Proteomic identification of fucosylated haptoglobin alpha isoforms in ascitic fluids and its localization in ovarian carcinoma tissues from Mexican patients

Abstract

BackgroundOvarian cancer is the most lethal gynecologic disease due to delayed diagnosis, and ascites production is a characteristic of patients in advanced stages. The aim of this study was to perform the proteomic analysis of ascitic fluids of Mexican patients with ovarian carcinoma, in order to detect proteins with a differential expression pattern in the continuing search to identify biomarkers for this disease.MethodsSamples were collected from 50 patients from the Instituto Nacional de Cancerología of México under informed consent and with approval of the bioethics and scientific committees. After elimination of abundant proteins (Albumin/IgGs) samples were processed for 2D electrophoresis and further protein identification by Mass Spectrometry (MALDI-TOF). Molecules of interest were followed by western blot and lectin binding assays, and their tissue location by histo-immunofluorescence and confocal analysis.Results and discussionAn area with a differential expression pattern among samples was located in the 2D gels. Identified proteins were 6 alpha 1 isoforms and 1 alpha 2 isoform of Haptoglobin, and 2 isoforms of Transthyretin. While Transthyretin isoforms were constitutively expressed in all samples, clear differences in the expression pattern of Haptoglobin alpha isoforms were found. Moreover, increased levels of fucosylation of Haptoglobin alpha isoforms analyzed in 40 samples by Aleuria aurantia lectin binding by 1D overlay assay showed a positive correlation with advanced stages of the disease. Tissue detection of Haptoglobin and its fucosylated form, by histo-immunofluorescence in biopsies of ovarian cancer, also showed a correlation with ovarian cancer progression. Moreover, results show that fucosylated Haptoglobin is produced by tumor cells.ConclusionsIncreased numbers of highly fucosylated Haptoglobin alpha isoforms in ascitic fluids and the presence of fucosylated Haptoglobin in tumor tissues of ovarian cancer Mexican patients associated with advanced stages of the disease, reinforce the potential of fucosylated Haptoglobin alpha isoforms to be characterized as biomarkers for disease progression.

Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

“…Our previous studies have demonstrated the presence of a personalized signature of Hp α in the ascitic fluid of Mexican patients; moreover, we also showed that Hp is expressed in the cells from ovarian tumors at different stages. 19 Therefore, it was important to establish whether the cancer cell itself is capable of synthesising this protein to further understand its biological function within the tumoral environment.…”

“… 35 However, our group has demonstrated differences in both the expression level and the number of Hp α isoforms in ascitic fluid from patients who were diagnosed with different histological grades of ovarian carcinoma. 19 …”

“…In previous work in our laboratory, we detected the expression of cytoplasmic Hp within the tumors of patients with ovarian carcinoma, whereas this protein was absent from cancer-free ovarian epithelium. 19 With these findings, we wonder what could be the role of a protein that is naturally produced in the liver in the microenvironment of the tumor cells, such as ascitic fluid from ovarian carcinoma.…”

Ascites from Ovarian Cancer Induces Novel Fucosylated Proteins

“…Additional studies on this antigen have shown that the overexpression of Lewis Y antigen promotes the human epididymis protein 4-mediated invasion and metastasis of ovarian cancer cells [15], p27 degradation by regulating ubiquitin-proteasome activity in the CAOV-3 and SKOV-3 cell lines [16], and chemotherapeutic resistance in ovarian cancer [17]. A few examples of modified fucosylated proteins are haptoglobin in several cancers, including ovarian cancer [18][19][20], transferrin and alpha-fetoprotein in liver cancer [21,22] and thyroglobulin in thyroid cancer [23]. In particular, previous results from our group have shown that the level of fucosylation of haptoglobin increases with stage progression [19] and was found to be highly fucosylated in different subtypes of epithelial ovarian cancer [20].…”

Improvement of core-fucosylated glycoproteome coverage via alternating HCD and ETD fragmentation

“…Another two fucosylated glycoproteins, kininogen and alpha-1-antitrypsin (a1AT), have been identified as candidates of hepatic tumor markers [7, 8]. In addition, it has been proven that core-fucosylated haptoglobin is a much better biomarker in ovarian carcinoma than simply monitoring the expression of the haptoglobin [9]. All the examples show that, during pathological processes, CF-glycoprotein changes occur not only in protein abundance but also in the occupancy of CF-glycosylation.…”