BCL11B expression in hepatocellular carcinoma relates to chemosensitivity and clinical prognosis

Abstract Introduction B‐cell lymphoma/leukemia 11B (BCL11B) is a subunit of SWI/SNF chromatin remodeling complexes and functions in cell cycle regulation and apoptosis upon DNA replication stress and damages via transcription. Many malignancies were reported to exhibit changes in BCL11B gene expression; however, no study has focused on the relationship between BCL11B and hepatocellular carcinoma, which potentially exhibits DNA replication stress and damages upon its oncogenesis. Thus, in this study, we examined the molecular characterization of BCL11B expression in hepatocellular carcinoma. Methods and Results The cumulative progression‐free survival and overall survival were significantly longer in the clinical cases of BCL11B‐negative hepatocellular carcinoma than BCL11B‐positve cases. Microarray and real‐time PCR analyses in hepatocellular carcinoma cell lines indicated a correlation between BCL11B and GATA6, a gene reported to be correlated with oncogenic activities and resistance to anthracycline, which is often used for hepatocellular carcinoma chemotherapy. Consequently, BCL11B‐overexpressing cell lines exhibited resistance to anthracycline in cell growth assays and the resistance has been evidenced by the increased expression of BCL‐xL in cell lines. The results were supported by the analyses of human HCC samples showing the correlation between BCL11B and GATA6 expressions. Discussions and Conclusion Our results indicated that overexpression of BCL11B amplifies GATA6 expression in hepatocellular carcinoma in vitro and in vivo that leads to anti‐apoptotic signal activation, and induces resistance to chemotherapy, which influenced the postoperative prognosis.


| INTRODUCTION
The B-cell lymphoma/leukemia 11B (BCL11B) gene encodes a lineage-specific C2H2-type zinc finger transcription factor protein. BCL11B expressed in various types of cells and contributes to the development of neuron, T cells, and others. [1][2][3][4][5][6][7][8][9] Furthermore, BCL11B is known as an adipogenesis regulator 10,11 and a haploinsufficient tumor suppressor. [12][13][14][15] The BCL11B allele loss results in the occurrence of human T-cell acute lymphoblastic leukemia and mouse thymic lymphoma. 1,16,17 On the other hand, several studies have reported the effect BCL11B on cell proliferation and chemoresistance in cancer as an oncogene. For instance, BCL11B upregulation has been reported in Ewing sarcoma to maintain its oncogenic character 18 ; the expression of BCL11B has been found to be correlated with the poorly differentiated tumor status of head and neck squamous cell carcinoma 19 ; enriched expression of BCL11B in glioma cells promotes cell growth 20 ; and BCL11B suppression using RNA interference technique inhibited leukemic T cells proliferation by apoptosis. 21 This oncogenic mechanism might involve the apoptosis resistance accompanied by the delay of cell cycle by accumulating cells at G1 associated with the upregulation of p27, p57, and p18. 21 The mechanism is also supported by the report that BCL11B contributes to the maintenance of genomic integrity at cell cycle as a result of the lack of the gene that causes the failure of checkpoint kinase 1 activity in thymocytes. 22 These data support the fact that BCL11B-overexpressing malignant cells may obtain resistance characteristics to chemotherapy and radiotherapy to induce apoptosis. As a fact, various cancer cells activate DNA repair pathways to gain chemo-and radio-resistance characteristics. [23][24][25] Based on this evidence, BCL11B could be a new therapeutic target in the refractory of malignancy to the conventional therapeutics. [26][27][28] Hepatocellular carcinoma (HCC) is a malignancy related to DNA damage and mutagenesis caused by various hepatitis caused by virus, fatty infiltration, and alcoholic liver disease, which leads to carcinogenesis. 29 Some studies have recently reported that DNA repair signaling pathway in HCC cells contributed to worse prognosis [30][31][32][33] ; however, no detailed mechanisms have been reported in this regard. Based on the role of BCL11B in biology, it is reasonable to hypothesize that BCL11B is involved in the clinical course of HCC as it is treated with various DNA-damaging chemotherapy and radiotherapy agents. Regarding the relationship between BCL11B and HCC, only a few studies have reported BCL11B gene modification by mutation, deletion, amplification, truncation, gain, and copy number aberrations, 34,35 immune evasion mechanisms, 36 and retention of cancer stem cell traits in HBV-related HCC. 37 Based on the aforementioned backgrounds, we have investigated the effect of BCL11B expression in HCC cells on resistance to various therapies and on HCC prognosis.

