Y-box Protein-1 Research Articles

Y-box protein-1/p18 as novel serum marker for ovarian cancer diagnosis: A study by the Tumor Bank Ovarian Cancer (TOC)

Introduction: The cold shock Y-box binding protein-1 (YB-1) fulfills important roles in regulating cell proliferation and differentiation. Overexpression occurs in various tumor cells. Given the existence of extracellular YB-1 we set out to determine the diagnostic, predictive and prognostic role of serum YB-1/p18 for patients with primary epithelial ovarian cancer (EOC).

Therapeutic nuclear shuttling of YB-1 reduces renal damage and fibrosis

Virtually all chronic kidney diseases progress towards tubulointerstitial fibrosis. Invitro, Y-box protein-1 (YB-1) acts as a central regulator of gene transcription and translation of several fibrosis-related genes. However, it remains to be determined whether its pro- or antifibrotic propensities prevail in disease. Therefore, we investigated the outcome of mice with half-maximal YB-1 expression in a model of renal fibrosis induced by unilateral ureteral obstruction. Yb1+/- animals displayed markedly reduced tubular injury, immune cell infiltration and renal fibrosis following ureteral obstruction. The increase in renal YB-1 was limited to a YB-1 variant nonphosphorylated at serine 102 but phosphorylated at tyrosine 99. During ureteral obstruction, YB-1 localized to the cytoplasm, directly stabilizing Col1a1 mRNA, thus promoting fibrosis. Conversely, the therapeutic forced nuclear compartmentalization of phosphorylated YB-1 by the small molecule HSc025 mediated repression of the Col1a1 promoter and attenuated fibrosis following ureteral obstruction. Blunting of these effects in Yb1+/- mice confirmed involvement of YB-1. HSc025 even reduced tubulointerstitial damage when applied at later time points during maximum renal damage. Thus, phosphorylation and subcellular localization of YB-1 determines its effect on renal fibrosis invivo. Hence, induced nuclear YB-1 shuttling may be a novel antifibrotic treatment strategy in renal diseases with the potential of damage reversal.

Cold shock protein YB-1 is involved in hypoxia-dependent gene transcription

Hypoxia-dependent gene regulation is largely orchestrated by hypoxia-inducible factors (HIFs), which associate with defined nucleotide sequences of hypoxia-responsive elements (HREs). Comparison of the regulatory HRE within the 3′ enhancer of the human erythropoietin (EPO) gene with known binding motifs for cold shock protein Y-box (YB) protein-1 yielded strong similarities within the Y-box element and 3′ adjacent sequences. DNA binding assays confirmed YB-1 binding to both, single- and double-stranded HRE templates. Under hypoxia, we observed nuclear shuttling of YB-1 and co-immunoprecipitation assays demonstrated that YB-1 and HIF-1α physically interact with each other. Cellular YB-1 depletion using siRNA significantly induced hypoxia-dependent EPO production at both, promoter and mRNA level. Vice versa, overexpressed YB-1 significantly reduced EPO-HRE-dependent gene transcription, whereas this effect was minor under normoxia. HIF-1α overexpression induced hypoxia-dependent gene transcription through the same element and accordingly, co-expression with YB-1 reduced HIF-1α-mediated EPO induction under hypoxic conditions. Taken together, we identified YB-1 as a novel binding factor for HREs that participates in fine-tuning of the hypoxia transcriptome.

Abstract 2451: Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2

Abstract Sonic hedgehog (Shh)-mediated medulloblastoma growth requires IGF2 (Insulin-like Growth Factor 2) and we recently showed that Yes Associated Protein (YAP1) induces IGF2 expression by Y-box protein 1(YB1) in Shh-stimulated cerebellar granule neural precursors (CGNPs), proposed cells-of-origin for the Shh molecular subclass of medulloblastoma, and mouse Shh-medulloblastoma cells. We found elevated levels of YAP1, YB1 and IGF2 in tumor cells occupying the peri-vascular niche, a microenvironmental niche proposed to house so-called tumor re-populating cells that survive radiation and contribute to medulloblastoma recurrence, which is fatal. We have developed an ex vivo approach using organotypic brain tumor slice cultures to better understand how YAP1, YB1, and IGF2 regulate peri-vascular niche cell survival post-radiation. We observed that the perivascular niche cell population expressing stem cell markers increases markedly following exposure to radiation. Additionally, on targeting any component of the YAP1-YB1-IGF2 axis we observed increased level of cell death within the niche and compromised cancer stem cell niche expansion post-radiation. These findings strongly indicate that therapeutic approaches intended to impair the function of this pathway could be used to reduce the use of cranio-spinal radiation of medulloblastoma patients, which causes life-long side effects that drastically impair quality of life. Citation Format: Abhinav Dey, Caroline Maier, Anshu Malhotra, Anna Kenney. Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2451.

