CBX4 Expression and AFB1-Related Liver Cancer Prognosis CBX4 Expression and AFB1-Related Liver Cancer Prognosis

Background : Previous studies have shown that chromobox 4 (CBX4) expression may involve in the progression of liver cancer, however, it is unclear whether it affects the prognosis of hepatocellular carcinoma (HCC) related to aflatoxin B1 (AFB1). Methods : A retrospective study was conducted in the high AFB1 exposure areas and a total of 428 patients with HCC were included in the final survival analyses. AFB1 exposure levels and CBX4 expression in the tumor tissues were tested using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. The effects of AFB1 and CBX4 on HCC out come were elucidated by Kaplan–Meier survival method and Cox regression model. Results : We found that the levels of AFB1 exposure and CBX4 expression in tumor tissues were significantly associated with some clinicopathological features such as microvessel density and tumor stage. Furthermore, both AFB1 and CBX4 significantly modified overall survival and tumor reoccurrence-free survival status of HCC. Additionally, some evidence of CBX4-AFB1 interaction affecting HCC prognosis was observed, with an interactive value of 1.98 for overall survival and 1.94 for tumor reoccurrence-free survival, respectively. Conclusion : These results suggest that CBX4 expression might be a useful marker for AFB1- related HCC prognosis.

The chromobox 4 (Cbx4) (GenBank accession NO. 8535) consists of six exons and spans about 6.26 kb on chromosome 17q25. 3. This gene encodes a 560-amino acid protein which is the important component of polycomb repressive complex 1 (PRC1) [19][20][21][22]. Functionally, CBX4 involves in PRC1-regulated transcription repression and post-translation modification [19][20][21][22]. Recently, increasing evidence has exhibited that the dysregulation of this gene may affect the carcinogenic process of some tumors such as HCC, colorectal cancer, breast cancer, and so on, and may be a significant prognostic biomarker [19,21,[23][24][25][26][27][28][29]. However, it is not clear whether CBX4 modify the prognosis of AFB1-related HCC. Here, we conducted a hospital-based retrospective study to investigate whether the CBX4 expression in the cancerous tissues is associated with the outcome of HCC related to AFB1 expression in the Guangxi Region, a high AFB1 exposure area.

Study population
Between January 2009 and December 2012, 428 consecutive patients with histopathologically confirmed hepatocarcinoma were recruited at the Divisions of Oncology and Pathology, the affiliated Hospitals of Guangxi Medical University and Youjiang Medical University for Nationalities. During the recruitment phase, only 5 cases refused to participate in the study (response rate 98.8%). All cases were from high AFB1 exposure areas, including Nanning, Bose, Tiandong, and Tianyang. After informed consent was obtained, surgically removed tumor samples were collected to analyze the amounts of AFB1-DNA adducts and CBX4 protein in the cancerous tissues. Additionally, all corresponding clinicopathological and survival following-up data were also collected in the hospitals as previously described methods [30][31][32]. In this study, the status of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection was evaluated using serum hepatitis B surface antigen (HBsAg) and anti-HCV, respectively; whereas the grade and stage of tumor was elucidated using the Edmondson and Steiner (ES) grading system and the Barcelona Clinic Liver Cancer (BCLC) staging system, respectively. For survival analyses, the last follow-up day was set on December 31, 2017. The study protocol was carried out according to the approved guidelines by the Institutional Ethics Committee from the Affiliated Hospitals of Youjiang Medical University for Nationalities and Guangxi Medical University.

Microvessel density (MVD) assay
MVD in the cancerous tissues was assessed using the immunohistochemistry staining of CD31 as our previously descripted [30]. In this study, positive status of MVD was defined as microvessel counts more than 50 per ×200 magnifications.

AFB1 exposure data
AFB1 exposure levels were evaluated using the amounts of AFB1-DNA adducts in the cancerous tissues as our previously descripted [31,32]. The amounts of AFB1-DNA adduct were tested using the competitive enzyme-linked immunosorbent assay. In this study, a value than less 1.00 μmol/mol DNA was considered as negative status for AFB1 exposure.

CBX4 expression assays
The level of CBX4 protein expression in cancerous tissues was elucidated using our previously published immunohistochemistry method [33,34]. Briefly, the amounts of CBX4 protein were tested using anti-CBX4 antibody and calculated using immunoreactive score system (IRS). In the present study, positive CBX4 protein in cancerous tissues was define as IRS > 4.

Statistical analysis
Logistic regression model with enter method for variables (including all known clinicopathological features) was used for statistical comparison between groups. The odd ratios (ODs) and corresponding 95% confidence intervals (CIs) were calculated in this model for evaluating the association between clinicopathological features of HCCs and either AFB1 exposure or CBX4 expression. Kaplan-Meier survival method with log-rank test was used for statistical comparisons between different levels of AFB1 expression and CBX4 expression. Multivariate Cox regression model (with retread method based on likelihood ratio test) analyses were performed to calculate the risk strength of independent variates and prognostic values. In this study, all analyses were finished using the SPSS soft version 18.0 (SPSS Inc. Chicago, IL), and a P-value less than 0.05 was defined as statistical significance. Table 1 gave the clinicopathological characteristics of all cases, and a total of 428 patients with HCC were included in the final analyses. All cases were followed-up more than 5 years to obtain median survival time. During the follow-up period, 261 patients with

