MATERIALS AND METHODS

Patients and tissue samples

A total of 224 cases of treatment-naïve HCCs (n=224) were retrieved from the files of the Department of Pathology, Asan Medical Center, Seoul, Korea. All cases were surgically resectable (R0). Early recurrence was defined as a recurrence of the tumor within 2 years after surgery. All patients provided written informed consent, and this study was approved by the Institutional Review Board of Asan Medical Center, Seoul, Korea.

Tissue microarray and immunohistochemistry

Tissue microarrays were prepared using representative formalin-fixed, paraffin-embedded blocks of HCC cases. Two 2-mmthick tissue cores were obtained from the donor blocks and transferred onto the recipient blocks after reviewing hematoxylin and eosin–stained slides. Four-micrometer-thick tissue microarray sections were immunostained using a rabbit polyclonal antiCRP antibody (1:1,000, AbCam, Cambridge, UK). The sections were transferred onto silanized slides, and heat-induced epitope retrieval was performed by treating the slides with Cell Conditioning 1 buffer for 32 minutes in a BenchMark XT automatic immunostainer (Ventana Medical Systems, Tucson, AZ, USA). The signals were detected using the OptiView DAB IHC Detection Kit (Ventana Medical Systems). The immunoreactivity was evaluated by a pathologist (C.J.K.) blinded to clinical information, using a 4-tier grading system: negative, grade 0; positive in less than 10% of tumor cells, grade 1; positive in less than 50% of tumor cells, grade 2; diffusely positive, grade 3.

Cell culture

Human HCC cell lines, Hep3B and SNU-449, were used for the analysis of CRP mRNA regulation by IL-6. Hep3B cells were cultured in Dulbecco’s modified Eagle medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. SNU-449 cells were cultured in RPMI 1640 medium (GE Healthcare Life Sciences, Pittsburgh, PA, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. For IL-6 treatment, 5×10⁵ cells were seeded into 100-mm dishes and were treated with 50 ng/mL of IL-6 (Cell Sciences, Canton, MA, USA) on the following day for 6 hours.

Quantitative real-time polymerase chain reaction

Total RNA was isolated from liver tissues (n=12) and cultured Hep3B and SNU-449 cells using Trizol and the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted RNA (1 µg) was reversetranscribed using the Reverse Transcription System (Promega, Madison, WI, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of CRP mRNA expression were performed using a TaqMan Gene Expression Assay (Hs00357041_ m1, Applied Biosystems, Carlsbad, CA, USA) and an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The human RPLPO (Large Ribosomal Protein) was used as an endogenous control for normalization.

Immunoblotting

Immunoblotting was done in 12 cases. Liver tissues were pulverized in liquid nitrogen, and total protein lysates were obtained using RIPA lysis buffer. Thirty micrograms of protein were electrophoresed in 12% sodium dodecyl sulfate–polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (GE Healthcare Life Sciences). The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and incubated at 4˚C overnight with mouse monoclonal primary antibodies against CRP (1:1,000, AbCam, Cambridge, MA, USA) and vinculin (1:1,000; SigmaAldrich, St. Louis, MO, USA), respectively. The blots were subsequently incubated at room temperature with horseradish peroxidase–conjugated secondary antibody for 1 hour (Cell Signaling Technology, Danvers, MA, USA). The signals were detected using a SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Statistical analyses

The analyses of continuous variables and proportions were done using Pearson’s chi-square test and Fisher exact test. The survival analysis was done using the Kaplan-Meier method. Independent variables and groups were compared using the MannWhitney U test. SPSS ver. 18.0 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

RESULTS

CRP expression in HCC

The demographics of the study population are summarized in Table 1. CRP immunoreactivity was observed as diffuse cytoplasmic immunostaining. Overall, 59.4% (133/224) of HCC cases were immunopositive for CRP, with grades 1, 2, and 3 immunoreactivity in 25.9% (58/224), 20.1% (45/224), and 13.4% (30/224) of the cases, respectively (Fig. 1). When we analyzed the relationship between multiple clinicopathologic parameters and CRP immunoreactivity, we found a significant difference in cancer-specific survival between CRP-negative or non-diffuse immunopositive cases (grades 0, 1, and 2) and grade 3 immunopositive cases (Fig. 2A). Grade 3 CRP immunopositive cases showed significantly shorter survival compared to CRP-negative or non-diffuse immunopositive cases (median of 50.7 months and range of 6-58 months vs. median of 58.5 months and range of 5–88 years; p=.0047). However, no significant correlation was found between the degree of CRP immunoreactivity and other clinicopathologic parameters including recurrence-free survival (Fig. 2B).

CRP mRNA expression in HCC

We further analyzed CRP protein and mRNA expression in HCCs (n=12) to see whether CRP expression is regulated at the transcriptional level. Immunoblotting for CRP confirmed variable expression of CRP in HCC cases (Fig. 3A). CRP signals were readily detected in six cases, but not in the remaining six cases. qRT-PCR analysis demonstrated a higher CRP mRNA expression in CRP immunopositive cases than in immunonegative cases (p=.046; ΔCt median of 3.62, range of 2.8–5.98 vs ΔCt median of 8.56, range of 1.07–15) (Fig. 3B).

After confirming the relationship between CRP protein and mRNA expression, we tested if CRP mRNA transcription was dependent on pro-inflammatory mediators. The treatment of Hep3B and SNU-449 cells with IL-6 at the concentration of 50 ng/mL for 6 hours induced a 17.5-fold increase in CRP mRNA expression in Hep3B cells, but CRP mRNA expression was not detected in SNU-449 cells even after IL-6 treatment (Fig. 4).

