Bo Peng

Autoantibodies to tumor-associated antigens as biomarkers in cancer immunodiagnosis.

Cancer sera contain antibodies that react with a unique group of autologous cellular antigens called tumor-associated antigens (TAAs), and therefore these autoantibodies can be considered as reporters from the immune system, to identify authentic TAAs involved in the malignant transformation. Once a TAA is identified, different approaches would be used to comprehensively characterize and validate the identified TAA/anti-TAA systems that are potential biomarkers in cancer immunodiagnosis. In this manner, several novel TAAs such as p62 and p90 have been identified in our previous studies. p62, a member of IGF-II mRNA binding proteins (IMPs), is an oncofetal protein absent in adult tissues, the presence of anti-p62 autoantibodies relates to abnormal expression of p62 in tumor cells. p90 was recently characterized as an inhibitor of the tumor suppressor PP2A (protein phosphatase 2A), and an autoantibody to p90 appears in high frequency in prostate cancer. The present review will focus on the recent advances in studies mainly associated with these two novel TAAs as biomarkers in cancer immunodiagnosis.

Autoantibodies against glucose-regulated protein 78 as serological diagnostic biomarkers in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a type of cancer with a very poor prognosis. Although α-fetoprotein (AFP) is the most effective marker available to detect HCC, the sensitivity and specificity are not optimal. Therefore, there is a need for the development of more sensitive and specific methods that can supplement AFP in the early detection of this cancer. In this study, autoantibody responses to glucose-regulated protein 78 (GRP78) were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting and indirect immunofluorescence assay in sera from patients with HCC, liver cirrhosis (LC) and chronic hepatitis (CH), as well as from normal human individuals. Immunohistochemistry (IHC) with tissue array slides was also preformed to analyze protein expression profiles of GRP78 in HCC and control tissues. The prevalence of autoantibodies against GRP78 was 35.5% (27/76) in HCC, which was significantly higher than that in LC, CH and normal human sera (NHS; P<0.01). The average titer of autoantibodies against GRP78 in HCC sera was higher compared to that in LC, CH and NHS(P<0.01). When both autoantibodies against GRP78 and AFP were used simultaneously as diagnostic markers, sensitivity reached 71.4%. Our data indicate that anti-GRP78 autoantibodies may be potential diagnostic markers for HCC, especially in conjunction with AFP.

Mini-array of multiple tumor-associated antigens (TAAs) in the immunodiagnosis of breast cancer

Sera from patients with cancer contain antibodies which react with a unique group of autologous cellular antigens called tumor-associated antigens (TAAs). This study aimed to determine whether a mini-array of multiple TAAs would enhance antibody detection and be a useful approach in breast cancer detection and diagnosis. The mini-array of multiple TAAs was composed of ten TAAs, including Imp1, p62, Koc, p53, c-myc, survivin, p16, cyclin B1, cyclin D1 and CDK2 full-length recombinant proteins. An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against these ten TAAs in 41 sera from patients with breast cancer, as well as 82 sera from normal individuals. The antibody frequency of the individual TAAs in breast cancer was variable and ranged between 7.3 and 22.0%. With the successive addition of TAAs to a final total of ten antigens, there was a stepwise increase in positive antibody reactions, reaching a sensitivity of 61.0% and a specificity of 86.6% in breast cancer. The positive and negative likelihood ratios were 5.545 and 0.438, respectively, which showed that the clinical diagnostic value of a parallel assay of eight TAAs was high. The positive and negative predictive values were 73.5 and 82.0%, respectively, indicating that the parallel assay of eight TAAs raised the diagnostic precision significantly. The agreement rate and κ-value were 79.7% and 0.52, respectively, while the Youden’s Index (YI) was 0.5, indicating that the observed value of this assay had a middle range coincidence with the actual value. The data from the present study further support our previous hypothesis that the detection of autoantibodies for the diagnosis of certain types of cancer may be enhanced using a mini-array of several TAAs as target antigens. A customized antigen mini-array using a panel of appropriately selected TAAs is able to enhance autoantibody detection in the immunodiagnosis of breast cancer.

Peroxiredoxin 1 is a tumor-associated antigen in esophageal squamous cell carcinoma

Peroxiredoxin 1 (Prdx1) is an antioxidant and plays an important role in H2O2-mediated cell signaling. We previously found that the expression level of Prdx1 was elevated in esophagus squamous cell carcinoma (ESCC) tissue using a proteomics approach. Since overexpressed protein can induce an autoimmune response, to further examine whether serum from ESCC patients exhibits immunoreactivity against Prdx1, autoantibody responses to Prdx1 were evaluated by ELISA, western blotting and indirect immunofluorescence assay in sera from patients with ESCC and normal individuals. Immunohistochemical study with tissue array slides and western blot analysis with cancer cell lines were also performed to analyze the protein expression profiles of Prdx1 in ESCC tissues and cancer cell lines. The results demonstrated that the positive rate of autoantibody against Prdx1 in ESCC sera was 13.2% (9/68), whereas this rate was 0% (0/89) in normal individuals. Data also showed that expression of Prdx1 was significantly increased in ESCC tissues when compared to expression in paired adjacent normal tissues (P<0.05). The data indicate that Prdx1 may contribute to malignant transformation of the esophagus, and may be used as a biomarker in the immunodiagnosis of ESCC.

