Background
A pivotal role for cold shock proteins has been originally described in breast cancer, as these proteins relate to cell transformation and confer aggressive tumor growth [
1,
2]. The overexpression of one prototypic member of this evolutionarily conserved protein family, denoted Y-box (YB) protein-1, in the mammarian gland results in breast carcinomas with 100% penetrance due to genetic instability, mitotic failure and centrosome amplification [
2]. It is known that YB-1 expression levels significantly correlate with growth "aggressiveness" and consequently with prognosis [
3]. Similar studies have been performed with prostate, epithelial ovarian [
4], cisplatin-resistant ovarian [
5] and non-small cell lung cancers [
6] as well as synovial sarcomas and osteosarcomas [
7]. The uniform finding is a correlation of upregulated, mostly nuclear YB-1 expression with poor outcome, e.g. early relapses and tumor growth, suggesting high YB-1 expression is an independent negative prognostic marker for many solid tumors. In particular, nuclear YB-1 overexpression was associated with more advanced diseases stages and poor prognosis in hepatocellular carcinoma [
8].
Pleiotropic cellular functions have been attributed to YB-1, including regulation of gene transcription, mRNA processing, transport and stabilization as well as translation [
9]. Transcription rates of proliferation-associated genes are upregulated by YB-1, e.g. DNA-polymerase-α [
10], epidermal growth-factor receptor [
11], platelet-derived growth factor [
12] and matrix metalloproteinase-2 [
13]. YB-1 facilitates wt-p53 binding to DNA motifs, however not of mutated p53, and represses cell death-associated
fas gene transcription [
14]. The YB-1 potency in conferring tumor growth or cell survival is underscored by in vitro-studies showing that YB-1 activates the EGFR signaling pathway in human breast epithelial cells resulting in EGF-independent growth [
15] and epithelial-mesenchymal transition [
16]. Adenocarcinoma, hepatoma, fibrosarcoma, and colon cancer cells die with YB-1 knock-down [
17,
18].
Recent findings from our laboratory indicate additional extracellular function(s) of YB-1. The protein is actively secreted by non-transformed and transformed cells following challenge with cytokines, e.g. PDGF-BB and TGF-β, and under oxidative stress [
19,
20]. The observation was unexpected as YB-1 lacks an N-terminal signal peptide motif. In this regard, YB-1 has similarities to other leaderless proteins, including interleukin-1β, high mobility group box protein (HMGB1) and macrophage migratory inhibitory factor (MIF). Besides full-length YB-1 protein, we observed secreted protein fragments in conditioned cell culture medium [
19,
20].
The universal role of YB-1 in various solid tumors and its secretion by tumor cells prompted the current study in which we tested for the first time the potential clinical and diagnostic value of YB-1 or its fragments in human plasma in cohorts of healthy volunteers and patients with malignant as well as non-malignant diseases, using a novel immunoblotting system.
Methods
Study populations
In this survey, five independent cohorts were tested for the diagnostic value of YB-1/p18 in human plasma: (i) healthy volunteers (n = 33), (ii) patients with chronic renal disorders and/or acute infections (n = 60), (iii) patients with solid tumors with hepatic metastases (n = 20), (iv) patients with known and histologically proven non-metastasizing hepatocellular carcinoma (n = 25), and (v) patients with chronic liver diseases (n = 111) undergoing extensive evaluation before potential liver transplantation.
The control population comprised 33 healthy blood donors (19 male, 14 female, median age 39, range 22-67 years) who had normal blood counts, normal aminotransferase activities and who tested negative for viral hepatitis markers and HIV. In order to determine renal function and acute or chronic inflammation as potential confounding variables for YB-1/p18, 60 patients from the Nephrology Department (37 male, 23 female, median 47 age, range 19-84 years) who presented to the Outpatient Clinic for a follow-up of chronic renal disorders (n = 33, 55%) or who presented due to acute infections (n = 27, 45%) were included. All populations were age- and sex-matched with the liver disease study population.