Y-box protein-1/p18 as novel serum marker for ovarian cancer diagnosis: A study by the Tumor Bank Ovarian Cancer (TOC)

IntroductionThe cold shock Y-box binding protein-1 (YB-1) fulfills important roles in regulating cell proliferation and differentiation. Overexpression occurs in various tumor cells. Given the existence of extracellular YB-1 we set out to determine the diagnostic, predictive and prognostic role of serum YB-1/p18 for patients with primary epithelial ovarian cancer (EOC). MethodsThe protein fragment YB-1/p18 was quantified by sandwich ELISA in serum samples from 132 healthy female volunteers and 206 patients with histological diagnosis of primary EOC. The ELISA sensitivity and specificity to detect EOC were calculated using receiver operating curves. Survival data were calculated using Kaplan Maier curves. ResultsMedian age at the time of diagnosis was 60years and follow-up ended with a mean of 44.8month. 188 (91%) patients were diagnosed at advanced stages (FIGO III/IV) and 188 patients (91%) suffered from high-grade serous ovarian carcinoma. YB-1/p18 levels were significantly decreased in older patients (p=0.021). Significantly lower serum levels of YB-1/p18 were detected in the EOC cohort when compared to the control group (p<0.0001, AUC=0.827; 95% CI, 0.787–0.867). Using the expression of serum YB-1/p18 in early stages I and II cases these could be differentiated from control cases (p<0.0001, AUC=0.816; 95% CI 0.704–0.929). No other significant associations between clinical prognostic factors and YB-1/p18 serum levels were detected. Immunoblotting results with serum samples suggest that masking of epitopes by the YB-1/p18 fragment in multiprotein-complexes under non reducing conditions leads to the observed reduced ELISA readings in the EOC cohort. ConclusionsThe quantification of fragment YB-1/p18 derived from cold shock protein YB-1 in serum samples could be useful for the early diagnosis of EOC.

YB-1, a new biomarker of glioma progression, is associated with the prognosis of glioma patients.

Y box protein 1 (YB-1) is a multifunctional cellular protein expressed in various cancers, and is a potential target in cancer therapy. Although there is evidence showing that YB-1 plays a role in human cancers, the clinical significance of YB-1 expression in glioma has not been established. In the present study, we investigated the YB-1 level in glioma tumors and analyzed the relationship between the YB-1 level and the grade of malignant glioma, with the aim of providing new ideas for the diagnosis and treatment of gliomas in clinical and basic research settings. A total of 108 patients, comprising 14, 31, 30, and 33 with gliomas of Grades I, II, III, and IV, respectively, were included in this study. The mRNA and protein levels of YB-1 were found to be significantly different between Grade IV and lower-grade tumors. The YB-1 levels in cerebrospinal fluid were significantly higher in Grades III and IV glioma patients than in Grades I and II patients. Immunofluorescence staining was used to detect the levels of YB-1 in the cytoplasm and the nucleus, and results indicated that the intracellular distribution was significantly associated with the pathological grade of glioma. A higher level of YB-1 was associated with shortened survival, suggesting that YB-1 plays a role in the progression of human glioma.

Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1

The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.

Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma.

Transforming growth factor β (TGF-β) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-β role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-β inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-β effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-β-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-β actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.