CIP2A regulates cell proliferation via the AKT signaling pathway in human lung cancer

Cancerous inhibitor of PP2A (CIP2A) is an intracellular endogenous protein phosphatase 2A (PP2A) inhibitor with oncogenic activities. Initially identified as a tumor-associated antigen (TAA) in gastric and liver cancer patients, CIP2A was overexpressed in a variety of cancer types. The overexpression of CIP2A in cancer cells is associated with increased cell proliferation. However, the mechanism of CIP2A in cancer cell proliferation remains poorly understood. In the present study, we reported that CIP2A can regulate AKT phosphorylation at S473 under growth factor stimulation and our results also showed that CIP2A may promote cell proliferation through the AKT signaling pathway. Notably, depletion of CIP2A did not induce a global change of AKT phosphatase activity, which indicated that CIP2A may recognize specific AKT targets and play certain roles in the signaling pathway. In addition, we detected that CIP2A expression was associated with mTOR phosphorylation. Our further analysis corroborated the relationship between CIP2A and AKT-mTOR signaling pathway. Therefore, our study addressed a novel role of CIP2A in mediating cancer progression through interacting with the AKT-mTOR signaling pathway.

Identification of glutathione S-transferase omega 1 (GSTO1) protein as a novel tumor-associated antigen and its autoantibody in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the main form of esophageal malignancy. The approach for early diagnosis of this malignancy is very limited. In the present study, we first evaluated glutathione S-transferase omega 1 (GSTO1), a protein related to metabolism, as a tumor-associated antigen in ESCC, and we also evaluated its autoantibody as a potential biomarker in early detection of ESCC. First, immunohistochemistry (IHC) analysis of GSTO1 protein expression in esophageal tissues showed that the percentage of positive staining of GSTO1 in ESCC tissues was 87.5xa0% while there was no positive staining in adjacent tissues or normal tissues, indicating that overexpression of GSTO1 is closely related to ESCC. Then, enzyme-linked immunosorbent assay (ELISA) showed that the frequency of detectable autoantibody against GSTO1 in patients’ sera totals 44.8xa0%. In contrast, the frequency of detectable autoantibody was only 6.7xa0% in normal human sera (pu2009<u20090.01). To further evaluate our ELISA results, western blotting and immunofluorescence assay were also performed. The results were consistent with the data from ELISA. In conclusion, the current study has demonstrated that GSTO1 protein is overexpressed in ESCC and can induce a detectable autoantibody response, which may serve as a potential biomarker in the early detection of ESCC.

Autoantibodies Response to MDM2 and p53 in the Immunodiagnosis of Esophageal Squamous Cell Carcinoma

The human homologue of the mouse double minute 2 (MDM2) is known to be overexpressed in a variety of human malignancies. As one of E3 ubiquitin–protein ligases, MDM2 interacts with the tumour suppressor p53 by mediating ubiquitination and degradation of p53. Since abnormally expressed proteins can induce autoimmune response, to further examine whether sera from patients with esophageal squamous cell carcinoma (ESCC) exhibited immunoreactivity against MDM2 and p53, autoantibody responses to MDM2 and p53 were evaluated by enzyme‐linked immunosorbent assay (ELISA) in sera from patients with ESCC and normal individuals. Positive results were also confirmed by Western blotting and indirect immunofluorescence assay. The results demonstrated that the positive rate of autoantibody against p53 and MDM2 in ESCC sera was 22.9% (36/157) and 14.0% (22/157), whereas this rate was 0% (0/85) and 1.2% (1/85), respectively, in normal individuals. Some of the sera with antibodies specific for MDM2 also contained antibodies against p53. And there was an increase of positive antibody reactions reaching a frequency of 35% (55/157) combination with MDM2 and p53. This was significantly higher than the frequency of antibodies in normal individuals (P < 0.01). Our preliminary results suggest that autoantibodies against MDM2 and p53 may be useful serum biomarkers in the immunodiagnosis of ESCC.