The liver disease study population consisted of 111 patients (66 male, 45 female, with a median age of 46 years; range 18-70 years) with chronic liver diseases who were evaluated as inpatients for potential liver transplantation. Patient data, blood samples and anamnestic information were collected prospectively. The study was done according to the ethical guidelines of the Declaration of Helsinki, approved by the local ethics committee and after obtaining written informed consent. The evaluation for a potential liver transplantation is a highly standardized routine procedure including a broad variety of clinical, laboratory and other diagnostic measures [
21]. Furthermore, the established tumor-markers α-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA) were assessed in all patients.
With regard to the underlying etiology of the liver disease, patients were assigned to the following groups: (a)
virus hepatitis (
n = 32) with chronic hepatitis B (
n = 14) or C (
n = 18) virus infection; (b)
biliary or autoimmune (
n = 27) with primary (
n = 4) or secondary (
n = 2) biliary cirrhosis, primary sclerosing cholangitis (
n = 17) or autoimmune hepatitis (
n = 4); (c)
alcohol-toxic or cryptogenic (
n = 29) with alcohol-toxic (
n = 20) and cryptogenic (
n = 9) cirrhosis; and (d)
other origins (
n = 23) with malignant liver tumors (
n = 4), liver metastases (
n = 3), liver cysts or benign tumors (
n = 5) or hereditary metabolic or vascular disorders (
n = 11). According to Child-Pugh's criteria [
22], patients were found to have no cirrhosis (
n = 18), Child A (
n = 35), B (
n = 44) or C (
n = 14) cirrhosis.
Blood samples were collected in EDTA plasma separator tubes, centrifuged at 2000 × g, and plasma was stored at -80°C. The scientist who performed the YB-1 blotting was blinded to the samples origin.
Antibodies
Monoclonal antibody generation for YB-1 has recently been described in detail [
23]. Polyclonal antibodies were generated by immunization of rabbits with synthesized polypeptides corresponding to the indicated domains (details about peptide sequences are available on request) and affinity purified before usage for immunoblotting.
YB-1 immunoblotting
0.1 μl human plasma was separated on 12.5% SDS-PA gels, transferred to nitrocellulose, blocked with 2.5% milk in TBST and incubated overnight at 4°C with the primary monoclonal anti-YB-1 antibody (biotinylated Portugal; II 2C-5, 1:1000) [
23]. Peroxidase-conjugated streptavidin (Dianova) and the ECL system (Amersham) were used for detection.
In each blot, one positive control sample obtained from a patient with metastasized small cell lung cancer was run in parallel, that was strongly YB-1/p18 positive. The YB-1/p18 signals were quantified by densiometry (NIH imager) and compared to the positive control signal which was assigned the optical density of "1.0". The relative optical density of the sample signals was calculated accordingly. All samples (patients and controls) were tested at least on two independent blots, with no mismatches (one positive, one negative) being detected.
Recombinant YB-1 protein purification and MS/MS analysis
A pRSET vector (Invitrogen) containing an insert coding for a hexahistidine T7 epitope-YB-1 fusion protein was a kind gift from Dr. Chien (University of California, San Diego). The recombinant YB-1 was synthesized with the pRSET prokaryotic coding sequence of rat YB-1 (also denoted EFIA). For expression in Escherichia coli, bacteria were induced with isopropyl-β-d-thiogalactoside, followed by addition of M13/T7 helper phage encoding T7 polymerase. Expressed protein was released by sonication, and the purification of recombinant YB-1 was performed with Ni+ affinity columns as outlined by the manufacturer (Invitrogen). Purity of the expressed YB-1 fusion product was ascertained by analytic SDS-polyacrylamide gel electrophoresis.
In-gel digest
The bands of interest were excised and in-gel digested in an adapted manner according to Shevchenko et al. [
24].
