Autoantibodies against glucose-regulated protein 78 as serological biomarkers in metastatic and recurrent hepatocellular carcinoma

Purpose To identify Heptocellular carcinoma (HCC) associated antigens by proteomics, and validate whether autoantibodies against tumor-associated antigens (TAAs) could be used for diagnosis and conditional monitoring. RESULTS The 78 kDa glucose regulated protein (GRP78) was selected as a candidate TAA. The titers of autoantibodies against 78 kDa glucose regulated protein (GRP78) from patients with HCC, liver cirrhosis (LC), and chronic hepatitis (CH) were significantly higher than that from normal controls (P<0.05, P<0.001, and P<0.01, respectively). The expression of autoantibodies against GRP78 was associated with clinical stage (P<0.01), portal vein invasion (P<0.05), and metastasis (P<0.05). The expression of anti-GRP78 antibodies was significantly higher 1 month after surgery in recurrent patients who had accepted hepatic resection 1 month after surgery compared to patients who had surgery before surgery or within 1 week after surgery (P<0.01 and P<0.001). Immunohistochemistry (IHC) showed higher expression of GRP78 in HCC compared to the non-HCC liver tissues (P <0.05). Materials and Methods HCC serum with high titer of autoantibodies against TAAs were screened and used for a proteome-based approach to identify HCC associated antigens. Indirect enzyme-linked immunoassay (ELISA) was used to detect the corresponding autoantibodies against TAAs. Conclusion GRP78 is an autoantigen that could stimulate autoimmune responses and serve as a potential marker for recurrent and metastatic progression in HCC.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide [1]. The majority of patients with HCC are usually in advanced stages of disease with poor prognosis. Alpha fetal protein (AFP) is a common serum marker of HCC, however, its sensitivity and biomarkers of HCC is of great clinical importance. Studies have demonstrated that antigenic changes in cells can be recognized by the immune system of patients and elicit an immunoreaction [4,5]. Many autoimmune diseases such www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 15), pp: 24828-24839

Research Paper
as systemic lupus erythematosus (SLE) [6], diabetes [7], and rheumatoid arthritis (RA) [8] have been shown to have autoantibody responses. Many nonautoimmune diseases such as cancer [9][10][11] also have autoantibody responses-TAAs are cellular proteins whose aberrant regulation of function is linked to malignancy. Autoantibodies stimulated by TAAs are involved in tumorigenesis. Therefore, these autoantibodies could be used as probes to identify antigens that are potentially involved in malignant transformation.
of TAAs in cancer. For instance, serum antibodies from patients can be used to screen cDNA libraries to identify TAAs with potential value as biomarkers for cancer diagnosis. Several studies have used this approach to to the cDNA expression library immunoscreen approach, proteomics technology can be used to identify a large number of antigens and distinguish isoforms and detection of autoantibodies directed against post-translational serum with high titer of autoantibodies against TAAs were whether autoantibodies to TAAs could be used for diagnosis and conditional monitoring in HCC.

Prevalence of autoantibodies in patients with hepatocellular carcinoma
serum samples from HCC patients with high titers of autoantibodies. Some patients' sera contained high titer of proteins, and 76-kDa proteins. The HCC sera contained a higher frequency of autoantibodies to these antigens than that in LC, CH, and normal human sera (NHS) groups.

by proteomic approach
HCC serum with high titer of autoantibodies was selected to identify candidate proteins. Serum samples and HepG cell extractions were subjected to immunoprecipitation followed by SDS-PAGE and coomassie blue staining. Target gels were analyzed including Alpha-actinin-4 (ACTN4), ATP-citrate synthase 78 kDa glucose regulated protein precursor (GRP78), and heat shock protein 75 kDa (TRAP1) were selected based on bioinformation standards, test reliability, and targeted molecular weight (Table 1). These proteins were primarily located in the cytoplasm and involved in biological processes such as protein transport, localization, programmed cell death, and associated with molecular functions such as stress response, and antigen processing

Detection of autoantibodies in sera from 173 HCC patients
Eight recombinant candidate proteins were commercially purchased and further used as coating GRP78, ACTN4, and HSPH1 had different expression levels when compared among HCC, LC, CH, and NHS groups (P was higher than in the NHS (P with early-HCC, LC, CH, and NHS group, the titer of autoantibodies against GRP78 in early stage of HCC, LC, serum autoantibodies against GRP78. The prevalence of anti-GRP78 autoantibodies in the HCC (P and LC (P anti-GRP78 autoantibody detection in HCC were 7.5% and 94.4%. Meanwhile, the positive predictive value and negative predict value of anti-GRP78 autoantibody of NHS samples was used as a cutoff value). The titer of anti-GRP78 autoantibodies was different in sera of HCC patients at different stages of disease (P anti-GRP78 autoantibodies showed a decreasing tendency with clinical and pathological characteristics (both P autoantibody were used simultaneously as biomarkers, 98

