BolA family member 2 enhances cell proliferation and predicts a poor prognosis in hepatocellular carcinoma with tumor hemorrhage

Objective: BolA family member 2 (BOLA2) is a novel gene highly associated with human hepatocellular carcinoma (HCC) progression. Tumor hemorrhage (TH) acts as a poor marker for HCC patients and is a community affair in the tumor microenvironment. In the present study, we examined a possible association between BOLA2 levels and HCC patients with TH. Methods: The mRNA and protein levels of BOLA2 were determined in two independent cohorts of HCC specimens by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) analysis, respectively. Survival curves and Cox regression models were used to evaluate the prognosis of HCC patients. The CRISPR/Cas9 system was used to knock out BOLA2 in HCC cells, and the functional role of BOLA2 in HCC cell proliferation in vitro and growth in vivo was examined. Results: BOLA2 mRNA expression is significantly higher in HCC tumour tissue than in nontumour tissue. Immunohistochemistry analysis of HCC tissues showed that BOLA2 protein was significantly correlated with TH, a more metastatic phenotype and worse HCC survival. The potential clinical relevance of BOLA2 expression and TH was validated by a Cox regression model. Furthermore, loss-of-function studies determined that BOLA2 plays critical roles in promoting iron overload, tumor growth and TH. Bioinformatics analysis from Gene Expression Profiling Interactive Analysis (GEPIA) revealed that BOLA2 is closely associated with the activation of p62-Keap1 signalling and ATG4B expression. These results were confirmed by immunohistochemistry analysis in HCC tissues. Conclusions: Our results suggest that BOLA2 plays an important role in cancer biology and is an independent predictor of prognosis in HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most malignant tumors and has an extremely poor prognosis [1]. The tumor microenvironment plays a critical role in promoting HCC progression and metastasis [2]. As an underlying key indicator of a fast-growing tumor, tumor hemorrhage (TH) is associated with progression and metastasis [3]. TH has been demonstrated to be a poor marker for HCC patients [4]. Therefore, determining the underlying mechanistic basis of these pathogenetic changes in HCC could greatly benefit the efficacy of the clinical treatment of HCC.

Ivyspring International Publisher
BolA family member 2 (BOLA2) is a nonclassical secreted member of the BolA protein family that also contains BOLA1, BOLA2 and BOLA3 [5]. Previous studies have revealed that BOLA2 functions as a key regulatory for intracellular iron homeostasis in the microenvironment [6,7]. Increasing evidence has revealed that iron homeostasis dysregulation is linked to cancer initiation and development [8,9]. BOLA2 was also reported to potently promote c-Myc's oncogenic activity in liver cancer [10]. Although its expression was reported to be elevated during tumorigenesis, BOLA2 has not been extensively studied in cancer, including HCC.
For the first time, we investigated the expression of BOLA2 expression in both HCC recurrence and metastasis and evaluated the significance of BOLA2 in the prediction of HCC prognosis, especially TH. We analysed the expression and functions of BOLA2 in HCC cell growth and TH. Our findings suggest that BOLA2 can be a potential therapeutic target for the treatment of HCC metastasis.

Patient populations and follow-up
For the use of clinical HCC tissues for research purposes, informed consent was obtained from each HCC patient before hepatic resection. All patients and the study protocol were approved by the Ethics Committee of Central South University (Xiangya Hospital, China). Two independent cohorts of HCC specimens were examined in this study. In the training cohort (n=96), fresh matched specimens of HCC and adjacent non-tumor tissue (NT) were randomly collected from HCC patients undergoing hepatic resection between August 2011 and October 2011 in the Department of Hepatobiliary Surgery, Xiangya Hospital, China. In the validation cohort (n=175), paraffin-embedded specimens were randomly collected from HCC patients undergoing curative resection in Xiangya Hospital from March 2012 to November 2012. HCC specimens accompanied with TH were reviewed by two independent pathologists in our hospital. The following procedures from our previous study were adopted [11]. Follow-up of HCC patients was terminated on 31 January 2017. All research protocols strictly complied with REMARK guidelines for reporting prognostic biomarkers in cancer [12].

HCC cell lines
HepG2, Hep3B, SNU449 and PLC/PRF5 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). L02, SMMC7721, Bel7402 and Huh7 cells were purchased from the Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Science (Shanghai, China). L02 is a normal liver cell line that is usually used as a control cell in cancer research. MHCC97L cell lines were provided by the Liver Cancer Institute of Zhongshan Hospital of Fudan University (Shanghai, China). The characteristics and proteome analysis of MHCC97L have been previously described [13]. All cell lines were routinely authenticated by STR genotyping and mycoplasma detection. Cells were stored in liquid nitrogen and cultured in 5% CO2 at 37°C with high-glucose Dulbecco's modified Eagle media (GIBCO, Grand Island, NY) supplemented with penicillin (100 IU/mL), streptomycin (100 mg/mL) and 10% FBS (GIBCO).

