Xing-Yu Pu , b,# , Do-Feng Zheng , b,# , To Lv , b , Yong-Jie Zhou , b , Ji-Yin Yng , b , Li Jing , b, ?

a Department of Liver Surgery and Liver Transplantation Center, West China Hospital, Sichuan University, Chengdu 610041, China

b Laboratory of Liver Transplantation, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China

Keywords:Hepatocellular carcinoma Proliferation Cell and molecular biology

ABSTRACT Background: Transcription factor 3 (TCF3) plays pivotal roles in embryonic development, stem cell maintenance and carcinogenesis.However, its role in hepatocellular carcinoma (HCC) remains largely unknown.This study aimed to analyze the correlation between TCF3 expression and clinicopathological features of HCC, and further explore the underlying mechanism in HCC progression.

Introduction

Hepatocellular carcinoma (HCC) ranks the third leading cause of cancer-related death in the world and the second in China [1].Although considerable progress and improvements have been made in molecular pathogenesis, surgery and treatment strategies in the past two decades, the survival of HCC patients still remains unsatisfactory.More effort is required to disclose some novel factors which might participate in HCC development, thus demonstrating the related molecular mechanism.

Transcription factor 3 (TCF3), also called E2A, belongs to the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor family that functions as a critical transcription factor in embryonic development, stem cell maintenance and carcinogenesis [2–5].TCF3 is important to embryonic development by modulating pattering and cell fate specification, and loss of TCF3 leads to gastrulation defects and embryonic lethality [3].Increasing evidence reveals that TCF3 plays important roles in the pathogenesis of several types of human cancers, including prostate cancer, breast cancer, colorectal cancer and leukemia [4–7].It has been well illustrated that the binding of TCF3 with PBX1 andHLF induced by abnormal chromosomal translocation in patients with pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) is correlated with their prognosis and survival [ 8 , 9 ].In addition, it is reported that TCF3 is an epithelial-mesenchymal transition (EMT) signature gene, which mutually regulates with Oct4 in HCC [10–13].However, whether TCF3 plays a vital role in HCC development remains largely unknown.In the current study, we investigated the expression of TCF3 and analyzed the correlation between the expression of TCF3 and the clinical event in HCC patients, and further explored the internal mechanism by which TCF3 aggravates HCC development.

Fig.1.TCF3 is highly expressed in HCC tumor tissues.A: The expression of TCF3 in TCGA HCC dataset.B: The expression of TCF3 in GEO HCC datasets (GSE14520 and GSE63898); TPM, transcripts per million; ???P < 0.001.C: Paired immunostaining images of TCF3; scale bar, 100 μm.D: The expression of TCF3 detected by Western blotting and analyzed by Image J software; N: adjacent non-tumor tissues; T: tumor; ??: P < 0.01.

Methods

Public data source acquisition and analysis

The RNA-seq transcriptome data and their related clinical features of HCC specimens were obtained from The National Cancer Institute Genomic Data Commons (NCI-GDC) ( https://gdc.cancer.gov/ ) [14].Total 374 HCC tissues and 50 adjacent non-tumor tissues were included for consequent analysis.Another two HCC datasets were downloaded from the Gene Expression Omnibus, accession number GSE14520 and GSE63898 ( https://www.ncbi.nlm.nih.gov/geo/ ).The expression profile of cell lines was downloaded from Broad Institute Cancer Cell Line Encyclopedia (CCLE, https://portals.broadinstitute.org/ccle/about ) [15].Data were analyzed byRsoftware (Version 3.6.3) or GraphPad Prism (Version 9, San Diego, CA,USA).

Patients, tissue microarray and immunohistochemistry staining

Specimens were collected from 138 HCC patients who underwent resection of tumors at West China Hospital of Sichuan University between January 2014 and December 2016 with intact clin-ical information.Overall survival (OS) was defined as the time between the initial surgery and death.Disease-specific survival (DSS)was defined as the time between the initial surgery and HCC specific death.Tissue microarrays were originated from corresponding formalin-fixed, paraffin-embedded HCC samples.These tissue microarray sections were immunostained, and the slices were determined blindly by three experienced pathologists.TCF3 expression was evaluated in term of the signal positive rate and intensity.Concisely, at least five areas under 400 ×magnification per slice were tested and the percentage of positive cells was evaluated by the following criteria: 0, ≤ 5%; 1,>5%–25%; 2,>25%–75%; and 3,>75%, respectively.The staining intensity was evaluated as follows: 0–1, weak; 2, moderate; and 3, strong.The final immunostaining score for each patient was the intensity score multiplied by the positive rate score.Specimens were divided into lowexpression and high expression according to the immunostaining score of 0–3 and 4–9, respectively.To analyze the correlation of TCF3 with HCC, classic core clinical characteristics, such as sex, age,alpha-fetoprotein, TNM stage, liver cirrhosis, tumor size, and vascular invasion were included.This study was approved by the Ethics Committee of West China Hospital of Sichuan University, and each patient provided written informed consent.

