Protein isoform-specific validation defines multiple chloride intracellular channel and tropomyosin isoforms as serological biomarkers of ovarian cancer
ABSTRACT 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-M/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.
SourceAvailable from: Michele Mazzanti[Show abstract] [Hide abstract]
ABSTRACT: In recent decades growing scientific evidence supports the role of ion channels in the development of different forms of cancer. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration and invasiveness into non-neoplastic tissues, are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channel (CLIC), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma, but in certain cases they change localization and function from hydrophilic cytosolic proteins to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression. These changes in intracellular localization, tissue compartments and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. Copyright © 2014. Published by Elsevier B.V.Biochimica et Biophysica Acta (BBA) - Biomembranes 12/2014; DOI:10.1016/j.bbamem.2014.12.012 · 3.43 Impact Factor
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ABSTRACT: Copyright: © 2014 Poersch A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Journal of Proteomics & Bioinformatics 04/2014; DOI:10.4172/jpb.S5-004
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ABSTRACT: Chloride intracellular channel 1 (CLIC1) has been shown to be up-regulated in various malignancies but its exact function remains unclear. Here, it is revealed that CLIC1 is critical for the stability of invadopodia in endothelial and tumor cells embedded in a 3-dimensional (3D) matrix of fibrin. Invadopodia stability was associated with the capacity of CLIC1 to induce stress fiber and fibronectin matrix formation following its beta3 integrin (ITGB3)-mediated recruitment into invadopodia. This pathway, in turn, was relevant for fibrin colonization as well as slug (SNAI2) expression and correlated with a significant role of CLIC1 in metastasis in vivo. Mechanistically, a reduction of myosin light chain kinase (MYLK) in CLIC1- as well as beta3 integrin-depleted cells suggests an important role of CLIC1 for integrin-mediated actomyosin dynamics in cells embedded in fibrin. Overall, these results indicate that CLIC1 is an important contributor to tumor invasion, metastasis and angiogenesis. Implications: This study uncovers an important new function of CLIC1 in the regulation of cell-extracellular matrix interactions and ability of tumor cells to metastasize to distant organs.Molecular Cancer Research 09/2014; 13(2). DOI:10.1158/1541-7786.MCR-14-0249 · 4.50 Impact Factor