Protein isoform-specific validation defines multiple chloride intracellular channel and tropomyosin isoforms as serological biomarkers of ovarian cancer
Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA. Journal of proteomics
(Impact Factor: 3.89).
06/2013; 89. DOI: 10.1016/j.jprot.2013.06.016
New serological biomarkers for early detection and clinical management of ovarian cancer are urgently needed, and many candidates have been reported. A major challenge frequently encountered when validating candidates in patients is establishing quantitative assays that distinguish between highly homologous proteins. The current study tested whether multiple members of two recently discovered ovarian cancer biomarker protein families, chloride intracellular channel (CLIC) proteins and tropomyosins (TPM), were detectable in ovarian cancer patient sera. A multiplexed, label-free multiple reaction monitoring (MRM) assay was established to target peptides specific to all detected CLIC and TPM family members, and their serum levels were quantitated for ovarian cancer patients and non-cancer controls. In addition to CLIC1 and TPM1, which were the proteins initially discovered in a xenograft mouse model, CLIC4, TPM2, TPM3, and TPM4 were present in ovarian cancer patient sera at significantly elevated levels compared with controls. Some of the additional biomarkers identified in this homolog-centric verification and validation approach may be superior to the previously identified biomarkers at discriminating between ovarian cancer and non-cancer patients. This demonstrates the importance of considering all potential protein homologs and using quantitative assays for cancer biomarker validation with well-defined isoform specificity.
This manuscript addresses the importance of distinguishing between protein homologs and isoforms when identifying and validating cancer biomarkers in plasma or serum. Specifically, it describes the use of targeted in-depth LC-MS/MS analysis to determine the members of two protein families, chloride intracellular channel (CLIC) and tropomyosin (TPM) proteins that are detectable in sera of ovarian cancer patients. It then establishes a multiplexed isoform- and homology-specific MRM assay to quantify all observed gene products in these two protein families as well as many of the closely related tropomyosin isoforms. Using this assay, levels of all detected CLICs and TPMs were quantified in ovarian cancer patient and control subject sera. These results demonstrate that in addition to the previously known CLIC1, multiple tropomyosins and CLIC4 are promising new ovarian cancer biomarkers. Based on these initial validation studies, these new ovarian cancer biomarkers appear to be superior to most previously known ovarian cancer biomarkers.
Available from: Bing Shen
- "Because growing evidence has indicated that CLIC4 is involved in the development of different cancers, including cutaneous cancer, breast cancer, ovarian cancer and others[11,12,15], we also investigated CLIC4 expression in surgical specimens from HNSCC patients. Immunohistochemical staining showed that CLIC4 expression in HNSCC tissue was obviously higher than that in normal gingival tissue (Fig. 1). "
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ABSTRACT: Human head and neck squamous carcinoma is the 6th most prevalent carcinoma worldwide. Although many novel therapies have been developed, the clinical treatment for patients remains non-ideal. Chloride intracellular channel 4 (CLIC4), one of the seven members of the CLIC family, is a newly found Cl − channel that participates in various biological processes, including cellular apoptosis and differentiation. Accumulating evidence has revealed the significant role of CLIC4 in regulating the apoptosis of different cancer cells. Here, we investigated the functional role of CLIC4 in the apoptosis of HN4 cells, a human head and neck squamous carcinoma cell line.
In the present study, we used immunohistochemical staining to demonstrate that the expression level of CLIC4 is elevated in the tissue of human oral squamous carcinoma compared with healthy human gingival tissue. Specific CLIC4 small interfering RNA was used to knockdown the expression of CLIC4. The results showed that knockdown of CLIC4 with or without 100 μM adenosine triphosphate (ATP) treatment significantly increased the expression of Bax, active caspase 3, active caspase 4 and CHOP but suppressed Bcl-2 expression in HN4 cells. Moreover, the results from the TdT-mediated dUTP nick end labeling assay indicated that CLIC4 knockdown induced a higher apoptotic rate in HN4 cells under the induction of ATP. In addition, knockdown of CLIC4 dramatically enhanced ATP-induced mitochondrial membrane depolarization in HN4 cells. Moreover, intracellular Ca 2+ measurement revealed that Ca 2+ release induced by ATP and thapsigargin, a Ca 2+ -ATPase inhibitor of the endoplasmic reticulum, was significantly enhanced by the suppression of CLIC4 in HN4 cells.
Knockdown of CLIC4 enhanced ATP-induced apoptosis in HN4 cells. Both the pathways of mitochondria and endoplasmic reticulum stress were involved in CLIC4-mediated cell apoptosis. Based on our finding, CLIC4 may be a potential and valuable target for the clinical treatment of head and neck squamous carcinoma.
Available from: Xin Liu
- "The list was filtered for known cancer biomarkers. CS and ES shared four known cancer biomarkers (hemoglobin alpha 1/2 [HBA1/2], galectin- 3-binding protein [LGACS3BP], serum amyloid A1 [SAA1], and thrombospondin [THBS1])     , and separately they had three (APOE, tropomyosin alpha-3 chain [TPM3], and transthyretin [TTR])    and one (C-reactive protein [CRP])  marker(s), respectively (Fig. 1C). APOE was selected for further verification because it has been shown to be upregulated in various cancers including lung cancer  . "
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ABSTRACT: Biomarkers to identify subjects at high-risk for developing lung cancer will revolutionize the disease outlook. Most biomarker studies have focused on patients already diagnosed with lung cancer and in most cases the disease is often advanced and incurable. The objective of this study was to use proteomics to identify a plasma biomarker for early detection of lung lesions that may subsequently be the harbinger for cancer. Plasma samples were obtained from subjects without lung cancer grouped as never, current, or ex-smokers. An iTRAQ-based proteomic analysis was performed on these pooled plasma samples. We identified 31 proteins differentially abundant in current smokers or ex-smokers relative to never smokers. Western blot and ELISA analyses confirmed the iTRAQ results which demonstrated an increase of apolipoprotein E (APOE) in current smokers as compared to both never and ex-smokers. There was a strong and significant correlation of the plasma APOE levels with development of pre-malignant squamous metaplasia. Additionally, we also showed that higher tissue levels of APOE are seen with squamous metaplasia, supporting a direct relationship. Our analysis reveals that elevated plasma APOE is associated with smoking, and APOE is a novel predictive protein biomarker for early morphological changes of squamous metaplasia in the lung. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Available from: PubMed Central
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ABSTRACT: Understanding the protection mechanism of 5'-AMP requires comprehensive knowledge of the proteins expressed during the period that the body is exposed to irradiation. Proteomics provides the tools for such analyses. Here, the experimental ICR mice were divided into three groups (normal group, model group and 5'-AMP + irradiation group). After different treatment, the hepatic total protein of each animal in three groups was separated by two-dimensional gel electrophoresis (2-DE). 2-DE analysis revealed fifty-eight protein spots were differentially expressed in comparison to three groups. From 58 protein spots, we selected nine spots to identify by MALDI-TOF-MS and received credible results. They were determined to be type I arginase, annexin A5, regucalcin, catalase, Tpm3 protein, Pdia4 protein, 14-3-3 protein epsilon, NAD-Malate dehydrogenase and heat shock protein 90. Considering the characteristic of these proteins, we proposed a possible protection pathway.
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