Fig.2.TCF3 expression is highly associated with prognosis of HCC patients in TCGA dataset.A: The expression of TCF3 in different tumor stage of HCC patients, compared with the expression of TCF3 in stage I.B: The expression of TCF3 in different tumor grade of HCC patients, compared with the expression of TCF3 in grade 1.C-E: The correlation of TCF3 expression with OS, DSS and PFS.?: P < 0.05; ??: P < 0.01; ???: P < 0.001; NS: not significant; HCC: hepatocellular carcinoma; OS: overall survival; DSS:disease-specific survival; PFS: progression-free survival.

Cell culture, transfection and biological behavior assessment

The human HCC cell lines Hep3B and SNU398 were originated from American Type Culture Collection (ATCC, Manassas, VA,USA), both of which were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), and cultured in a humidified incubator with 5% CO 2.Plasmids encoding shTCF3(sh1, 5’-CAC CGG AAA TCT CTT TGC AGG ATT CCG AAG AAT CCT GCA AAG AGA TTT CC-3’; sh2, 5’-CAC CGC TTC CTA CTT GGT AGA ATG GCG AAC CAT TCT ACC AAG TAG GAA GC-3’; sh3, 5’-CAC CGC ATC GAG CAC AGA TGT TAA GCG AAC TTA ACA TCT GTG CTC GAT GC-3’) were packaged by Lipofectamine 30 0 0 and transfected into cells referring to the manufacture’s instruction (Invitrogen, Carlsbad, CA, USA).After 72 h of transfection, Western blotting was used to test the knockout efficiency.Cell viability and proliferation were measured by CCK-8 (Sigma-Aldrich, St.Louis, MO, USA)and EdU (RiboBio, Guangzhou, China) incorporation assays according to the manufactures’ instruction.Matrigel invasion assay was performed to evaluate cell migration and invasion ability as described [16].

Total proteins isolation and Western blotting

Fresh HCC tissues and cells were lysed with RIPA referring to the manufacture’s instruction (Beyotime Biotechnology, Shanghai, China).Protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.22 μm PVDF membranes (Millipore, Billerica, MA, USA).Furthermore, membranes were blocked with phosphate buffered saline containing 5% skim milk, and incubated with antibodies at 4 °C overnight.The antibodies were as follows: TCF3 (#ab69999, 1:10 0 0, Abcam, Cambridge, UK), CDK4 (#ET1612-1, 1: 30 0 0, Huabio,Hangzhou, China), E2F4 (#ER65549, 1: 50 0 0, Huabio), Histone H3(#4499, 1: 50 0 0, CST, Boston, MA, USA), DVL2 (#ER63295, 1: 10 0 0,Huabio), MAPK9 (#ab76125, 1: 10 0 0, Abcam), CCND3 (#2936, 1:20 0 0, CST).Protein expression levels were visualized using an enhanced chemiluminescence detection system (4A Biotech, Beijing,China) and semi-quantified by Image J software (National Institutes of Health, Bethesda, Maryland, USA).

Statistical analysis

All statistical analyses were conducted by the SPSS software(Version 21.0, SPSS Inc., Chicago, IL, USA) or GraphPad Prism 9.0(GraphPad, San Diego, CA, USA).The relationship between TCF3 expression and the corresponding clinicopathological parameters in HCC patients was analyzed using Chi-square test or Fisher’s exact test.Survival was assessed by the Kaplan-Meier method, and survival curves were analyzed by the log-rank test.Cox regression was used to analyze the relationship between TCF3 expression and clinicopathological prognosis in HCC patients.Pearson’s correlation was applied to analyze expression between TCF3 and other genes.APvalue of less than 0.05 was considered statistically significant.

Results

TCF3 is significantly highly expressed in HCC tissues

TCF3 was significantly elevated in HCC tumor tissues compared with that in corresponding tumor adjacent tissues ( Fig.1 A, B).High expression of TCF3 in tumor tissues was further confirmed by immunostaining and Western blotting assays ( Fig.1 C, D), which indicated that TCF3 may play a certain role in HCC development.

