Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer

Abstract

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n ¼ 6), grade 2 (n ¼ 8) and grade 3 (n ¼ 24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4 -7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (Po0.05), 31 in grade 2 (Po0.05) and 25 in grade 3 (Po0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.

Serum Protein Profiling to Identify High-Risk Neuroblastoma: Preclinical Relevance of Blood-Based Biomarkers

“…ApoA-IV is a 46 kD plasma glycoprotein with limited associations with cancer; Kawakami and colleagues showed ApoA-IV to increase in sera from hepatocellular cancer after radiofrequency ablation [20]. HP is a well characterized APP with increasing evidence as a cancer biomarker [21][22][23][24][25]. Lastly, SAA is an acute phase apolipoprotein associated as a marker of cancer progression [26][27][28][29][30].…”

Postoperative serum proteomic profiles may predict metastatic relapse in high-risk primary breast cancer patients receiving adjuvant chemotherapy

“…Interestingly, some of the identified biomarkers in our study were found to be modulated by various cancer diseases in other serum-based proteomic studies. Thus, haptoglobin was found upregulated in serum of ovarian, oesophageal and small-cell lung cancer patients (Steel et al, 2003;Ahmed et al, 2004;Bharti et al, 2004), while complement C3 and apolipoprotein A1 (Steel et al, 2003;Zhang et al, 2004) were found downmodulated in liver or ovarian cancer. However, these studies were dedicated to the identification of serum biomarkers associated to cancer diagnosis, rather than prediction of clinical outcome.…”

“…However, some of them may yet potentially directly impact on the metastatic process. For example, haptoglobin, an acutephase protein mainly produced in the liver has been shown to be upregulated in the serum of patients with various solid tumors (Ahmed et al, 2004;Bharti et al, 2004;Kwak et al, 2004;Tolson et al, 2004), and has also been demonstrated to participate in angiogenesis, tissue remodeling and cell migration (Cid et al, 1993;De Kleijn et al, 2002). Similarly, a proposed role for transferrin signalling has been suggested in regulating the metastatic capacity of various solid tumors including breast cancer (Nicolson et al, 1992;Inoue et al, 1993;Cavanaugh and Nicolson, 1998;Cavanaugh et al, 1999).…”

Recent advances in blood‐related proteomics

“…It was shown that at least three structurally different protein species can be differentiated and accounted for the most commonly observed spot patterns of haptoglobin a chains. As shown in Table 2, haptoglobin has been shown to be a potential marker of cancer [37][38][39]. Watanabe et al [40], after having analyzed serum samples from pregnant women by 2-DE, were able to identify in women presenting with preeclampsia a group of overexpressed spots corresponding to clusterin.…”