| Clinical course and BCL11B expression in vivo
With a written informed consent, tissue samples were collected from the HCC cases who were diagnosed with imaging studies including magnetic resonance imaging and computed tomography, and underwent surgical resection in Niigata University Hospital. The tissues were stained with hematoxylin and eosin or immunohistochemical stains: Rat anti-Ctip2 (Bcl11b) antibody (ab18465; Abcam), Rabbit anti-GATA6 antibody (ab175349; Abcam), Vectastain Elite ABC Rat immunoglobulin G kit (PK-6104; Vector Laboratories), Vectastain Elite ABC Rabbit immunoglobulin G kit (PK-6101; Vector Laboratories, Burlingame, CA), and 3,3′-diaminobenzidine chromogen tablets (Muto Pure Chemicals). The expression of BCL11B and GATA6 was confirmed via RT-PCR in recently resected 70 cases using the aforementioned procedure. The tumor tissues of these cases were immunohistochemically stained to determine the relationship between BCL11B and GATA6. Images were captured randomly, and analyzed quantitatively, using the ImageJ software (version 1.8.0_172; National Institutes of Health, Bethesda, MD). 38

| Microarray and bioinformatic analyses
Mock-and BCL11B-transfected HLE cell lines were compared for the gene expression using SurePrint G3 Human Gene Expression (v2) Microarray Kit and GeneSpring GX version 14.5.1 (Agilent Technologies, Inc.). Among the 50,599 genes analyzed, 1052, which exhibited >2fold differences in expression, were assessed with related gene expressions using the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database. The gene orthology terms were selected based on Fisher's exact test, followed by the Benjamini-Yekutieli correction method.

| Cells
HLE and HepG2 cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan). The cells were cultured in Minimum Essential Medium with fetal bovine serum (10%) and of penicillin and streptomycin (100 U/mL). The BCL11B complementary DNA was cloned in pCMV6-Entry Tagged Cloning Vector (OriGene Technologies, Inc.). Either mock or BCL11B-cloned vectors were transfected into the cells using FuGENE HD Transfection Reagent (Promega) and selected using G418 sulfate. Three independent clones were isolated from each of the two cell lines for the analyses.

| Gene and protein expression
in the cells BCL11B and GAPDH genes expression was confirmed via reverse transcription-polymerase chain reaction (RT-PCR). RNA was prepared from the cells (RNA Easy Mini Kit) and the complementary DNA was synthesized from 1 to 5 μg of RNA (SuperScript II Reverse Transcriptase, Invitrogen). Complementary DNA products (1-2 aliquots) were used for PCR. The primers used are (F, forward; R, reverse): PCR protocol: 94°C for 10 min, followed by 30 cycles of (94°C for 30 s, 54°C for 30 s, and 72°C for 1 min) followed by 72°C for 7 min of extension.

| Cell growth assay
The cells were plated in 96-well tissue culture dishes at a concentration of 2 × 10 4 cells per well in 100-μL medium. Then, they were treated with CDDP or epirubicin using doses determined based on previously reported studies. 39,40 The cell growth was assessed using the Premix WST-1 Cell Proliferation Assay System (Takara Inc.).

| Statistical analyses
Data were analyzed by paired t-test or one-or two-way ANOVA followed by Bonferroni's multiple comparison test. The cumulative PFS and OS curves were generated by the Kaplan-Meier method, and the occurrence rates were compared using a log-rank test. GraphPad Prism 9 software (version 9.3.1; GraphPad) was used for the analyses. p < 0.05 was considered statistically significant.

| Effect of BCL11B expression in HCC tumor on prognosis
To examine the effect of BCL11B expression in HCC tumor cells on the prognosis of the reported cases, the PFS and OS were assessed in BCL11B-positive (>5% positive in IHC analyses, n = 67) and BCL11B-negative (n = 98) groups. The characteristics of the patients and their clinical information are summarized in Table 1. No significant differences were observed in age, gender, body mass index, etiology of the liver disease, histological classification of HCC, complication of liver cirrhosis, hepatic reserve function, tumor markers, nutrition, and number of postoperative treatments. The PFS (2608 vs. 480 days, p < 0.0001) and OS (3570 vs. 1564 days, p < 0.01) were significantly longer in the BCL11B-negative tumor cell group ( Figure 1). These results indicated that the BCL11B expression in HCC helps gain resistance to various postoperative therapeutic options and leads to poor prognosis.