Calcineurin-mediated YB-1 Dephosphorylation Regulates CCL5 Expression during Monocyte Differentiation

Y-box (YB) protein-1 serves as a master regulator in gene transcription and mRNA translation. YB-1 itself is regulated at various levels, e.g. through post-translational modifications. In our previous work, we identified RANTES/CCL5 as a transcriptional target of YB-1. We previously demonstrated that YB-1 protein is transiently up-regulated during monocyte/macrophage differentiation evidenced in monocytic cells (THP-1 cells) that were differentiated using phorbol myristate acetate (PMA). Here we provide evidence that YB-1 phosphorylation, specifically at its serine residue 102 (Ser-102), increases early on in THP-1 cells following PMA treatment as well as in differentiated primary human monocytes. This process is mediated through the Akt signaling pathway. Ser-102-phosphorylated YB-1 displays stronger binding affinity and trans-activating capacity at the CCL5 gene promoter. Notably, Ser-102-phosphorylated YB-1 disappears at later stages of the monocyte/macrophage differentiation process. We demonstrate that serine-threonine phosphatase calcineurin (CN) dephosphorylates YB-1 preventing it from binding to and trans-activating the CCL5 promoter. Co-immunoprecipitation assays prove a direct YB-1/CN interaction. Furthermore, analyses in kidney tissues from mice that were treated with the CN inhibitor cyclosporine A revealed an in vivo effect of CN on the YB-1 phosphorylation status. We conclude that YB-1 phosphorylation at Ser-102 is an important prerequisite for CCL5 promoter activation during macrophage differentiation. Our findings point to a critical role of YB-1 in the resolution of inflammatory processes which may largely be due to CN-mediated dephosphorylation.

High prevalence of Y-box protein-1/p18 fragment in plasma of patients with malignancies of different origin

BackgroundExpression of the cold shock protein Y-box protein 1 (YB-1) is associated with deleterious outcome in various malignant diseases. Our group recently showed that the detection of an 18 kDa YB-1 fragment (YB-1/p18) in human plasma identifies patients with malignant diseases. We now tested the prevalence, clinical, and diagnostic value of YB-1/p18 detection in common tumors.MethodsA newly established monoclonal YB-1 antibody was used to detect YB-1/p18 by immunoblotting in plasma samples from 151 unselected tumor patients, alongside established tumor markers and various diagnostic measures, during evaluation for a cancerous disease and in follow-up studies after therapeutic interventions.ResultsCirculating YB-1/p18 was detected in 78% of patients having a tumor disease. YB-1/p18 positivity was highly prevalent in all examined malignancies, including lung cancer (32/37; 87%), breast cancer (7/10; 70%), cancer of unknown primary (CUP; 5/5, 100%) or hematological malignancies (42/62; 68%). Positivity for YB-1/p18 was independent of other routine laboratory parameters, tumor stage, or histology. In comparison to 13 established tumor markers (cancer antigens 15–3, 19–9, 72–4, and 125; carcinoembryonic antigen; cytokeratin fragments 21–1; neuron-specific enolase; alpha-fetoprotein; beta-2-microglobulin; squamous cell carcinoma antigen; thymidine kinase; tissue polypeptide antigen; pro-gastrin-releasing peptide), YB-1/p18 detection within serum samples was the most sensitive general parameter identifying malignant disorders. YB-1/p18 concentrations altered during therapeutic interventions, but did not predict prognosis.ConclusionsPlasma YB-1/p18 detection has a high specific prevalence in malignancies, thereby providing a novel tool for cancer screening independent of the tumor origin.

Cold shock Y-box protein-1 proteolysis autoregulates its transcriptional activities

BackgroundThe Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s).ResultsTo address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter.ConclusionCompartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation.