TCF3 and clinicopathological features

The expression of TCF3 was positively correlated with tumor stage and grade ( Fig.2 A, B).According to Kaplan-Meier Plotter ( http://kmplot.com/analysis/index.php?p=service ) [17], patients with high TCF3 expression had shorter OS (P= 0.012),DSS (P= 0.022) and progression-free survival (PFS) (P= 0.013)( Fig.2 C–E).According to the immunostaining score, 56 samples (40.6%) were identified as lowexpression subtype (TCF3low)and the other 82 samples (59.4%) were high expression subtype(TCF3high ) ( Fig.3 A,B).The association of TCF3 expression and the baseline clinical characteristics was shown in Table 1.The results showed that high expression of TCF3 was more likely to be observed in HCC patients with large tumor size (63.4%,P= 0.001)and more advanced TNM stage (67.1%,P= 0.002) ( Table 1 ).In univariate analysis, high TCF3 expression (P= 0.004), tumor size>5 cm (P= 0.011) and TNM stage III/IV (P= 0.013) were significantly associated with OS.The multivariate analysis revealed that high TCF3 expression (P= 0.008), tumor size>5 cm (P= 0.017)and clinical TNM stage III/IV (P= 0.019) were associated with OS ( Table 2 ).In addition, patients with high TCF3 expression had shorter OS (P= 0.014) and DSS (P= 0.007) ( Fig.3 C, D), indicating that TCF3 is a potential prognostic marker for HCC patients.

Table 1Association between TCF3 and clinicopathological features of HCC patients.

Table 2Univariate and multivariate analysis of different prognostic variables influencing overall survival in HCC patients.

Fig.3.The high TCF3 expression is associated with poor prognosis.A: Representative images of different TCF3 immunohistochemistry scores, and the proportions of each score in 138 HCC patients; scale bar, 100 μm.B: The representative images of high and lowexpression of TCF3; scale bar, 50 μm.C and D: The correlation of TCF3 expression with OS and DSS.HCC: hepatocellular carcinoma; OS: overall survival; DSS: disease-specific survival.

TCF3 is critical for HCC cancer cell proliferation

Gene set enrichment analysis (GSEA) showed that the genes highly correlated with TCF3 were enriched in pathway in cancer, cell cycle and Wnt signaling pathway based on TCGA HCC dataset (TCF3high vs.TCF3 low ; Fig.4 A).The co-expression ofTCF3and core genes in these signaling pathways were shown in Fig.4 B.The cell cycle critical regulator genes,E2F4andCDK4,and core Wnt pathway genes,DVL2andMAPK9(also called JNK2),were significantly correlated withTCF3(r>0.50,P<0.0 0 01)( Fig.4 C–F).

TCF3 was highly expressed in Hep3B and SNU398 cell lines,both of which were selected for further study ( Fig.5 A).Knockdown of TCF3 was achieved by specific short-hairpin RNAs (shRNAs)( Fig.5 B).The CCK8 assay demonstrated that knockdown of TCF3 remarkably reduced Hep3B and SNU398 cell viability ( Fig.5 C).More importantly, EdU incorporation assay results showed that less proliferation rates of both Hep3B and SNU398 were observed in the shTCF3-treated group (30.40% ± 4.81% vs.62.20% ± 9.46%,P= 0.0 0 02; 29.61% ± 4.04% vs.53.71% ± 5.60%,P<0.0 0 01)( Fig.5 D).However, no obvious difference was found in cell migration ability after TCF3 silencing ( Fig.5 E).

TCF3 enhances cell proliferation by activating Wnt signaling pathway

The expression of E2F4 and CDK4 were reduced by more than half after TCF3 knockdown ( Fig.6 A).In addition, Wnt sig-naling pathway regulator DVL2 and downstream effector MAPK9 were both reduced to varying degrees ( Fig.6 A).Consistent with the GSEA results, the level of CCND3, a cell cycle regulator and Wnt signaling pathway effector, was reduced after TCF3 silencing.Moreover, similar expression pattern of these corresponding proteins was found in low TCF3 expression (TCF3 low ) and high TCF3 expression (TCF3high ) HCC samples ( Fig.6 B,C).Finally, we examined the proliferation rate of TCF3 lowand TCF3highHCC samples by Ki-67 immunostaining.More proliferating tumor cells were found in TCF3hightissue slides (17.77% ±4.22% vs.11.24% ±2.60%,P<0.001) ( Fig.6 D).All these results indicated that TCF3 promotes cell proliferation via the activation of Wnt signaling pathway.

Fig.5.Silencing TCF3 inhibits cell proliferation.A: The relative expression of TCF3 in HCC cell lines in Cancer Cell Line Encyclopedia (CCLE) database.B: TCF3 was effectively knocked down by shRNAs in Hep3B and SNU398.C: Cell viability was measured by CCK-8 assays at indicated timepoints.D: Cell proliferation ability was measured by EdU incorporation assay; scale bar, 50 μm.E: Cell migration ability was measured by transwell assay.??: P < 0.01; ???: P < 0.001.NS: not significant.

