Underexpression of Deleted in liver cancer 2 (DLC2) is associated with overexpression of RhoA and poor prognosis in hepatocellular carcinoma

The study protocol was approved by the Ethics Committee of the Central South University. Fresh samples of HCC tissue and pericarcinomatous liver tissue (PCLT, 1 cm away from the carcinoma) were obtained from 53 (46 male and 7 female) patients with primary hepatocellular carcinoma who underwent curative hepatectomy at the Third Affiliated Hospital of Central South University (CSU) during 2000 to 2003. The specimens were immediately frozen in liquid nitrogen and stored at -80°C for western blotting. The median age of these patients was 55 year, ranging from 19~75 year. All specimens obtained from hepatic resection were confirmed by pathological examination and clinicopathological parameters such as tumor diameter, number of tumor node, tumor capsule, histopathological classification, venous invasion, extrahepatic metastatic lesion and hepatitis virus infection were obtained.

Because there is no commercial DLC2 antibody at present, a mouse anti-human DLC2 monoclonal antibody was developed through ProMab Company. Brief, a 15aa polypeptide representing a unique epitope was synthesized according to the full-length DLC2 sequence [15], then the polypeptide was coupled with KLH. After injection in mouse, immune serum was obtained following standard techniques. Western blot confirmed the specificity of the antibody for DLC2. The quality control examination showed that the antibody is valid.

Tissues from HCC and PCLT were lysed in a lysis buffer (Sigma, St. Louis, MO, USA) and protease-inhibitor (Promega, Madison, USA). The lysates were centrifuged at 13,000 g for 20 min at 4°C and the supernatants were stored at -80°C. Extracts equivalent to 50 μg of total protein were separated by SDS-PAGE gel and were blotted to polyvinylidene fluoride membrane (Sigma, St. Louis, MO, USA). After they were blocked in 4% nonfat dry milk in PBS containing 0.1% Tween-20 for 1 h at room temperature, the membranes were incubated with primary antibody (mouse anti-human DLC2 monoclonal antibody, ProMab, China, diluted at 1:500) for 1 h at 37°C. After washing, the membranes were incubated with a 1:5000 dilution of horseradish peroxidase-linked rabbit anti-mouse antibody (ProMab, China) for 30 mins at 37°C. Then the membranes were washed and treated with western blotting luminal reagent (Santa Cruz, California, USA) to visualize the bands, the results were obtained on Kodak film and quantified by densitometry (Beckman, South Pasadena, Canada) [16].

One hundred and twenty-eight HCC deparaffinized specimens, including the 53 cases of HCC fresh specimens used for Western blotting, were evaluated for immunohistochemistry. All the specimens were collected from the Third Affiliated Hospital of Central South University (CSU) between 1991 and 2003. The clinicopathological parameters of all the specimens, including tumor diameter, number of tumor node, tumor capsule, histopathological classification, venous invasion, extrahepatic metastatic lesion and hepatitis virus infection were obtained. Follow-up data was obtained following the operations for all patients; the endpoint of the follow-up was set at the patients' death. In brief, tissue sections of 4 μm thick were cut and baked at 60°C for 2 h, deparaffinized in xylene and rehydrated through graded ethanol. Next, 3% hydrogen peroxide was applied to block the endogenous peroxidases for 20 mins, and the sections were subjected to heat-induced antigen retrieval in 0.01 M citrate buffer (PH = 6.0). The sections were incubated with normal rabbit serum to reduce non-specific binding. Then, they were incubated at 37°C for 1 h with specific antibodies (mouse anti-human DLC2 monoclonal antibody, ProMab, China) used at a 1:50 dilution. The second antibody was applied for 45 min at 37°C. The Streptavidin-biotin-peroxidase complex (SABC) tertiary system (Boster, Wuhan, China) was used according to the manufacturer's instruction for 20 min at room temperature. The tissues were visualized by applying 3, 3-diaminobenzidine tetrahydrochloride (DAB) for 3 min. Sections were counterstained using haematoxylin, dehydrated through gradient alcohols and mounted for viewing. Negative controls were done by omitting the primary antibody, whereas, HCC specimens of high DLC2 expression confirmed by Western blotting were used as positive controls. The intensity of cytoplasmic staining was scored as 0 to 3+ by comparison to the positive controls. DLC2-negative expressions were devoid of any cytoplasmic staining or contained faint, equivocal staining (scores 0 and 1+) [12].

Expression of DLC2 protein was detected in all HCC and PCLT. The relative expression of DLC2 protein was 0.40 ± 0.06 in HCC and 0.57 ± 0.08 in PCLT respectively. HCC tissues revealed significantly lower levels of DLC2 protein than PCLT (P = 0.02) (Figure 1).

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The distribution pattern of DLC2 was determined by dividing the expression levels of DLC2 protein in subgroups by clinicopathological parameters. HCC with poor cell differentiation showed a lower expression level of DLC2 protein than those with well cell differentiation. There were no significant associations between expression of DLC2 gene and other clinicopathological parameters such as sex, hepatitis virus infection, liver cirrhosis, tumor size and capsular condition, venous invasion and nodular number (Table 1). The same result was observed based on the correlation between immunohistochemical study and clinicopathological parameters of 128 cases of HCC specimen (Table 2).

The protein expressions of DLC2 and RhoA were examined simultaneously. The protein expressions of DLC2 and RhoA showed strongly negative correlation. (correlation coefficient = -0.447, P = 0.01) (Figure 2).

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To further investigate the correlation between DLC2 expression level and prognosis, HCCs were divided into the DLC2 negative expression group (immunohistochemistry score 0 and 1+; n = 95) and DLC2 positive expression group (immunohistochemistry scores 2+ and 3+; n = 33). DLC2 expression level and the prognosis of HCC patients were analyzed by Kaplan-Meier method. The result showed the mean overall survival of patients with DLC2-negative expression (414 days) was significantly lower than those of patients with DLC2-positive expression (697 days, P = 0.003). HCC Patients with DLC2-negative expression revealed significantly poorer prognosis than those with DLC2-positive expression (Figure 3).

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