Validation of Anti-GRP78 antibodies as biomarkers for monitoring curative effects in HCC patients
The expression of anti-GRP78 autoantibodies the expression of anti-GRP78 autoantibodies in patients surgery in recurrent patients who accepted hepatic resection compared to patients before surgery or within 1 differences in the titer of anti-GRP78 autoantibodies in patients were tested for AFP before and after 1 week of AFP, which was different from that of anti-GRP78 patients declined one month after surgery compared to recurrent HCC patients who had detectable levels of both AFP and GRP78 autoantibodies before surgery and 1 month after surgery while only the titer of autoantibodies against GRP78 was elevated 1 month after surgery. GRP78 staining. The positive rate of GRP78 in the HCC group was higher than in the non-HCC group (P expression of GRP78 in the HCC, LC, CH and normal liver

DISCUSSION
GRP78, also known as immunoglobulin heavy chain (ER) and is involved in proper protein folding and assembly, proteosome degradation of misfolded proteins, ER Ca binding, and the activation of transmembrane ER GRP78 has a causal relationship with tumor occurrence expression of GRP78 was higher in HCC than non-HCC groups, suggesting that GRP78 may be associated with that GRP78 expression in gastric carcinoma was positively linked to tumor size, depth of invasion, lymphatic and expression of GRP78 and clinicopathological features of HCC. This may be due to the limited number of specimens. This study revealed a high expression level of GRP78 in be regarded as a biomarker for different types of cancer warrants further investigation. autoantibodies against GRP78 in sera from patients in sera from normal controls. Further analysis indicated that the expression level of autoantibodies against GRP78 was associated with clinical stage, portal vein of anti-GRP78 autoantibodies increased in metastasis carcinogenesis have been shown to provoke autoantibody GRP78 auto-antibody titer preceded the detection of a palpable tumor mass. This study showed the average titer of anti-GRP78 autoantibodies was higher in the early stage of HCC. Although our study showed the titer of autoantibodies against GRP78 in early-HCC, LC, and there was no difference among early-HCC, LC, and CH groups. The study also showed that the titer of anti-GRP78 before the detection of cancer. This is supported by a anti-GRP78 autoantibody for HCC diagnosis were 7.5% GRP78 autoantibodies in HCC and LC groups were anti-GRP78 autoantibody may not be used for diagnosis of HCC because of its low sesitivity in HCC.
To compare changes in anti-GRP78 autoantibodies before and after treatments, HCC patients were divided into surgery and non-surgery groups. Anti-GRP78 but no differences were observed in the non-surgery group. Previous reports found that chemotherapy and radiotherapy differentially affect the anti-GRP78 immune response. Moreover, radiation increased the concentration of GRP78 auto-Ab and chemotherapy reduced the concentration of GRP78 auto-Ab. Different treatments may have different effects on anti-TAA autoantibodies. and reduces the production of GRP78 antigens and anti-GRP78 antibodies.

NHS
This study also showed that the expression level of anti-GRP78 autoantibodies differed before surgery and after surgery in recurrent HCC patients. The titer of anti-GRP78 autoantibodies before surgery was higher than that within 1 week after surgery, and lower than that1 month after surgery. The expression level of anti-GRP78 autoantibodies was detected in HCC serum at 1-15 months after surgery. Therefore, we speculated that the decrease then increase titer of anti-GRP78 autoantibodies after surgery may predict recurrence or metastasis. The fact that the titer of anti-GRP78 autoantibodies was more sensitive than AFP suggests that anti-GRP78 autoantibodies may serve as a potential marker for tumor recurrence and metastatic progression.

Patient characteristics
The number of total participants was 561, including HCC patients with high titers of autoantibodies against patients were recruited to detect the serum levels of autoantibodies against eight candidate TAAs (cohort changes of autoantibodies against TAAs before and after treatment such as hepatic resection, liver transplantation, transcatheter arterial chemoembolization, radiofrequency Jiaotong University.

Serum samples and tissue specimens
Serum and liver tissue specimens from patients patients were treated with chemotherapy, radiotherapy collected before and after treatment. All blood samples

Cell culture and cell extracts
Jiaotong University.

Western blotting
sulfate polyacrylamide gel electropheresis (SDS-PAGE) membranes to screen the autoantibody-positive serum.
human serum for 8 h, and then incubated with horseradish MA, USA) was used to detect immunoreactive bands. Immunoprecipitation suspending cells in 1.5 ml of radioimmunoprecipitation serum was immobilised on dynabeads. The antigen (whole antibody gel mixture together with binding buffer on a rocking platform at room temperature. The antigens were eluted from the beads into the elution buffer using microcentrifuge spin cups.

SDS-PAGE electrophoresis and in-Gel digestion
Following gel electrophoresis, gels were stained

Absorption of antibodies with recombinant protein
in HCC serum samples and the intracellular location of assay.

Statistics
of autoantibodies against candidate proteins in each group was compared using analysis of variance. The autoantibody frequency to GRP78 of patients' sera in each test. Comparison of the clinicopathological parameters with GRP78 expression was conducted by the two-tail GRP78 before and after treatment and AFP were compared rank sum test. A P www.impactjournals.com/oncotarget markers for aggressive behavior and poor prognosis in progression and evasion from immune surveillance. Asian immune system in pancreatic cancer progression and immune modulating treatment strategies. Cancer treatment protein-treated peripheral blood monocyte-derived dendritic cells are refractory to maturation and induce regulatory T-cell induces cell surface localized glucose-regulated protein anticancer treatments and angiogenesis on the seric titer of autoantibody used as tumor and metastasis biomarker.
antigens as diagnostic biomarkers in hepatocellular carcinoma and other solid tumors. Expert review of