DISCUSSION

The primary findings of this study are (1) CRP expression is relatively common in HCCs, with variable immunoreactivity in nearly 60% of the cases tested, (2) there is an inverse relationship between diffuse and strong CRP immunoreactivity and cancer-specific patient survival (p=.0047), (3) CRP-positive cases in immunoblotting showed significantly higher CRP mRNA expression, suggesting that increased CRP production is a consequence of transcriptional activation of CRP, and (4) CRP mRNA transcription is induced by IL-6 in Hep3B cells, but not in SNU- 449 cells, suggesting that CRP expression marks distinct molecular phenotypes among HCCs.

Hepatocellular carcinogenesis represents a classic model of viral etiology associated with chronic inflammation [23]. The elevation of inflammatory biomarkers such as CRP, IL-6, C-peptide, and adiponectin is associated with a higher risk of HCC [24]. Several investigations have addressed the clinical significance of serum CRP. Serum CRP has been consistently shown to be a key component of inflammation-based prognostication of HCC. Mori et al. [25] proposed that a preoperative scoring system based on the preoperative serum concentration of CRP and alpha-fetoprotein has a prognostic value in patients with HCC after hepatectomy. Although the contribution of CRP produced by tumor cells to the elevation of serum CRP cannot be directly assessed, it is very probable that CRP of tumor origin is released into the systemic circulation. Although we expected certain differences in clinicopathologic characteristics between CRP immunopositive and immunonegative HCCs, a clear difference was found only in the cancer-specific survival of strong CRP immunopositive cases.

One of the major potential drives for CRP overexpression is IL-6, and a role of IL-6 in hepatocellular carcinogenesis has been strongly suggested. When compared to healthy controls, cirrhotic patients and HCC patients had serum IL-6 concentrations that were 4-fold and 25-fold higher, respectively [26]. IL-6 signaling mediated via IL-6 receptors activates STAT3, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. IL-6–mediated STAT3 activation is known to be a biological link between chronic inflammation and carcinogenesis [27], and IL-6 confers anti-apoptotic effects to cells. Blocking STAT3 activation using STAT3 siRNA or small molecular STAT3 inhibitor LLL 12 has been shown to abrogate the anti-apoptotic effects of IL-6 against doxorubicin-induced apoptosis in SNU- 449 HCC cells, which express a higher level of endogenous IL-6 compared to Hep3B cells [28]. We thought that CRP would be induced in both Hep3B and SNU-449 cells by IL-6 treatment. However, CRP mRNA expression after IL-6 treatment was significantly different between SNU-449 cells and Hep3B cells. Therefore, the observations in HCC tissue samples and the cell lines in vitro indicate that the degree of CRP expression involves several mechanisms, which need further elucidation.

A major drawback of this study is that serum CRP or IL-6 levels could not be determined due to a lack of blood samples. Future analyses of the relationship between the CRP-positive HCC phenotype and systemic inflammatory profile will provide a more comprehensive understanding of the biology of HCC. We also could not determine the underlying biochemical mechanisms involved in the differential IL-6-induced responses between Hep3B and SNU-449 cells in terms of induction of CRP mRNA expression. Considering the complexities of IL-6 signaling and CRP transcription, more in vitro studies are necessary.

In summary, we report the expression of CRP in HCC for the first time, and provide evidence to support the significance of CRP in HCC based on the clear difference in cancer-specific survival between CRP-positive and -negative cases. Overall findings strongly suggest that CRP is a marker for future molecular phenotyping of HCC.

Acknowledgments

Fig. 1.

Cytoplasmic C-reactive protein (CRP) immunoreactivity in hepatocellular carcinoma cases. Immunoreactivity is analyzed using a 4-tier grading system: grade 0 (A), grade 1 (B), grade 2 (C), and grade 3 (D).

Fig. 2.

Clinical significance of C-reactive protein (CRP) immunoreactivity. (A) There is a significant difference in cancer-specific survival between patients with CRP grade 3 hepatocellular carcinomas (HCCs) and those with CRP grade 0, 1, and 2 HCCs. (B) There is no difference in recurrence-free survival between the two groups.

Fig. 3.

Correlation between C-reactive protein (CRP) mRNA and protein expression in hepatocellular carcinomas (HCCs). (A) Immunoblotting for CRP protein shows variable expression in HCCs (N, non-neoplastic liver; T, HCC). CRP protein expression is not found in the tumor of case 4, while CRP bands are readily detectable in both non-neoplastic and HCC samples of cases 1, 2, and 3. (B) Quantitative real-time polymerase chain reaction results are shown in box plots of ΔCt for CRP mRNA expression (Ct_CRP–Ct_RPLPO).

Fig. 4.

Induction of C-reactive protein (CRP) mRNA in Hep3B and SNU-449 hepatocellular carcinoma cell lines after interleukin 6 (IL-6) treatment. (A) There are no significant changes in cellular morphology in either cell line after IL-6 treatment. (B) There is a 17.5-fold increase in CRP mRNA expression in IL-6–treated Hep3B cells (50 ng/mL for 6 hours), while CRP mRNA expression is not detected in SNU-449 cells. The yaxis represents fold-changes in CRP mRNA expression following IL-6 treatment.

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Figure & Data

References

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