Fig.6.TCF3 promotes cell proliferation by activating Wnt signaling pathway.A : Silencing of TCF3 in Hep3B and SNU398 resulted in the reduced expression of E2F4, CDK4(cell cycle), DVL2, MAPK9 and CCND3 (Wnt signaling pathway).B and C: These proteins shared similar expression pattern with TCF3 in HCC tumor tissues.D: HCC patients with high TCF3 expression have higher tumor cell proliferation rate.?: P < 0.05; ??: P < 0.01; ???: P < 0.001.Scale bar, 100 μm.

Discussion

HCC development is a multi-factor and multi-step process.Genetic and epigenetic changes of critical transcription factors result in dysregulation of cancer-related genes, which endows malignant characteristics of cancer cells, such as unlimited proliferating ability and high invasion ability [18].As a critical transcription factor, the biological function of TCF3 in embryonic development and stem cell self-renewal or differentiation has been well documented [ 19 , 20 ].In the past decade, accumulating evidence has been found that abnormal expression or mutation ofTCF3plays pivotal roles in tumorigenesis [4–9].It has been reported thatTCF3, acting as the downstream targeted gene of miR-449a, maintains self-renewal in liver cancer stem-like cells [21],and participates in tumor growth and metastasis regulation in a study focusing on the biologic role of HN1L [12].At the same time,TCF3was found to be anEMTgene in HCC patients infected with HCV [10].However, there is no systematic analysis of the relationship between the expression of TCF3 and the prognosis of HCC patients and the detailed role of TCF3 in HCC remains largely unknown.

According to the ciBioPortal online website ( https://www.cbioportal.org/ ), only 1.1% HCC patients harboredTCF3mutation(4/366).Therefore, in the present study, we only examined the expression pattern of TCF3 in HCC.Firstly, elevated expression of TCF3 was found in HCC tumor tissues compared with tumor adjacent tissues in both mRNA and protein level based on TCGA database.And the mRNA level of TCF3 was positively correlated with tumor grade and stage, and patients with high TCF3 expression had shorter OS.In addition, according to our immunohistochemistry staining results, TCF3 was highly expressed in 59.4% HCC patients (82/138).Our further analysis of the correlation between TCF3 expression and clinicopathological features revealed that the expression of TCF3 was closely correlated with tumor size, TNM stage and OS of HCC patients, and that TCF3 is a potential prognostic marker for HCC patients.

Wnt signaling pathway has been reported as one of the most important pathways widely involved in all the biological processes,such as embryonic development, stemness maintenance, differentiation, cell proliferation and cancer development, which largely relays on the binding ofβ-catenin to TCF/LEF transcription factor family, including TCF1, TCF3 and TCF4 [22–25].TCF3/β-catenin complex has been verified to modulate gene expression in carcinogenesis [ 26 , 27 ], and the dysregulation of TCF3 found in colorectal cancer, breast cancer, cervical cancer and leukemia, can lead to cell proliferation, invasion and stemness alteration [ 4 , 5 , 28–30 ].Consistent with these findings, we found that TCF3 expression was positively correlated with cell cycle regulation genes and Wnt signaling pathway genes at both mRNA and protein levels, and that the silencing ofTCF3substantially reduced the expression of Wnt target genes.These results indicated that TCF3 promoting cell proliferation is mediated via Wnt signaling pathway.Although the existing evidence proves the importance of TCF3 in the prognosis of HCC patients, more studies are needed to further investigate the role of TCF3 in the development of HCC.

In conclusion, this study reported the expression pattern and prognostic value of TCF3 in HCC patients for the first time.Specifically, overexpression of TCF3 may drive hepatocarcinoma development by enhancing cell proliferation via activating Wnt signaling pathway.

Acknowledgments

None.

CRediT authorship contribution statement

Xing-Yu Pu:Data curation, Formal analysis, Writing –original draft.Dao-Feng Zheng:Data curation, Resources, Writing –original draft.Tao Lv: Methodology, Writing –original draft.Yong-Jie Zhou:Data curation, Formal analysis, Investigation.Jia-Yin Yang:Methodology, Validation, Writing –review & editing.Li Jiang:Conceptualization, Funding acquisition, Supervision, Writing –review& editing.

Funding

This study was supported by a grant from the Key Research and Development Program of Sichuan Provincial Department of Science and Technology ( 2020YFS0134 ).

Ethical approval

This study was approved by the Ethics Committee of the West China Hospital of Sichuan University.Written informed consent was obtained from the patients.

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.