Ningjing Lei

Abstract 2808: Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450

Esophageal squamous carcinoma (ESCC) may be developed through a progressive sequence from mild to severe dysplasia, carcinoma in situ, and finally, invasive carcinoma. Chemoprevention can block or weaken the influence of development. Recent study has shown luteolin, a bioflavonoid, possesses anti-inflammatory, antioxidant, and anti-proliferative effects, and it might have a preventive effect in this progress. In this study, we focused on the effect of luteolin on cell cycle regulation in human Esophageal Squamous Carcinoma Cell Line EC1 and KYSE450 Cells in vitro and its potential mechanisms. Observations by flow cytometer showed that luteolin inhibited cell cycle progression at G2/M phase in a dose- and time-dependent manner. We also found that luteolin could induce cell apoptosis via decreasing activation of caspase-3 and down-regulation of mitochondrial membrane potential. Western blot results showed the protein expression of cycle related protein CyclinD1 and apoptosis related proteins caspase-3, caspase-9, and Bak were also significantly decreased in luteolin treated cells compared with the non-luteolin treated cells. Our results suggest that the proper use of luteolin might be a practical approach to the prevention of esophageal carcinoma via the inhibition of cell proliferation and other potential high risk regions for this disease. Citation Format: Ping Chen, Tao Hu, Yane Ma, Xiaoyu Chen, Liping Dai, Ningjing Lei, Ziming Dong, Pei Li. Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450. [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 2808. doi:10.1158/1538-7445.AM2015-2808

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.

Autoantibodies to tumor-associated antigens as biomarkers in human hepatocellular carcinoma (HCC)

Tumor-associated antigens (TAAs) recognized by cellular and/or humoral effectors of the immune system are attractive targets for diagnostic and therapeutic approaches to human cancer. Different approaches can be used to comprehensively characterize and validate the identified TAA/anti-TAA systems, which are potential biomarkers in cancer immunodiagnosis. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The high fatality rate of HCC within one year after its detection might be partly attributed to a lack of diagnostic methods that enable the early detection. Our previous studies have shown that novel autoantibodies can appear which are not detected prior to pre-malignant conditions during transition from chronic liver disease to HCC. The hypothesis we advance is the transition to malignancy can be associated with autoantibody response to certain cellular proteins that might have some role in tumorigenesis. We propose that the information that the cancer patient’s immune system is conveying in the form of autoantibodies to tumor-associated antigens (TAAs) should be utilized to a greater extent in identifying early signs of tumorigenesis. In this review, we will focus on the important features of TAA and the possibility that autoantibodies to TAAs can be used as biomarkers in immunodiagnosis and prognosis of HCC.

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.

CIP2A regulates cancer metabolism and CREB phosphorylation in non-small cell lung cancer.

The cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized endogenous inhibitor of the phosphatase activity of protein phosphatase 2A (PP2A), which extends the half-life of oncogenic protein c-myc and promotes in vivo tumor growth. The function of CIP2A in cancer progression is still poorly understood. To uncover the underlying mechanism of CIP2A-mediated cell proliferation, we implemented a two-dimensional electrophoresis (2DE)-based proteomic approach to examine lung cancer cell H1299 with and without CIP2A. We found 47 proteins differentially expressed where 19 proteins were upregulated and 28 proteins were downregulated. These were categorized into functional groups such as metabolism (25%), transcriptional and translational control (23%), and the signaling pathway and protein degradation (20%). On one hand, we validate our proteomic work by measuring the metabolic change. The knockdown of CIP2A decreased the expression of LDH-A as well as the enzymatic activity, accompanying with a decreased lactate production, an increased NADH/NAD+ ratio and ROS production. On the other hand, we found that CIP2A may regulate CREB activity through bioinformatics analysis. Our following experiments showed that, CIP2A positively regulated the phosphorylation of CREB in response to the serum treatment. Therefore, our proteomic study suggested that CIP2A mediates cancer progression through the metabolic pathway and intracellular signaling cascade.

