KDM5B expression in HBV-related HCC
We performed transcriptomic analysis of 139 retrospectively collected HCC (cohort 1) patients to assess whether KDM5B was associated with hepatocarcinogenesis. We evaluated and compared the expression of KDM5B in HCC cases developed from various backgrounds: chronic hepatitis B, chronic hepatitis C, alcoholic liver disease, and nonalcoholic steatohepatitis. As shown in Figure 1A, KDM5B was significantly highly expressed in HBV-related HCC cases compared to other etiological-related HCC cases. Moreover, for HBV proteins, only HBx up-regulated KDM5B in HepG2 cells and immortalized human hepatocytes (TTNT16 and T5b cells) (Figure 1B). These results indicated that HBx was associated with HBV-related hepatocarcinogenesis through the activation of KDM5B.
Figure 1 Heterogeneity of KDM5B expression in various hepatocellular carcinoma cases.
A: Expression analysis of KDM5B-based quantitative RT-PCR data in 139 HCC samples. The horizontal lines in the boxplots represent the median, the boxes represent the interquartile range, and the whiskers represent the 10th and 90th percentiles. A nonparametric test was used to compare the 5 patient groups of hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholic liver disease (ALD), nonalcoholic steatohepatitis (NASH) and others respectively as indicated by P values; B: Expression of KDM5B gene in HepG2, TTNT16, and T5B cells. HBV proteins were introduced into these cells by a retroviral system; C: Kaplan-Meier plot of the overall survival of 238 hepatocellular carcinoma (HCC) cases based on their classification by KDM5B expression; D: Kaplan-Meier plot of recurrence-free survival of HCC cases based on their classification by KDM5B expression.
To understand the effect of KDM5B on the survival of HBV-related HCC cases, we analyzed the microarray data of 238 HCC cases (cohort 2). Kaplan-Meier survival analysis revealed that the KDM5B high expression group had a significantly shorter overall survival (P < 0.05) and recurrence-free survival (P < 0.05) than the KDM5B low expression group (Figure 1C and 1D).
KDM5B regulates hepatic stem cell-like features in HCC
Previously, we identified two HCC subgroups, one resembling the gene expression signatures of hepatic stem cells (HpSCs) (referred to as HpSC-HCC) and the other similar to mature hepatocytes (referred to as MH-HCC). HpSC-HCC had stem cell-like features and a poorer prognosis than MH-HCC cases. We hypothesized that KDM5B induced hepatic progenitor-like features in HBV-related HCC cases.
To determine whether KDM5B was functionally linked to the HpSC-like phenotype, we analyzed representative expression levels of HpSC markers in cohort 2 HCC cases. Consistently, HpSC markers, such as EpCAM, AFP, PROM1, and NANOG, were more abundantly expressed in KDM5B high expression HCC cases as compared to KDM5B low expression cases (Figure 2A). Moreover, KDM5B levels were positively correlated with EpCAM or AFP levels in 238 HCC cases (Figure 2B and C).
Figure 2 KDM5B is associated with HpSC markers in hepatocellular carcinoma cases.
A: Expression analysis of HpSC markers (EpCAM, AFP, PROM1, and NANOG) in cohort 2 cases based on their classification by KDM5B expression; B: Spearman correlation analysis of KDM5B and EpCAM expression data as determined by mRNA arrays of cohort 2 cases; C: Spearman correlation analysis of KDM5B and AFP expression in 238 HCC cases.
Next, to confirm that HBx upregulated KDM5B and HpSC markers in HCC cells, we introduced HBx-wt in HepG2 and Huh7 cells and evaluate the expression of KDM5B and HpSC markers. Introduction of HBx up-regulated the expression of KDM5B, EpCAM, AFP, PROM1 and NANOG in these cells (Figure 3A and B).
Figure 3 Introduction of hepatitis B virus X protein is associated with KDM5B and HpSC markers expression.
A: Expression analysis of KDM5B and HpSC markers (EpCAM, AFP, PROM1, and NANOG) in HepG2 cells; B: Expression analysis of KDM 5B and HpSC markers in Huh7 cells.
In addition, inhibition of KDM5B suppressed cell invasion (Figure 4A and B) and spheroid formation (Figure 4C) in Hep3B and Huh7 cells. However, KDM5B did not affect cell proliferation or apoptosis in these cells as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and transferase-mediated dUTP nick-end labeling assays (data not shown). These data indicated that KDM5B is an important molecule for maintaining HpSC-like features in HBV-related HCC cases.
Figure 4 Inactivation of KDM5B is associated with stem-like features.
Effect of KDM5B siRNA on KDM5B expression (A); Cell invasion of Huh7 cells transduced with KDM5B siRNA as determined by the Boyden chamber cell invasion assay (B); Spheroid formation of Huh7 cells transduced with KDM5B siRNA. A vertical axis indicates a total number of spheroids from 1000 cells (C); c-Myc mediated silencing of KDM5B and HpSC markers (D). Effect of c-Myc siRNA on the endogenous levels of c-Myc (D), KDM5B (E), and EpCAM (F) in Huh7 cells.
The available Chip-Seq data (http://genome.ucsc.edu/ ) revealed that several transcriptional factors, such as c-Myc and TCF, preferentially bound to the immediate 5′ upstream sequence of the predictive transcription initiation site of KDM5B. Moreover, c-Myc is one of the transcription factors known to be activated by HBx. To determine whether c-Myc regulates KDM5B expression, we silenced c-Myc expression with a c-Myc-specific siRNA in Hep3B and Huh7 cells. Consistently, we found that inhibition of c-Myc resulted in the suppression of KDM5B and EpCAM expression (Figure 4D-F).
KDM5B predicts poor prognosis in HCC cases
We performed IHC analysis of cohort 3 cases. We confirmed the nuclear accumulation of KDM5B stained by an anti-KDM5B antibody (Figure 5A). After evaluating the clinicopathological characteristics of EpCAM-positive and -negative HCC cases, we found that EpCAM-positive HCCs were associated with a significantly high frequency of KDM5B-positive cases (Figure 5B). We further identified the survival outcome of these cases by Kaplan-Meier survival analysis. KDM5B-positive HCCs were associated with significantly lower overall survival outcomes within three years compared with KDM5B-negative HCCs (P < 0.01) (Figure 5C).
Figure 5 KDM5B protein expression is associated with EpCAM protein expression and poor prognosis.
A: Photographs of immunohistochemistry staining with anti-KDM5B and EpCAM antibodies in 89 Japanese hepatocellular carcinoma (HCC) cases; B: Summary of KDM5B and EpCAM expression; C: Kaplan-Meier plot of overall survival of cohort 3 cases based on their classification by KDM5B expression.