YB-1 immunization combined with regulatory T-cell depletion induces specific T-cell responses that protect against neuroblastoma in the early stage

Neuroblastoma is the most common extracranial solid cancer in childhood and the most common cancer in infancy. Currently, no effective clinical treatments are available for advanced neuroblastoma. In a previous study, we screened Y Box protein 1 (YB-1) as a potential neuroblastoma-associated antigen from sera of AGN2a-immunized mice by serological analysis of recombinant cDNA expression libraries technique. The aim of this study is to explore if YB-1 immunization in the context of Treg depletion could induce protective immune response against the neuroblastoma in mice. YB-1 was expressed and purified by pET-15b prokaryotic expression system. It was demonstrated that anti-YB-1 CD8(+) T-cell responses could be induced by AGN2a immunization, and the strongest CD8(+) T-cell responses against AGN2a were induced by YB-1-immunized mice in the context of Treg depletion compared with YB-1 only immunization group and control group. Importantly, the survival rate of mice treated with YB-1 immunization combined with Treg depletion was 80% when challenged by 1 × 10(4) AGN2a cells, significantly higher than that of mice immunized with YB-1 alone (P < 0.01). Furthermore, T-cell adoptive therapy showed that the neuroblastoma growth was inhibited when T cells or splenic cells from YB-1-immunized mice with Treg depletion were transferred to AGN2a bearing mice. Both CD4(+) and CD8(+) T cells were involved in the anti-neuroblastoma responses induced by YB-1 immunization combined with Treg depletion. These results indicated that YB-1 immunization combined with Treg depletion could induce specific T-cell responses against neuroblastoma and could be a potential strategy for the prevention and treatment of neuroblastoma in the early stage.

Stable expression of Y-box protein 1 gene in early development of the abalone Haliotis diversicolor

Abalone animals are import models for the study of the early development of marine invertebrates. However, systematical evaluations of internal control genes (ICG) have seldomly been performed. In this study, ten candidate genes were cloned and surveyed for their stability throughout the early developmental period of H. diversicolor using qPCR. In a period from fertilized egg to postlarva, three genes, Y-box protein 1 (YB1), ornithine decarboxylase antizyme 1 (OAZ1) and eukaryotic translation initiation factor 5A (EIF5A), were found to be the most stable and could be used as ICGs. It is suggested that using two genes jointly, such as YB1 and OAZ1, could be sufficiently reliable to normalize the temporal dynamics of other genes. Normalized by YB1/OAZ1, some rough features of early development of a small abalone were characterized. This is the first report of the temporal dynamics of metabolic activities and overall mRNA abundance of abalone animals in early stages. It is also the first time the multi-functional gene YB1 has been described as an internal control for early developmental biology studies. Phylogeny and function of YB1 are further discussed.

Cold shock Y-box protein-1 participates in signaling circuits with auto-regulatory activities

The cold shock protein Y-box (YB) binding-1 is an example of a highly regulated protein with pleiotropic functions. Besides activities as a transcription factor in the nucleus or regulator of translation in the cytoplasm, recent findings indicate extracellular effects and secretion via a non-classical secretion pathway. This review summarizes regulatory pathways in which YB-1 participates, all iterating auto-regulatory loops. Schematics are developed that elucidate the cold shock protein activities in (i) fine-tuning its own expression level following platelet-derived growth factor-B-, thrombin- or interferon-γ-dependent signaling, (ii) as a component of the messenger ribonucleoprotein (mRNP) complex for interleukin-2 synthesis in T-cell commitment/activation, (iii) pro-fibrogenic cell phenotypic changes mediated by transforming growth factor-β, and (iv) receptor Notch-3 cleavage and signal transduction. Emphasis is put forward on subcellular protein translocation mechanisms and underlying signaling pathways. These have mostly been analysed in cell culture systems and rarely in experimental models. In sum, YB-1 seems to fulfill a pacemaker role in diverse diseases, both inflammatory/pro-fibrogenic as well as tumorigenic. A clue towards potential intervention strategies may reside in the understanding of the outlined auto-regulatory loops and means to interfere with cycling pathways.

Role of cold shock Y-box protein-1 in inflammation, atherosclerosis and organ transplant rejection

Chemokines (chemoattractant cytokines) are crucial regulators of immune cell extravasation from the bloodstream into inflamed tissue. Dysfunctional regulation and perpetuated chemokine gene expression are linked to progressive chronic inflammatory diseases and, in respect to transplanted organs, may trigger graft rejection. RANTES (regulated upon activation, normal T cell expressed and secreted (also known as CCL5)) is a model chemokine with relevance in numerous inflammatory diseases where the innate immune response predominates. Transcription factor Y-box binding protein-1 (YB-1) serves as a trans-regulator of CCL5 gene transcription in vascular smooth muscle cells and leucocytes. This review provides an update on YB-1 as a mediator of inflammatory processes and focuses on the role of YB-1 in CCL5 expression in diseases with monocytic cell infiltrates, albeit acute or chronic. Paradigms of such diseases encompass atherosclerosis and transplant rejection where cold shock protein YB-1 takes a dominant role in transcriptional regulation.

