the development and progression of cancers.41 PAKs are positioned at the intersection of a number of signaling pathways required for oncogenesis. When activated by mutations, overexpression, or upstream elements such as Rac or Cdc42, most PAK isoforms have oncogenic signaling effects in cells, including the acquisition of growth signal autonomy, evasion of apoptosis, and promotion of invasion and metastasis.41 Since cytoskeleton protein EPB41 also impacts cancer cell growth, apoptosis, invasion, and metastasis, it would be interesting to examine whether altered EPB41 protein levels may play a part in the PAK oncogenic signaling pathway in the future. In all, we developed a research strategy using integrative functional genomics to reveal potential missing heritability and causative mechanisms of malignancies including HCC. Using c-Myc as an example, we identified a SNP whose functional change led to HCC predisposition through differential c-Myc-mediated EPB41 expression. Based on functional-based assays, we first disclose EPB41 as a HCC tumor suppressor dysregulated in an allelic-specific manner by c-Myc. Our results suggest prevalent involvement of regulatory genetic variations in cancers and provide pathogenic insights into HCC development. Supplemental Data Supplemental Data include six figures and five tables and can be found with this article online at http://dx.doi.org/10.1016/j. ajhg.2016.05.029.

Acknowledgments This work was supported by National Natural Science Foundation of China (81201586 and 31271382), the National High-Tech Research and Development Program of China (2015AA020950), and the open project of State Key Laboratory of Molecular Oncology (SKL-KF-2015-05). The authors would like to thank the many individuals who participated in the study. We thank Dr. Gwo-Shu Mary Lee of Dana-Farber Cancer Institute, Harvard Medical School for comments and for critically reading and English editing the manuscript. We also thank Dr. Yi-Ran Cai of the Department of Pathology, Beijing Chest Hospital, Capital Medical University for his assistance in tissue IHC analyses and Huaijin Zhang, Honglei Pu, Huijiuan Su, Jing Zhang, and Lingchen Xiao for excellent technical support. Received: January 16, 2016 Accepted: May 30, 2016 Published: July 21, 2016

Web Resources 1000 Genomes, http://www.1000genomes.org dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/ GEO, http://www.ncbi.nlm.nih.gov/geo/ hmChIP, http://jilab.biostat.jhsph.edu/database/cgi-bin/ hmChIP.pl

International HapMap Project, http://hapmap.ncbi.nlm.nih.gov/ OMIM, http://www.omim.org/

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