Mass spectrometric analysis
Dried samples were dissolved in 10 μl 2% ACN/0.1% trifluoroacetic acid (TFA) and applied to an Ultimate 3000 Nano-HPLC (Dionex, Germany), respectively. Each sample was first trapped on a 1 mm PepMap-trapping column (Dionex, Germany) for 10 min at 30 μl/min 2%ACN/0.1% TFA and subsequently subjected to a 75 μm ID, 5 cm PepMap C18-column (Dionex, Germany). Peptide separation was performed by an ACN- gradient at 300 nl/min. The separation column outlet was online coupled to a nano-spray interface (Bruker, Germany) of an Esquire HCT ETDII-Iontrap mass spectrometer (Bruker, Germany). Mass spectra were acquired in positive MS-mode, tuned for tryptic peptides. MS/MS-precursor selection was performed in an optimized automatic regime, with preference for double and triple charged ions. Every selected precursor was fragmented by collision induced dissociation (CID) and electron transfer dissociation (ETD), respectively. MS/MS spectra were processed by the Data Analysis and BioTools software from Bruker, Germany. Combined CID/ETD-derived fragment lists were analyzed by the MASCOT algorithm on in-house- and swissprot-databases.
Statistics
Results are reported as median and range, and differences between groups were assessed by Mann-Whitney U-test, Kruskal-Wallis-ANOVA or chi-square-test [
25]. Correlation analyses were performed by Spearman rank correlation test. Receiver operating characteristic (ROC) curve analysis and the derived c-statistic were calculated to assess the accuracy of a marker for predicting an event [
26]. All statistical analyses were performed using SPSS 12 (SPSS, Chicago, IL, USA).
Discussion
In this work, we identified circulating 18 kDa fragments of YB-1 protein (YB-1/p18) in human plasma using immunoblotting with a monoclonal antibody. The presence of YB-1/p18 in plasma samples may indicate malignant disorders of different origin. We found YB-1/p18 in about 80% of patients with advanced carcinomas and hepatic metastases, but in none of healthy volunteers. Potential confounding variables such as acute inflammations, renal or hepatic dysfunction could be excluded in non-cancerous cohorts. In a very well characterized group of 111 patients with chronic liver diseases, YB-1/p18 had a high sensitivity and reasonable specificity to identify patients with malignant tumors, suggesting its clinical potential as a tumor marker for screening 'high-risk' patient populations.
Although many 'tumor markers' are widely used in monitoring cancer patients during therapeutic interventions, lack of sensitivity and specificity preclude the use of most existing markers for the early detection of malignancy [
35]. The clinically best established general screening tumor marker at present is prostate-specific antigen (PSA), which is considered a specific indicator of prostate cancer, but still about 15% of 'PSA-negative' men have biopsy-detected prostate cancer [
36].
However, some markers can be useful in patient cohorts at high risk of developing certain carcinomas, e.g. AFP in patients with chronic virus hepatitis or liver cirrhosis at risk for HCC [
28]. Several new markers have been suggested that could, alone or in combination with AFP, improve the accuracy of diagnosing HCC, such as AFP-L3, DCP/PIVKAII, CA242 or AAG [
37‐
40]. In our study, AFP proved again to be superior in detecting HCC compared with CEA, CA19-9 and YB-1/p18 (Figure
4B). However, AFP or other 'established' markers were not powerful as screening tools for malignancies other than HCC. In fact, AFP and especially CEA or CA19-9 were elevated in a high number of patients with viral hepatitis without detectable malignancy, in line with previous reports [
31,
32]. YB-1/p18, on the other hand, was independent of the degree of liver cirrhosis and had a low rate of 'false-positive' results in liver disease patients as well as in patients with acute inflammatory or renal diseases, suggesting its usefulness in principle as a screening tool. YB-1/p18 furthermore tested negative in all blood donors ('healthy controls') and was not associated with inflammation or renal impairment ('non-malignant control patients'); all control populations were strictly age- and sex-matched to the liver patient cohort (Figure
3). In contrast to YB-1/p18, renal function is a well appreciated confounding variable for most established tumor markers [
41,
42].