Immunohistochemistry (IHC) analysis
IHC analysis and scoring were performed as previously described [14]. A BOLA2 polyclonal antibody (GeneTex, MA, USA, Cat. # GTX51984) diluted at 1:1000 was used as the primary antibody. A peroxidase-labelled polymer was conjugated to goat anti-rabbit IgG (DAKO Corporation, Carpinteria, CA, Cat. # K5007) for 30 minutes at room temperature, and color was developed using 3, 3'-diaminobenzidine (DAB) solution. BOLA2 expression was assessed when cytoplasmic or membranous staining was observed.

Establishment of BOLA2-knockout HCC cell lines
The characteristics of HCC Hep3B cells with high intracellular iron content have been described in the literature [16]. BOLA2-deficient Hep3B cells were established using the CRISPR/Cas9 system. The gRNA was selected with the assistance of the CRISPR design tool according to a standard protocol [17]. The sgRNA sequences of BOLA2 were 5'-CATGGCAAGC GCGAAAAGCC-3'; and bottom strand: 5'-TCACCA CATGCTCCGCCTCC-3'. For control cells, no guide RNA was inserted. Each experimental replicate was performed using the HCC cell line Hep3B.

Cell proliferation assay
Eight hundred Hep3B cells per well were grown in 96-well plates, and a cell proliferation assay was performed using a Cell Counting Kit-8 (Med Chem Express, USA, Cat. # HY-K0301) according to the manufacturer's protocol. Each experiment was repeated three times.

Colony-forming assay
Hep3B cells were seeded into six-well plates (1000 cells/well). After incubation for 14 days, the colonies that formed were washed and then stained with 0.2% crystal violet (Fisher Scientific, Asheville, NC, Cat. # R40053). The colonies were quantified from three independent experiments per treatment group.

Tumor xenograft model
All animal work was conducted in accordance with protocols approved by the Animal Care and Use Committee at Central South University, China. A subcutaneous HCC mouse model was established according to an existing protocol [18]. BOLA2-knockout Hep3B cells (2×10 6 cells/mouse) were injected subcutaneously into the right axilla (200 μl). All nude mice were sacrificed six weeks after tumor implantation. Prussian blue staining (Sigma-Aldrich, Cat. # 03899) was performed on sections of each transplanted tumor lesion. In addition, IHC staining against BOLA2, p62 and Ki-67 was performed on paraffin sections.

Bioinformatics prediction
We used the Oncomine database (www.oncomine.com) to predict the levels of BOLA2 mRNA in HCC and normal tissues. Then, Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) was employed to forecast a potential correlation between BOLA2 mRNA expression levels and the overall survival of HCC patients [19]. Images of BOLA2 protein expression in HCC specimens and normal liver tissues were obtained from the Human Protein Atlas (www.proteinatlas.org) online database [20]. Tumor Immune Estimation Resource (TIMER) (https://cistrome.shinyapps.io/timer/) was used to study the correlation between the BOLA2 gene and a pair of user-defined genes in liver hepatocellular carcinoma (LIHC) [21]. A protein-protein interaction network of BOLA2 highly correlated genes was generated by the STRING database (http:// string-db.org).

Statistical analysis
Statistical analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL, USA) and GraphPad Prism software (GraphPad Software, San Diego, CA). Quantitative values are presented as the mean ± SD or median (range). Paired t tests and Student's tests were used for paired and unpaired continuous data, respectively. The χ 2 test was applied for categorical data. The cumulative overall survival (OS) and recurrence-free survival (RFS) were evaluated using the Kaplan-Meier method and the log-rank test. A Cox proportional hazards regression model was used to determine whether BOLA2 expression is an independent prognostic indicator. Statistically significant changes are indicated with asterisks. *= (P < 0.05); **= (P < 0.01); *** = (P <0.001).

Overexpression of BOLA2 correlated with poor survival in HCC
We performed a pan-cancer analysis of the expression of BOLA2 genes in normal and cancerous tissues by using the Oncomine database and found that BOLA2 was increased in most types of solid tumors, including breast, colorectal, pancreatic, and liver ( Figure 1). Similar results were found through the online GEPIA database ( Figure 2A). BOLA2 mRNA was significantly increased in HCC compared with normal liver tissues (P<0.05, Figure 2B). High levels of BOLA2 mRNA also strongly correlated with a higher HCC stage, worse survival (P = 0.0095) and disease-free survival (P = 0.047) ( Figure 2C). In addition, the human protein atlas verified a similar expression trend of BOLA2 protein within HCC samples ( Figure 2D).