overexpressing HCC cells
To examine the molecular function of BCL11B on the viability of HCC, we produced BCL11B-overexpressing cell lines by transfecting plasmid DNA-expressing human BCL11B into HCC cell lines, that are, HLE and HepG2. Figure 2A presents the BCL11B gene and BCL11B protein expression in the cells comparing with that of Molt4 cell line, which is known to express BCL11B. Quantitative analyses of multiple clones revealed a significantly higher expression of BCL11B in HLE and HepG2 cells by RT-PCR, WB, and IHC ( Figure 2B,C). Under normal cell culture condition with 10% fetal bovine serum, BCL11Boverexpressing cells exhibited no significant difference in growth rate compared with the mock-transfected cells ( Figure 2D).

| Effect of BCL11B expression on gene expression modification
To determine the molecular mechanism of BCL11B, the gene expressions in mock-transfected HLE and BCL11Boverexpressing HLE were compared via DNA microarray analyses ( Figure 3A,B). Analysis of KEGG orthology terms, after the hierarchical clustering of genes, revealed higher gene expression in terms of various oncogenic pathways, including the Jak-STAT, MAPK, Wnt, and Hippo signaling pathways, apoptosis, cell cycle, p53 signaling, and DNA replication in BCL11B-overexpresing cells than in mock-transfected cells. The genes upregulated and downregulated in BCL11B-overexpressing cells are presented in Tables 2 and 3. Based on the analyses, in order to examine the mechanisms of BCL11B on  prognosis of HCC cases, we have focused on the correlation of GATA binding protein 6 (GATA6), which showed 78905.9-fold increase upon BCL11B-overexpression, and apoptosis-related gene expression. Moreover, GATA6 has been reported to be related to the differentiation and progression of the malignant disease, such as pancreatic cancer, colorectal carcinoma, and HCC. [41][42][43][44][45][46] The BCL11B-overexpressing HLE and HepG2 cell lines demonstrated a significantly higher GATA6 expression than the mock-transfected cells ( Figure 4A,B). Furthermore, the BCL11B expression contributed to BCL-xL increase in both cell lines and BCL-2 increase in HLE but not to BAX expression. Altogether, these results indicated that BCL11B overexpression in HCC cells contributes to the induction of GATA6 expression and anti-apoptotic phenotype ( Figure 4C).

| Effect of BCL11B expression on cell growth under a cytotoxic condition
To examine the effect of BCL11B on cell viability under a cytotoxic condition, cell growth assay was conducted using BCL11B-overexpressing cells exposed to either epirubicin hydrochloride or cisplatin, which are often used in chemotherapy for HCC ( Figure 5). While mocktransfected HLE and HepG2 cell lines exhibited dosedependent cell growth inhibition when treated with epirubicin hydrochloride (Figure 5A,C) or cisplatin ( Figure 5B,D), BCL11B-overexpressing HLE and HepG2 showed a significantly milder inhibition with epirubicin hydrochloride (Figure 5A,C). These results indicated that BCL11B overexpression induced resistance to epirubicin hydrochloride in HCC cells.

F I G U R E 3
Microarray and bioinformatic analyses. KEGG orthology classification for the genes with more than 2-fold differences in the expression. Higher (A) and lower (B) expressions in BCL11B-overexpressing HLE than mock-transfected HLE.

| Expression of BCL11B and GATA6 in HCC tissues
To determine whether BCL11B-induced GATA6 expression contributes to cytotoxic agent tolerance and affects prognosis, the gene and protein expressions in the HCC and surrounding liver tissues were tested by RT-PCR, and immunohistochemical testing was conducted on the HCC tissues. RT-PCR revealed a significant correlation between the BCL11B and GATA6 expressions in tumor and non-tumor tissues ( Figure 6A,B), and the IHC analysis supported the results in tumor tissue ( Figure 6C,D). These findings indicated that GATA6 upregulation in the BCL11B-positive HCC tissue might be related to chemosensitivity to epirubicin hydrochloride, an anthracycline.