Y-box protein-1/p18 fragment identifies malignancies in patients with chronic liver disease

BackgroundImmunohistochemical detection of cold shock proteins is predictive for deleterious outcome in various malignant diseases. We recently described active secretion of a family member, denoted Y-box (YB) protein-1. We tested the clinical and diagnostic value of YB-1 protein fragment p18 (YB-1/p18) detection in blood for malignant diseases.MethodsWe used a novel monoclonal anti-YB-1 antibody to detect YB-1/p18 by immunoblotting in plasma samples of healthy volunteers (n = 33), patients with non-cancerous, mostly inflammatory diseases (n = 60), hepatocellular carcinoma (HCC; n = 25) and advanced solid tumors (n = 20). YB-1/p18 was then tested in 111 patients with chronic liver diseases, alongside established tumor markers and various diagnostic measures, during evaluation for potential liver transplantation.ResultsWe developed a novel immunoblot to detect the 18 kD fragment of secreted YB-1 in human plasma (YB-1/p18) that contains the cold-shock domains (CSD) 1-3 of the full-length protein. YB-1/p18 was detected in 11/25 HCC and 16/20 advanced carcinomas compared to 0/33 healthy volunteers and 10/60 patients with non-cancerous diseases. In 111 patients with chronic liver disease, YB-1/p18 was detected in 20 samples. Its occurrence was not associated with advanced Child stages of liver cirrhosis or liver function. In this cohort, YB-1/p18 was not a good marker for HCC, but proved most powerful in detecting malignancies other than HCC (60% positive) with a lower rate of false-positive results compared to established tumor markers. Alpha-fetoprotein (AFP) was most sensitive in detecting HCC, but simultaneous assessment of AFP, CA19-9 and YB-1/p18 improved overall identification of HCC patients.ConclusionsPlasma YB-1/p18 can identify patients with malignancies, independent of acute inflammation, renal impairment or liver dysfunction. The detection of YB-1/p18 in human plasma may have potential as a tumor marker for screening of high-risk populations, e.g. before organ transplantation, and should therefore be evaluated in larger prospective studies.

Y‐box protein‐1 is a transcriptional regulator of BMP7

Bone morphogenetic protein-7 (BMP7) is an endogenous antifibrogenic protein in the kidney which is down regulated in experimental chronic kidney diseases such as obstructive and diabetic nephropathy in parallel with progressively increasing TGFβ. In vitro studies were performed in Madin-Darby Canine Kidney (MDCK)-cells to identify transcriptional regulators of BMP7. Experiments with various BMP7 promoter fragments (465-4,267 bp) identify small proximal promoter segments that are transcriptionally activated by high glucose (3.2-fold) but down regulated by TGFβ (0.2-fold) compared to normal glucose. Protein binding to these DNA segments is increased by high glucose and decreased by TGFβ in a time-dependent, progressive manner. Analysis of BMP7 promoter-binding proteins with liquid chromatography/tandem mass spectrometry (LC/MS/MS) identifies seven unique, partially overlapping peptides, spanning 25% of the amino acid sequence of Y-box protein-1 (YB1). EMSA-Western blot combination experiments confirm that YB1 is a BMP7 promoter-binding protein. YB1 knock-down reduces transcriptional responses to high glucose and TGFβ by about one-half, respectively. In addition, high glucose induces but TGFβ reduces nuclear translocation of YB1 from the cytoplasm. These studies identify YB1 as a transcriptional activator of BMP7 and helps to explain the progressive decline in renal BMP7 in diabetic nephropathy and other kidney diseases.

