Ji-Run Peng

Large Scale Identification of Human Hepatocellular Carcinoma-Associated Antigens by Autoantibodies

Autoantibodies are often detected in hepatocellular carcinoma (HCC), and these responses may represent recognition of tumor Ags that are associated with transformation events. The identities of these Ags, however, are less well known. Using serological analysis of recombinant cDNA expression libraries (SEREX) from four HCC patients, we identified 55 independent cDNA sequences potentially encoding HCC tumor Ags. Of these genes, 15 are novel. Two such proteins, HCA587 and HCA661, were predominantly detected in testis, but not in other normal tissues, except for a weak expression in normal pancreas. In addition to HCC, these two Ags can be found in cancers of other histological types. Therefore, they can be categorized as cancer-testis (CT) Ags. Two other Ags (HCA519 and HCA90) were highly overexpressed in HCC and also expressed in cancer cell lines of lung, prostate, and pancreas, but not in the respective normal tissues. Four other Ags were identified to be expressed in particular types of cancer cell lines (HCA520 in an ovarian cancer cell line, HCA59 and HCA67 in a colon cancer cell line, HCA58 in colon and ovarian cancer cell lines), but not in the normal tissue counterpart(s). In addition, abundant expression of complement inactivation factors was found in HCC. These results indicate a broad range expression of autoantigens in HCC patients. Our findings open an avenue for the study of autoantigens in the transformation, metastasis, and immune evasion in HCC.

Association of Mortalin (HSPA9) with Liver Cancer Metastasis and Prediction for Early Tumor Recurrence

Hepatocellular carcinoma (HCC) is well known for poor prognosis and short survival because of high recurrence rate even after curative surgery. Today there is no available biomarker or biochemical test to indicate HCC recurrence, and this study aims to identify protein markers that can discriminate postoperative patients with early recurrence (ER), i.e. disease relapsed within the first year. In this study, 103 hepatitis B-related HCC patients were recruited, and 68 of them were used for ER-related biomarker discovery study. Proteomic expression patterns of matched tumor and adjacent non-tumor tissues from these patients plus 16 normal liver tissues were delineated by the two-dimensional gel electrophoresis differential profiling method. Significant protein spots were evaluated by hierarchical clustering analysis. SSP4612 that yielded the highest receiver operating characteristic (ROC) curve value for the ER subgroup of HCC was subsequently identified by tandem mass spectrometry, and the corresponding expression patterns were further confirmed by quantitative PCR, Western blot, and immunohistochemistry. Correlation analysis with clinicopathological data was also examined. Proteomic profiling analysis revealed overexpression of mortalin (gene HSPA9) in HCC when compared with the non-tumor and normal liver tissues (area under the curve (AUC) = 0.821). Furthermore, elevated mortalin level was also detected in the ER subgroup of HCC versus the recurrence-free state (where no cancer recurs for >1 year) (AUC = 0.833, sensitivity = 90.9%, specificity = 71.4%). Metastatic HCC cell lines also exhibited higher levels of mortalin and HSPA9 mRNA. Clinically, mortalin overexpression in HCC was closely associated with advanced tumor stages and venous infiltration, having implications for increased malignancy and aggressive behavior. Mortalin (HSPA9) is associated with HCC metastasis and thus suggested as a tumor marker for predicting early recurrence, which may have immediate clinical applications for cancer surveillance after curative surgery.

The Spontaneous CD8+ T-Cell Response to HLA-A2-Restricted NY-ESO-1b Peptide in Hepatocellular Carcinoma Patients

Purpose: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2–restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. Experimental Design: In HLA-A2+ patients with NY-ESO-1+ HCC, we analyzed T-cell antigen-dependent interferon (IFN)-γ and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-γ–producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. Results: The frequency of specific CD8+ T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA+HLA-A2+ HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8+ T cells was 0.3% (89 ± 59 per 2.5 × 104 CD8+ cells) and 1.2% as measured by IFN-γ release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b–specific CD8+ T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-γ and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b–specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer+CD8+ T cells were also present. The CD8+ T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8+ T-cell responses in HCC patients was observed. Conclusions: The elevated frequency of specific CD8+ T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA+HLA-A2+ HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.

PLAC1 is a tumor-specific antigen capable of eliciting spontaneous antibody responses in human cancer patients

Immunoselection and tumor evasion constitutes one of the major obstacles in cancer immunotherapy. A potential solution to this problem is the development of polyvalent vaccines, and the identification of more tumor‐specific antigens is a prerequisite for the development of cancer vaccines. To identify novel tumor‐specific antigens, suppression subtractive hybridization (SSH) was performed to isolate genes differentially expressed in human hepatocellular cancer (HCC) tissues. PLAC1 (PLACenta‐specific 1) was one of the genes identified highly expressed in HCC tissues but not in paired noncancerous tissues. Further analyses revealed its expression in several other types of cancer tissues as well as tumor cell lines, but not in normal tissues except for placenta. Among HCC samples tested, 32% (22/69) showed PLAC1 mRNA expression while the protein was detected in 23.3% (7/30). A serological survey revealed that 3.8% (4/101) of HCC patients had anti‐PLAC1 antibody response, suggesting the immunogenicity of PLAC1 in HCC patients. PLAC1 represents a new class of tumor associated antigen with restricted expression in placenta and cancer tissues, that may serve as a target for cancer vaccination.

The immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4+ T cells, CD8+, CD3−CD56+, CD3+CD56+, and γδT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3+CD56+ cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4+ cells in the tumor tended to produce more IL-10 but less IFN-γ, whereas CD8+ cells showed impaired capacity for the production of both IFN-γ and perforin. Consistent with previous reports, we observed a significant increase of Foxp3+ cells in the tumor tissue. Intriguingly, although over 90% of CD4+CD25high cells were found to be Foxp3+, the majority of Foxp3+ cells were identified in the CD4+CD25medium and CD4+CD25− subsets. In support of its role as a negative regulator, CD4+CD25high cells suppressed the proliferation of CD4+CD25− cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.

Specific CD8 + T cell responses to HLA-A2 restricted MAGE-A3 p271–279 peptide in hepatocellular carcinoma patients without vaccination

The MAGE-A3 protein, one of the promising tumor antigens for immunotherapy, is highly expressed in human hepatocellular carcinoma (HCC). In this study, we estimated the specific CD8+ T cell immune response to MAGE-A3 p271–279 peptide (M3271) in the peripheral blood of HCC patients without antigen vaccination in order to evaluate its immunotherapeutic potential in these patients. After expansion in vitro, the functional IFN-γ producing M3271 specific CD8+ T cells were detected in 30.8% (8/26) of HLA-A2+MAGE-A3+ HCC patients. The effector CD8+ T cells could release cytotoxic molecules of granzyme B and perforin after restimulation with natural HLA-A2+MAGE-A3+ HCC cell lines in the samples tested. The functional supertype of HLA-A2 in the presentation of HLA-A*0201 restricted M3271 peptide has been identified in the Chinese HCC patients of Han ethnicity, that widely expanded the applicability of this tumor peptide vaccine in Chinese HCC patients. Thus, the functionally detectable pre-existence of M3271-specific CD8+ T cells in HCC patients makes M3271 a potential target for immunotherapy in these patients. The responsive CD8+ T cells to both NY-ESO-1 and MAGE-A3 antigens provide a rationale for the application of a bivalent vaccine in HCC patients with tumors expressing both antigens.

Identification of new cytotoxic T-lymphocyte epitopes from cancer testis antigen HCA587

The cancer testis (CT) antigen HCA587 is highly expressed in human hepatocellular carcinoma (HCC) and induces specific T-cell responses in a significant proportion of HCC patients. To explore its potential in cancer immunotherapy, a reverse immunology approach was adopted to identify HCA587-derived HLA-A( *)0201-restricted epitopes. Multiple peptides with a top ranking in various prediction programs were thus synthesized and three of them-p248-256, p140-149 and p144-152-were found to bind to HLA-A(*)0201 molecules with a high affinity and effectively induced a recall response of CD8+ T cells, which were either primed in vitro with the HCA587 antigen or directly isolated from HCC patients bearing HCA587+ tumors. Notably, these peptide-specific CD8+ T cells exhibited potent cytotoxic activity over HCA587+ tumor cells. Taken together, the present study has identified three new HLA-A(*)0201-restricted cytotoxic T cell epitopes in the CT antigen HCA587, which may serve as targets for peptide-based immunotherapy for HCC patients.

3-Bromopyruvic Acid, A Hexokinase II Inhibitor, is an Effective Antitumor Agent on the Hepatoma Cells : in vitro and in vivo Findings

Over-expressed in cancer cells, hexokinase II (HK II) forms a mitochondrial complex, which promotes cancer survival. 3- Bromopyruvic acid (3-BrPA) dissociates HK II from this complex, causing cell death, and thus, having an anti-tumor effect. The design of this study was to first analyze the expression of HK II in the hepatoma cell line, BEL-7402, then investigate the effects of 3-Br-PA on these cells, and finally, discuss its potential for clinical usage. HK II expression was detected in BEL-7402 cells by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). In vitro treatment of cells with 3-BrPA significantly inhibited their growth, as evaluated by MTT assay and adenosine triphosphate-tumor chemosensitivity assay (ATP-TCA). To analyze the in vivo function and safety of this drug, a tumor model was established by subcutaneously implanting hepatic cancer cells into nude mice. 3-BrPA treatment (50 mg/kg ip. daily, 6 days/week for three weeks) was effective in the animal model by attenuating tumor growth and causing tumor necrosis. Toxic signs were not observed. The acute toxicity study provided an LD50 of 191.7 mg/kg for 3-BrPA. Taken together, our in vitro and in vivo analyses suggest that 3-BrPA exerts anti-hepatoma effects, and may be an effective pharmacological agent for the treatment of hepatocellular carcinoma.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro.

Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

Overexpression of human telomerase reverse transcriptase promotes the motility and invasiveness of HepG2 cells in vitro

Recent studies have indicated that telomerase activity promotes cancer invasion and metastasis, but the underlying mechanism remains unclear. Several studies have shown that expression of exogenous human telomerase reverse transcriptase (hTERT) can promote motility and invasiveness among telomerase-negative tumor cells, and inhibition of endogenous telomerase activity can reduce invasiveness in tumor cells. However, whether overexpression of hTERT can further enhance the motility and invasiveness of telomerase‑positive tumor cells has yet to be determined. In the present study, we showed that stable overexpression of hTERT can increase telomerase activity and telomere length, which significantly promotes the invasive and metastatic potential of telomerase‑positive HepG2 cells but does not affect cell proliferation. Further analysis suggested that enhanced invasiveness and metastasis may act through corresponding upregulation of mRNA and protein expression of matrix metalloproteinase 9 (MMP9) and Ras homolog gene family member C (RhoC). Our study indicated that exogenous expression of hTERT may promote invasiveness and metastasis through upregulation of MMP9 and RhoC.