| DISCUSSION
Our study demonstrated the influence of the amplified expression of BCL11B upon prognosis. The BCL11B expression in HCC induces resistance to epirubicin hydrochloride, an anthracycline, and results in poor postoperative prognosis. The amplification of GATA6 in BCL11B overexpressed cells and BCL11B-positive HCC tissues might be related to the anthracycline resistance. The mechanisms involved activation of anti-apoptotic proteins, including BCL-xL. It is important to analyze factors involved in anthracycline chemosensitivity, since for intermediate stage HCC, chemoembolization is recommended 47,48 and anthracycline has been reported to be more effective than other chemicals for chemoembolization. [49][50][51] The results obtained in the current study are supported by previous study demonstrating that BCL-xL expression is inhibited in BCL11B knockdown cells and thymocytes of BCL11B knockout mice 22 due to transcriptional repression. A different expression changes between HLE and HepG2 cell lines might be due to the characteristics of the cells, that is, invasive and poorly differentiated HLE cells and noninvasive and hepatoblastoma-oriented HepG2 cells. Furthermore, our study demonstrated the increased expression of GATA6 in BCL11B-overexpressing cells and highly BCL11B-expressing human tissues of HCC and surrounding liver tissues. The GATA family consists of six members (GATA1 to GATA6), which are transcriptional factors with a zinc finger structure. 52 GATA1 to GATA3 are important for differentiation of hematopoietic stem cells and GATA4 to GATA6 are expressed in the various organs including heart, gut, pancreas, lung, and ovary. [53][54][55][56][57] Among them, the GATA6 gene is transcribed in a pattern overlapping GATA4, and its expression is additionally found in liver tissues. 57 Moreover, it works as an oncogenic factor in various types of tumors, including stomach, 58 pancreatic, [41][42][43] and colorectal cancers 44,45 and HCC. 46 Recently, the oncogenic role of GATA6 on HCC has been reported in an in vitro study to counter the tumorsuppressive effect of miR-143. 59 Interestingly, GATA4 and GATA6 have been reported to contribute to cardiac hypertrophy when overexpressed in cardiomyocytes, interacting with numerous cofactors under complex mechanisms. 52 Such mechanisms involve anti-apoptotic signal activation, supported by the findings that overexpression of GATA4 or GATA6 attenuates cardiac muscle cell apoptosis induced by anthracyclines. 60 In addition, GATA4 has been reported to positively regulate anti-apoptotic protein BCL-2. 61 These findings support the results of the present study, which indicated that GATA6 expression in hepatocytes may positively regulate BCL-xL and BCL-2 proteins to attenuate the apoptotic signal with epirubicin hydrochloride, and support HCC growth. The fact that BCL11B is also involved in the mechanisms of the cardiac hypertrophy 7 suggests the potential relationship between BCL11B and GATA6.
Regarding the relationship between BCL11B and the GATA family, it has been reported that BCL11B and GATA3 collaborate to play a crucial role in T-cell development by repressing cyclin-dependent kinase inhibitor 2b. 62 Moreover, it is known that the BCL11B/ GATA3 complex is involved in this sequence and that BCL11B controls the GATA3-mediated gene activation. 63 These reports support our present results indicating the BCL11B-associated GATA6 expression in HCC which activates anti-apoptotic characteristics and led to the poorer postoperative prognosis than non-BCL11B expressed HCC cases. These findings suggest that BCL11B expression in HCC might be the potential therapeutic target; however, further studies focusing on the direct relationship between BCL11B and GATA6 using Bcl11b conditional knockout mice and its contribution in HCC pathology are essential. In addition, as there are several genes up-and down-regulated by the BCL11B overexpression and involved in the key roles of various oncogenic pathways (Figures 3 and 4; Tables 2 and 3), further analyses are important to explore the therapeutic target in BCL11B-expressing HCC, which is also an important factor in various organs and cells.
The current study had several limitations: The number of cases involved is small, more than 50% of our HCC cases are associated with viral hepatitis. Therefore, based on the results obtained in this study, further analyses for larger number of HCC cases with various clinical stages and on various liver diseases including non-alcoholic fatty liver and autoimmune liver diseases, 64 in a multicenter study is encouraged. With the development of systemic treatment including immunotherapy and molecular target therapy for the advanced stage HCC, 65 combination of various agents and chemoembolization is considered to improve the prognosis of HCC. 66 Furthermore, the molecular mechanisms for these therapeutic impacts have been reported. For example, recent studies have shown the therapeutic effectiveness of capecitabine when used as a second-line treatment after sorafenib failure by inducing T-cell apoptosis. [67][68][69] Therefore, basic and clinical studies focusing on the chemosensitivity of agents used for HCC treatment could further contribute to therapeutic advances and a safer HCC treatment.

| CONCLUSIONS
Our results indicated that overexpression of BCL11B amplifies GATA6 expression in HCC in vitro and in vivo, leads to anti-apoptotic signal activation, and induces resistance to anthracycline used in chemotherapy for HCC, which influenced the postoperative prognosis.