Extracellular YB-1 Blockade in Experimental Nephritis Upregulates Notch-3 Receptor Expression and Signaling

Background: Notch receptors are involved in kidney development and pathogenesis of inflammatory glomerular diseases. Given the secretion of Y-box (YB) protein-1 following cytokine stimulation and subsequent extracellular association with membrane receptor Notch-3 in vitro, we elucidated functional effects of YB-1 targeting on the Notch-3 signaling pathway. Methods: Rat mesangial cells were challenged with a monoclonal anti-YB-1 antibody (YB-1-mAb) and analyzed for YB-1 and Notch-3 expression. Notch-3 expression in mice with a targeted disruption of one YB-1 allele (YB-1^+/d) was compared with their wild-type littermates. Furthermore, YB-1-mAb was applied during mesangioproliferative anti-Thy1.1 nephritis, and glomerular Notch-3, Notch target genes and YB-1 expression were analyzed by immunohistochemistry, quantitative real-time PCR and immunoblotting. Results: Upon challenge with YB-1-mAb, rat mesangial cells showed an increased expression of YB-1 and Notch-3 protein. Concordantly, we found a significant upregulation of Notch-3 expression in renal cells of YB-1^+/d mice. YB-1-mAb treatment in anti-Thy1.1 nephritis resulted in enhanced mesangial Notch-3 expression and differential Notch target gene activation (HES2/Hey-2). Notably, YB-1 mRNA content did not differ between groups; however, glomerular YB-1 protein was significantly increased, suggesting a posttranslational mechanism. Conclusion: Extracellular targeting of YB-1 potently induces glomerular Notch-3 receptor expression, Notch signaling and YB-1 stabilization, most likely via an autoregulatory feedback mechanism.

Nuclear detection of Y-boxprotein-1 (YB-1) closely associates with progesterone receptor negativity and is a strong adverse survival factor in human breast cancer

BackgroundY-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestrates transcription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA and directs translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival).MethodsHybridoma cell generation was carried out with recombinant YB-1 protein as immunogen and Mab characterization was performed using immunoblotting and ELISA with recombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breast cancer specimens. Breast tumor tissue array staining results were analyzed for correlations with receptor expression and outcome parameters.ResultsYB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping to two domains within the N-terminal half of the protein and detects nuclear YB-1 protein by immunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarray comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissue samples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overall survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection and absence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detection marks a specific subgroup of breast cancer. Likely due to limitation of sample size Cox regression models failed to demonstrate significance for nuclear YB-1 detection as independent prognostic marker.ConclusionMonoclonal YB-1 antibody F-E2G5 should be of great value for prospective studies to validate YB-1 as a novel biomarker suitable to optimize breast cancer treatment.

Y-Box Protein 1 Stimulates Mesangial Cell Proliferation via Activation of ERK1/2

Aims: To investigate the effects of Y-box protein 1 (YB-1) on the mitogen-activated protein kinase pathway to ascertain which signaling pathway is involved in YB-1-induced mesangial cell (MC) proliferation. Methods: The rat MC line HBZY-1 was transfected with pcDNA3.1-YB-1. Cell proliferation was determined by [³H]thymidine incorporation, cell counting and cell-cycle analysis. The expression of cyclins was examined by real-time RT-PCR and immunoblotting analysis. Phosphorylation of c-Raf, MEK1/2, and ERK1/2 was detected by immunoblotting analysis. Results: MCs transfected with YB-1 showed an increased DNA synthesis and number of cells in the S and G2 phases of the cell cycle. The expression of cyclins (cyclin A2, cyclin D1, cyclin E and p27) was increased, while p21 levels were decreased. The expression levels of phosphorylated c-Raf kinase, MEK1/2 and the ERK1/2 proteins were elevated in MCs transfected with YB-1. YB-1-stimulated cell proliferation was blocked by U0126, a specific inhibitor of ERK1/2. ERK1/2 regulated YB-1 expression in MCs stimulated with TGF-β, an effect that was inhibited by U0126. Conclusions: YB-1 has an apparent stimulatory effect on c-Raf, MEK1/2, ERK1/2 and cell-cycle progression in MCs, after which activated ERK1/2 goes on to upregulate YB-1 expression and augment the proliferative effect of YB-1.