Association between BOLA2 and TH
We examined the relative transcription levels of BOLA2 by qRT-PCR in tumor tissues and corresponding nontumour tissue (NT) in a cohort of 96 HCC patients. The mRNA expression of BOLA2 in the HCC tumor tissues of the training cohort significantly correlated with the clinicopathological features of tumor invasion and aggression, such as venous invasion, satellite nodules, tumor hemorrhage (TH), tumor differentiation and HCC stage (Table 1). Intriguingly, when 96 HCC tumors were classified into subgroups by TH or not ( Figure 3A), HCC tumors with TH showed even higher expression of BOLA2 (1.748 ± 0.118 versus 2.317 ± 0.135, P<0.01; Figure 3B). TH indicated a worse tumor immunological microenvironment [4,22]. As shown in Figure 3C, by using TIMER, a positive correlation was observed between BOLA2 mRNA expression and defined reactive oxygen species (ROS) modulator genes in HCC, such as p62, autophagy-related 4B (ATG4B) and Kelch-like ECH-associated protein 1 (Keap1), whereas BOLA2 mRNA expression was inversely correlated with nuclear factor erythroid 2-related factor 2 (NRF2). These results suggest that BOLA2 expression correlates with oxidative stress and TH.

BOLA2 expression predicts clinical outcome in HCC
To confirm the above results, in a training cohort of HCC samples, we validated the expression of BOLA2, p62, Keap1, NRF2, and ATG4B and the proliferation marker (Ki-67) using immunohistochemistry ( Figure 4A). HCC patients with TH and lower BOLA2 expression had higher levels of NRF2, whereas patients with higher BOLA2 expression had increased ATG4B, p62 and Keap1 levels ( Figure 4B). Furthermore, HCC patients with TH showed even higher Ki-67 expression ( Figure 4C). Moreover, the expression levels of BOLA2 protein were confirmed in another validation cohort containing 175 HCC patients ( Table 2). Cox's multivariate analysis indicated that BOLA2 expression was an independent positive prognostic factor in the training cohort (HR=2.108, 95% CI 1.541 to 6.067; Table 3). The BOLA2 high expression group exhibited worse overall survival (OS) and shorter recurrence-free survival (RFS) than the BOLA2 low expression group (Figure 4D and 4E). The 1-, 3-, and 5-year OS rates in the high expression group were significantly lower than those in the low expression group (60.2% versus 79.8%, 28.4% versus 48.5%, and 18.2% versus 45.7%, respectively). The 1-, 3-, and 5-year RFS rates were markedly lower in the high expression group than in the low expression group (52.2% versus 75.6%, 18.4% versus 39.7%, and 16.6% versus 43.4%, respectively). Taken together, these results suggest that the value of BOLA2 may be a clinical predictor of HCC prognosis.

BOLA2 knockout suppresses HCC cell proliferation, tumor growth and TH
The protein levels of BOLA2 among eight established HCC cell lines were compared with the immortalized human hepatocyte cell line L02. BOLA2 expression was higher in HCC cells than in L02 cells ( Figure 5). We knocked out BOLA2 in Hep3B cell lines using CRISPR/Cas9 technology and evaluated the effects of BOLA2 in these cells. BOLA2 modulation exhibited significant effect on the protein expression of p62 and Keap1 in BOLA2-deficient Hep3B cells ( Figure 6A). Cell proliferation assays revealed that BOLA2 knockout potently slowed cell proliferation compared with the WT cell group ( Figure 6B). Compared with WT cells at day 4, the suppressive rates were 37.5% (KO-1) and 33.3% (KO-3) in the three BOLA2-deficient colonies derived from Hep3B cells. Cell colony assays further confirmed the similar suppressive effects of BOLA2 knockout ( Figure 6C), with inhibitory rates of 45.3% and 33.6% in Hep3B KO-1 and KO-3 cells, respectively. These findings indicate the oncogenic role of BOLA2 in HCC tumor progression. Next, we attempted to determine whether BOLA2 knockout had the same suppressive effects on HCC tumor growth in vivo. Subcutaneous xenograft animal studies were performed to further investigate the tumorigenicity of BOLA2 in vivo. Compared to the WT group, the knockout of BOLA2 in HCC cells induced strong tumor growth suppression ( Figure  6D). The average tumor weights in the KO-1, KO-3, and WT groups were 0.61±0.08 g, 0.43±0.08 g, and 0.98±0.06 g, respectively ( Figure 6E). We found that xenografts derived from Hep3B-KO-BOLA2 cells exhibited decreased TH ( Figure 6F, left panel). Intracellular iron is essential for HCC cell growth [23]. Another intriguing finding was that after the injection of Hep3B-KO-BOLA2 cells, the iron scores decreased markedly in the transplanted tumors of nude mice according to the histological examination of Prussian blue staining (0.61±0.04 vs. 0.99±0.10 vs. 2.29±0.36, Figure 3F, right panel). This observation led us to further explore the mechanism by which BOLA2 regulates the growth of HCC ( Figure 6F, right panel).