p90/CIP2A mediates breast cancer cell proliferation and apoptosis

Cancerous inhibitor of PP2A (p90/CIP2A) was recently characterized as an innovative oncoprotein in human malignancies. p90/CIP2A inhibited c-Myc-associated PP2A phosphatase activity to promote cell proliferation and tumor growth. A growing number of studies have demonstrated that the overexpression of p90/CIP2A in various human malignancies. But the function of p90/CIP2A in cancer progression is still poorly understood. In the current research, we aim to explore the biological function of p90/CIP2A in breast cancer. shRNA knockdown was performed in MDA-MB-231 and LM2-4 cell lines. Cell proliferation assay, colony formation assay and flow cytometry were carried out to evaluate the role of p90/CIP2A in cell proliferation and apoptosis. p90/CIP2A depletion in breast cancer cells inhibited proliferation and increased paclitaxel-induced apoptosis. Furthermore, p90/CIP2A silencing down-regulated the expression of c-Myc and the level of p-ERK1/2. Taken together, our data suggest that p90/CIP2A as a crucial oncoprotein has been involved in cell proliferation and apoptosis, which may serve as a therapeutic target in breast cancer treatment.

Evaluation and characterization of anti-RalA autoantibody as a potential serum biomarker in human prostate cancer

Autoantibodies against intracellular tumor-associated antigens (TAAs) are commonly found in human cancers. In this study, we characterized the serum autoantibody response to the RalA, Ras-like GTPase, in patients with prostate cancer (PCa). The autoantibodies were detected by immunofluorescence assay in PCa cell lines, ELISA, and immunoblotting in 339 serum samples from patients with PCa and benign prostatic hyperplasia (BPH), and in normal human sera (NHS). The expression of RalA in prostate tumor tissues was evaluated by immunohistochemistry (IHC) in tumor microarrays. The autoantibody level to RalA (median) in NHS was significantly lower than in PCa (0.053 vs 0.138; P < 0.001) and BPH (0.053 vs 0.132; P < 0.005) groups. The circulating anti-RalA autoantibody could distinguish PCa patients from normal individuals with the area under the receiver operating characteristic (ROC) curve (AUC) performing at 0.861, with sensitivity of 52.9% and specificity of 91.0%. Elevation in serum immunoreactivity was observed in PCa patients after radical prostatectomy. The combined use of both anti-RalA autoantibody and PSA showed a significantly higher discriminatory ability compared with either of those markers alone. RalA protein expression was detected by IHC in 85.3% of tumor tissues from PCa patients, but without significant difference compared to BPH or normal control tissues. Together, our study shows the additional benefits of anti-RalA autoantibody as a potential serological biomarker for PCa, particularly in patients with normal PSA, and further demonstrate the utility of biomarker combinations in the immunodiagnosis of PCa.

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 4974: Using serological proteomics analysis to identify anti-nucleophosmin 1 autoantibody in sera from prostate cancer patients as potential diagnositic and prognostic biomarker

Prostate specific antigen (PSA) level test in blood has been widely implemented for the early detection and prediction of prostate cancer (PCa). However, lack of specificity led to overdiagnosis, following by unnecessary biopsies and overtreatment. Therefore, novel serological biomarkers are urgently needed to complement PSA by enhancing its sensitivity and specificity in the diagnosis of PCa. Autoantibodies against intracellular tumor associated antigens (TAAs) are commonly found in various types of human cancers. Their utility in providing insights into tumor biology and as potential cancer biomarkers is well documented. Previous studies have demonstrated that sera from patients with PCa contain autoantibodies that react with TAAs. In this study, we identified and characterized nucleophosmin 1 (NPM1) with 33 kDa molecular weight as an autoantigen by using serological proteomics analysis (SERPA) approach. In the further validation study, it was observed that the level of anti-NPM1 in sera from patients with PCa was significantly higher than that from benign prostatic hyperplasia (BPH) and healthy individuals and receiver operating characteristic (ROC) curve analysis showed high diagnostic value for PCa (area under the curve (AUC):0.805). Interestingly, when considering anti-NPM1 combined with PSA level in sera from PCa patients at the early stage as well as BPH, 100% PCa patients could be identified correctly, while 69.2% patients with BPH who has elevated PSA level were found anti-NPM1 negative. The data suggested that anti-NPM1 can elicit humoral immune response in PCa and might be a potential biomarker for the immunodiagnosis of PCa, and it would be a supplementary of PSA examination to distinguish PCa from BPH. Key words: prostate cancer; nucleophosmin 1 (NPM1); tumor-associated antigens (TAAs); autoantibodies; serological proteomics analysis (SERPA) Citation Format: jitian li, Liping Dai, Ningjing Lei, Mengtao Xing, Carlos A. Casiano, Jianying Zhang. Using serological proteomics analysis to identify anti-nucleophosmin 1 autoantibody in sera from prostate cancer patients as potential diagnositic and prognostic biomarker. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4974.