CIP2A overexpression induces autoimmune response and enhances JNK signaling pathway in human lung cancer

BackgroundCancerous inhibitor of PP2A (CIP2A) is a recently characterized oncoprotein, which promotes cancer cell proliferation. But the role of CIP2A in lung cancer progression is still not well understood.MethodsThe expression level of CIP2A in lung cancer tissues was examined by immunohistochemistry. CIP2A-associated cell proliferation was performed by knock down or overexpression of CIP2A in lung cancer cells. Phospho-array was used to screen kinase candidates related to expression change of CIP2A. Western-blot and luciferase reporter assay were used to validate phospho-array results.ResultsOverexpression of CIP2A in lung cancer not only triggers immune response in lung cancer patients but also promotes lung cancer cell proliferation. By phospho-array, several kinase candidates were identified, one of which is c-Jun activated kinases (JNK). The knock down of CIP2A decreased JNK phosphorylation, and the phosphorylation of downstream transcriptional factors, ATF2 and c-Jun, whose transcriptional activity were decreased as well. Furthermore, the expression level of CIP2A also affected the phosphorylation of the upstream kinase of JNK, MKK4/MKK7. At last, treatment with JNK inhibitor partially abolished CIP2A-induced cell proliferation.ConclusionCIP2A is a tumor-associated autoantigen in lung cancer, which promote lung cancer proliferation partially through MKK4/7-JNK signaling pathway.

Abstract 5381: p62/IMP2 promotes hepatocellular carcinoma (HCC) progression.

Autoantibodies to autologous cellular antigens in cancer patients have been considered as reporters identifying antigenic changes in cellular factors involved in the transformation process. We have made use of such autoantibodies from hepatocellular carcinoma patients to immunoscreen a cDNA expression library and have identified a cytoplasmic RNA-binding protein p62. Autoantibodies to p62 have been detected in up to 21% of a cohort of hepatocellular carcinoma (HCC) patients. Further study has demonstrated that p62 is a member of insulin-like growth factor 2 mRNA binding protein family (IMPs), which is an isoform of IMP2. The three members of the IMP family (IMP1, 2 and 3) have been found to regulate translation of insulin-like growth factor 2 (IGF2) mRNA in a post-transcriptional manner. Since several studies have suggested that IMP1 and IMP3 play important roles in cancer progression, whether p62/IMP2 can also play a role in cancer formation remains to be investigated. In this study, we intend to interrogate the role of p62/IMP2 in HCC formation. We found that the expression of p62/IMP2 (57.5%) is significantly higher in HCC specimen than that in normal liver tissue (15.4%) by immunohistochemistry study. Furthermore, p62/IMP2 is detected to be overexpressed in several liver cancer cell lines. Depletion of p62/IMP2 via lentivirus-mediated knockdown in SNU449 liver cancer cell line decreased the cell proliferation. On the contrary, the ectopic overexpression of p62/IMP2 increased cell proliferation. Our data suggested that p62/IMP2 is overexpressed in liver cancer and is functionally correlated with cancer cell growth. Citation Format: Ningjing Lei, Bo Peng, Yang Li, Yurong Chai, Jianying Zhang. p62/IMP2 promotes hepatocellular carcinoma (HCC) progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5381. doi:10.1158/1538-7445.AM2013-5381

Abstract 340: Identification of p62/IMP2 novel targets in breast cancer metastasis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnAs one of the most common types of cancer in women, breast cancer is far from being well controlled, especially when the cancer spreads to other organs in the body. Therefore, it is important to understand the molecular and cellular mechanism of metastatic breast cancer, and to identify new proteins that regulate the metastatic process, as such proteins may be used as targets for possible therapeutic intervention. Our previous study showed that p62/IMP2 (IGF2 mRNA binding protein 2) is overexpressed in breast cancer tissues. In our present study, some variants were generated with stable overexpression of p62/IMP2 in MDA-MB-231 cells and LM2-4 cells, and we found that overexpression of p62/IMP2 can increase breast cancer cell migration and reduce cell adhesion. We also found that the p62/IMP2 can bind to c-myc mRNA and increase the expression of c-myc in the two cell lines. To identify new p62/IMP2 targets related with cell adhesion, we performed a Human Extracelluar Matrix & Adhesion Molecules PCR Array to see which gene expression changes in the p62/IMP2 overexpressed cells, compared to the control cells. Our results showed that 18 adhesion molecules are up-regulated with the overexpression of p62/IMP2 (Fold regulation>2, p<0.05) while 2 are down-regulated. We selected 7 up-regulated genes (such as CTGF, HTBS1 and COL15A1) and a down-regulated gene (ITGB4) as candidates for the further evaluation by real time PCR. All candidates genes were further verified byRNA immunoprecipitation (RIP). The data from RIP suggested that connective tissue growth factor (CTGF) may be a potential target of p62/IMP2. These results provided evidences for a better understanding of breast cancer metastasis and targets for future anti-metastatic drug design.nnNote: This abstract was not presented at the meeting.nnCitation Format: Yang Li, Bo Peng, Ningjing Lei, Wei Qian, Giulio Francia, Jianying Zhang. Identification of p62/IMP2 novel targets in breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 340. doi:10.1158/1538-7445.AM2015-340