The effects of AFB1 exposure on the clinicopathological features and the prognosis of HCC cases
In this study, the status of AFB1 exposure was elucidated using the amount of AFB1-DNA adducts in the cancerous tissues. Results from competitive ELISA exhibited the patients with HCC featured a 2.82 ± 1.60 μmol/mol DNA of AFB1 exposure level. To investigate the effects of AFB1 exposure on the clinicopathological features of HCC cases, we defined the amount of AFB1-DNA adducts ≤1.00 μmol/mol DNA as negative AFB1 exposure according to our previous published results [31,32]. Our results showed that these patients with positive AFB1 status (AFB1-DNA adducts: > 1.00 μmol/mol DNA) had higher BCLC stage (adjusted OR = 2.09 and adjusted 95% CI = 1.04-4.24), bigger tumor size (adjusted OR = 69.06 and adjusted 95% CI = 33.62-141.86), and higher MVD (adjusted OR = 2.56 and adjusted 95% CI = 1.36-4.81) compared with those without positive AFB1 status (OR = 1) ( Table 2). Additionally, we also found that the levels of AFB1 exposure were significantly associated with the age of patients with hepatocarcinoma (adjusted OR = 1.80, adjusted 95% CI = 1.22-2.66, and P = 3.07×10 −3 ). However, AFB1 exposure was not correlated with other clinicopathological features of HCCs ( Table 2).
Next, we investigated the effects of AFB1 exposure on the HCC prognosis using Kaplan-Meier survival model ( Figure 1A).

The effects of CBX4 expression on the clinicopathological features and the prognosis of HCC cases
In the present, the levels of CBX4 protein in the cancerous tissues were amounted using immunohistochemistry technique with IRS counting system and the median IRS value was 5.58 for  Figure 1B). Taken together, CBX4 expression in the cancerous might be an important biomarker for HCC prognosis.

The joint effects of AFB1 exposure and CBX4 expression on HCC prognosis
Given that both AFB1 exposure and CBX4 expression modified HCC outcome, we questioned whether CBX4 expression interacted with AFB1 expression, and whether this interaction affected the prognosis of hepatocarcinoma. First, we analyzed the joint effects of AFB1 exposure and CBX4 expression on the prognosis of patients with HCC using Kaplan-Meier survival model (Figure 2). In this model, the combination of AFB1 exposure and CBX4 expression was divided into four groups: cases with negative-AFB1 and negative-CBX4 status (AC-1), cases with negative-AFB1 and positive-CBX4 status (AC-2), cases with positive-AFB1 and negative-CBX4 status (AC-3), and cases with positive-AFB1 and positive-CBX4 status (AC-4). We found MST and MRT gradually decreased from AC-1 to AC-4 (89.00-11.00 months for MST and more than 125.00-7.00 months for MRT, respectively) (Figure 2A and B).  We next finished multivariable Cox regression analyses based on the retread method with likelihood ratio test (including significant variables and all kinds of possible interactive variables) ( Table 4), and found both AFB1 exposure and CBX4 expression in the cancerous tissues were independent prognostic factors. Furthermore, we also observed that AFB1 exposure significantly and multiplicatively interacted with CBX4 protein expression (interactive values, 1.98 for overall survival and 1.94 for tumor reoccurrence-free survival, respectively).

Discussion
In Guangxi Zhuang Autonomous Region, HCC is the most malignant disease. In the past decades, the annual incidence and death rate (AIR and ADR) of hepatocarcinoma in this area has been reported to remarkably increase (up to about 100-200 per 10,000 for AIR about 50 per 10,000 for ADR) [1]. Lots of epidemiological studies have shown that AFB1 exposure is   the most important cause for this high AIR and ADR [1]. AFB1 is a known I-type chemical carcinogen produced by Aspergillus parasiticus and Aspergillus flavus, and has been proved to involve in the carcinogenesis and progression of HCC [4][5][6][7][8][9][10]. This carcinogenicity of AFB1 mainly results from its metabolic product binding to DNA and inducing DNA damage. Among DNA damage types induced by AFB1, AFB1-DNA adducts are very important, because of its non-enzymatic, time-dependent, and apparent persistent characteristics in the genomic DNA strands [3,35]. Our previous studies have exhibited that AFB1-DNA adducts, especially from liver tissues, are highly associated not only with increasing HCC risk, but with the poor prognosis of HCC [2,31,[36][37][38][39]. Here, our data displayed that increasing levels of AFB1 exposure significantly correlated with higher tumor stage, increasing tumor size, and higher MVD; furthermore, AFB1 was also poor prognostic marker for HCC. Taken together, these data suggest that AFB1 may involve in the startup and progression of HCC.
Noticeably, some evidence of the joint effects of CBX4 and AFB1 on HCC outcome was observed in the prognostic analyses based on the gene-environmental joint effects. Our results showed that CBX4 expression significantly and multiplicatively interacted with AFB1 exposure levels, and that this multiplicative interaction remarkably increased the death risk and tumor reoccurrence risk of patients with HCC. Recently, two studies from high AFB1 exposure areas have also reported that the dysregulation of CBX4 in the cancerous tissues from patients with hepatocarcinoma increases MVD, promotes angiogenesis, and increases sensitivity of HCC cells on anti-cancer drugs [33,34]. Altogether, these results are indicative of the angiogenesis induced by CBX4 involving in the progression of AFB1-related HCC.  In summary, our present study proposes that CBX4 expression in the cancerous tissues can act as a valuable biomarker for AFB1-related HCC. However, several limitations confine the value of this study. First, because of the hospital-based retrospective design, selective bias may take place. Second, because liver damage itself affects AFB1 metabolite and may increase the amount of AFB1-DNA adducts, the prognostic and interactive values of AFB1 and CBX4 may be underestimated. Finally, we did not do functional and mechanical analyses. Therefore, detailed functional analyses deserve further evaluation on the basis of the foresighted design and the combination of AFB1 and CBX4.