Involvement of p62-Keap1 signalling in BOLA2-mediated HCC cell proliferation
IHC staining was performed on serial sections of Hep3B cell transplantation tumors, and the protein expression of BOLA2 and p62 was significantly lower in the Hep3B-KO-BOLA2 group than in the WT group ( Figure 7A). Proliferating cells were quantified by counting the number of Ki-67-positive cells; more Ki-67-positive cells were observed in WT tumors than in Hep3B-KO-BOLA2 tumors ( Figure 7B). STRING was used to reveal the interactions of the BOLA2 pathway in HCC (Figure 7C), and a subnetwork of 20 genes was presented that included mTOR, AKT1, and MAPKP1, which are implicated in regulating tumor growth [24,25]. High levels of p62 expression are needed for the activation of NRF2 or mTORC1, the induction of c-Myc, and the protection of HCC-initiating cells from oxidative stress-induced cell death [26]. Thus, we speculate that BOLA2 might promote the development of HCC and maintain cancer cell growth under metabolic stress conditions.

Discussion
HCC is highly lethal because of its aggressive metastasis and the minimally effective strategies available against metastasis [27]. HCC is characterized by its fast growth and multiple factors, including genetic and epigenetic alterations that cause uncontrolled cellular proliferation and metastasis. In the current study, BOLA2 overexpression was positively correlated with venous invasion, TH, satellite nodules, and TNM stage. Moreover, the survival analysis showed that HCC patients with BOLA2 high expression had shorter OS than those with BOLA2 low expression. We hypothesized that BOLA2 might act as a tumor promoter to enhance HCC development. To confirm this hypothesis, functional studies showed that the knockout of BOLA2 reduced HCC cell proliferation in vitro and decreased tumor growth subsequent to TH in vivo. Similar to other investigations that reported the rapid growth rate in HCC, BOLA2 overexpression enhanced proliferation and colony formation in HCC cells, highlighting its importance in HCC progression.
We previously demonstrated that TH was involved in fast-growth HCC. The precise mechanism underlying this BOLA2-mediated TH of tumor growth remains to be determined. BOLA2 expression is regulated by at least four mechanisms: (i) specific in-frame fusion transcript regulation, which controls the copy number; (ii) monothiol CGFS glutaredoxin binding partners [28]; (iii) a glutaredoxin-3 (GRX3)-dependent anamorsin maturation pathway [29]; and (iv) as a c-Myc-targeted gene in HCC [10]. In this work, we performed a TIMER analysis, and the results indicated that BOLA2 is highly associated with p62 expression in the tumor immunological microenvironment. A recent study showed that activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in HCC cells [30]. The Keap1-Nrf2 system and autophagy are both involved in the oxidative stress response, metabolic pathways, and innate immunity, and the dysregulation of these processes is associated with pathogenic processes [31]. Therefore, we speculate that BOLA2 contributes to uncontrolled cellular proliferation, which may cause HCC development.  It is widely accepted that carcinogenesis is a multistep process that involves cell proliferation, adhesion, growth, and metabolism [32]. Increasing evidence has revealed alterations in iron metabolism in HCC that facilitate cancer cell growth [33]. Recent evidence suggests that the iron-mediated production of reactive oxygen species promoted autophagy in cancer stem cells [34]. TH is a very complex process in the tumor microenvironment and involves hypoxia, cell death, necrosis, the immune response, angiogenesis and nutrient-generating autophagy, etc. [3]. We found the overexpression of BOLA2 in human HCC tumors with TH; these findings reveal BOLA2 as a potential target against HCC.
Iron is essential for the proliferation of normal and neoplastic cells. Many cancers exhibit an increased requirement for iron, because the need for iron as a regulator is essential for growth and proliferation [9]. Excess liver iron (iron overload) is one of the major risks leading to increased HCV hepatitis, fibrosis, cirrhosis, and HCC [35]. Recent studies have reignited interest in the deregulation of iron homeostasis as an anticancer therapy [36,37]. Further studies of BOLA2 in HCCs will be of interest to clarify its precise role in tumor growth and TH.

Conclusions
Collectively, the present results indicate that BOLA2 is highly expressed in tumor tissues and is significantly correlated with a poor prognosis in HCC patients. Moreover, BOLA2 serves as a key emerging molecule in HCC cell growth and TH. These findings have important implications for identifying new therapeutic targets in HCC.