Global proteomic profiling in multistep hepatocarcinogenesis and identification of PARP1 as a novel molecular marker in hepatocellular carcinoma

Abstract

The more accurate biomarkers have long been desired for hepatocellular carcinoma (HCC). Here, we characterized global large-scale proteomics of multistep hepatocarcinogenesis in an attempt to identify novel biomarkers for HCC. Quantitative data of 37874 sequences and 3017 proteins during hepatocarcinogenesis were obtained in cohort 1 of 75 samples (5 pooled groups: normal livers, hepatitis livers, cirrhotic livers, peritumoral livers, and HCC tissues) by iTRAQ 2D LC-MS/MS. The diagnostic performance of the top six most upregulated proteins in HCC group and HSP70 as reference were subsequently validated in cohort 2 of 114 samples (hepatocarcinogenesis from normal livers to HCC) using immunohistochemistry. Of seven candidate protein markers, PARP1, GS and NDRG1 showed the optimal diagnostic performance for HCC. PARP1, as a novel marker, showed comparable diagnostic performance to that of classic markers GS and NDRG1 in HCC (AUCs = 0.872, 0.856 and 0.792, respectively). A significant higher AUC of 0.945 was achieved when three markers combined. For diagnosis of HCC, the sensitivity and specificity were 88.2% and 81.0% when at least two of the markers were positive. Similar diagnostic values of PARP1, GS and NDRG1 were confirmed by immunohistochemistry in cohort 3 of 180 HCC patients. Further analysis indicated that PARP1 and NDRG1 were associated with some clinicopathological features, and the independent prognostic factors for HCC patients. Overall, global large-scale proteomics on spectrum of multistep hepatocarcinogenesis are obtained. PARP1 is a novel promising diagnostic/prognostic marker for HCC, and the three-marker panel (PARP1, GS and NDRG1) with excellent diagnostic performance for HCC was established.

Keywords: PARP1; biomarker; hepatocarcinogenesis; hepatocellular carcinoma.

Figure 1. Overview of the study design for marker discovery and verification

Normal livers, NL, hepatitis livers, HL, cirrhotic livers, CL, peritumoral livers, PL, HCC livers, HCC.

Figure 2. Global protein expression patterns on the spectrum of multistep hepatocarcinogenesis and biological functional annotation

(A) The protein expression distributions (upper) and densities representing the distribution (lower) of all proteins in HL (blue), CL (green), PL (yellow), and HCC (red). The dotted line represents threshold of 2 fold-change. (B) Hierarchical clustering of all identified proteins. HL and PL clustered together. HL and HCC comprised a major sample cluster. (C) Gene ontology function analysis of the differentially expressed proteins in HL, CL, PL and HCC groups compared with NL, the top 10 biological process were presented.

Figure 3. Diagnostic performance of FDPS, 14-3-3sigma, TPD52, HSP70, NDRG1, GS, and PARP1 for HCC in 40 nonmalignant nodules (7 NLs, 19 CLs, 14 DNs), 51 HCCs (24 grade 1-2, and 27 grade 3) and 23 ICCs

(A) ROCs of these candidate markers and three-marker panel (GS, NDRG1 and PARP1) for diagnosis of HCC. (B) The AUCs with 95% CI. AUCs of NDRG1, GS, and PARP1 were significantly higher than that for 14-3-3sigma, TPD52, and HSP70. AUCs of 14-3-3sigma, TPD52, and HSP70 were significantly higher than that of FDPS. The AUC value of panel significantly increased to 0.945, when PARP1, GS and NDRG1were combined. *p < 0.01. (C) The expression patterns of PARP1, GS and NDRG1 examined by immunohistochemistry in a series of liver tissues (40×). NL, CL, NL and ICC tissues negatively expresses PARP1, GS and NDRG1, while the HCC tissues show positive staining of the three markers. PARP1, GS and NDRG1 immunoreactivity were primarily examined in the nucleus, nucleocytoplasm and cytoplasm/cytomembrane, respectively. NL, normal liver; CL, cirrhotic liver; DN, dysplastic nodule; ICC, intrahepatic cholangiocarcinoma.

Figure 4. Kaplan-Meier survival curves with regard to overall survival according to PARP1, GS and NDRG1 protein expression in 180 patients with HCC (log-rank test)

(A) Overall survival of patients in high expression of PARP1 significantly worse than that in low expression of PARP1 (p = 0.005). (B) Overall survival of patients in high expression of GS significantly better than that in low expression of GS (p = 0.023). (C) Overall survival of patients in high expression of NDRG1 significantly worse than that in low expression of NDRG1 (p < 0.001).