YB-1/p18 was more powerful in detecting malignancies other than HCC compared to AFP, CEA or CA19-9 in patients with chronic liver disease (Figure
4C), and patients with a variety of malignancies with liver metastases tested predominantly positive for YB-1/p18 (Figure
1B,
3C). The relatively high sensitivity of YB-1/p18 for various malignant disorders may be explained by its unique role in tumor biology. Nuclear YB-1 overexpression is a common finding in a variety of solid tumors, including breast, prostate, ovarian or lung cancer [
4‐
7], and has been linked to tumor growth and survival of cancer patients in several studies. At present, it is unclear whether the detection of extracellular (circulating) YB-1/p18 fragment is associated with nuclear YB-1 overexpression in tumors. We therefore collected tumor tissue specimens (n = 20) and determined the subcellular YB-1 localization with monoclonal antibody by immunohistochemistry. At the same time we have determined the presence or absence of YB-1/p18 in corresponding serum samples. As a result, we were not able to find a positive correlation (unpublished observations). However, future studies will have to confirm these preliminary observations.
YB-1 appears to fulfill critical cellular functions such as transcriptional upregulation of proliferation-associated and downregulation of apoptosis-related genes or induction of drug-transporter genes (like MDR-1) involved in chemoresistance [
1,
7]. The exact function of extracellular YB-1 remains to be elucidated; however, by adding recombinant YB-1 protein to cell-lines
in vitro we were able to demonstrate a profound pro-mitogenic effect, suggesting that secreted YB-1 fragments could act as a tumor growth-promoting factor [
19]. Besides the abundant presence of full-length YB-1 protein with a relative mobility corresponding to 50 kDa in SDS-PAGEs that was detected in all (healthy, non-malignant, malignant) plasma samples, the YB-1 fragment identified here, corresponding to a relative MW of ~18 kDa (denoted YB-1/p18) and harboring the evolutionarily conserved cold shock domain appears to be associated with the presence of malignant disease. Antibody and MS/MS mapping of different recombinant YB-1 protein derivatives confirmed the cold-shock domain identity of the 18 kDa fragment (Figure
2). However, it is currently unclear whether YB-1/p18 is released from the tumor cells or from infiltrating stromal cells. Our own experiments indicated that YB-1 processing may occur intracellularly within the vesicles that release YB-1 from the cells, but it is unknown whether this is a phenomenon of cancer and transformed cells only [
19].
At present, the detection of YB-1/p18 can only be regarded as a "qualitative" tumor phenomenon, because we failed to quantitatively relate YB-1/p18 in plasma to either total full-length YB-1 in plasma or to nuclear YB-1 expression in the tumor (not shown). The development of a specific ELISA system for YB-1/p18 may in the future allow to more quantitatively measure YB-1/p18 levels. With such a more accurate quantification tool, new quantitative associations (e.g., between full-length YB-1 in plasma or nuclear YB-1 expression levels) may be explored.
Given our promising results, we set out to prospectively evaluate the clinical use of YB-1/p18 in larger cohorts of patients with diagnosed malignant disease. In patients with various solid or hematological malignancies, YB-1/p18 is detected in more than 75% of cancer patients (FT, NK, PRM, unpublished observations). Tumor-specific factors (origin, staging, grading, tumor mass, response to treatment) are currently evaluated for their impact on YB-1/p18. The presented results suggest that YB-1/p18 should be further investigated as a potential screening biomarker for malignant diseases, preferably in high-risk patient populations.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NK, AEN and CSE performed YB-1 immunoblots. TK and VS provided experimental tools and performed mass spectometric analyses. CT assisted in study design and patient recruitment. FT, NK and PRM designed the study, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.