Overexpression Of YB-1 Research Articles

Downregulation of Roundabout guidance receptor 2 suppresses hepatocellular carcinoma progression by interacting with Y-box binding protein 1

Roundabout guidance receptor 2 (Robo2) is closely related to malignant tumors such as pancreatic cancer and liver fibrosis, but there is no relevant research on the role of Robo2 in HCC. The study will further explore the function and mechanism of Robo2 and its downstream target genes in HCC. Firstly, Robo2 protein levels in human HCC tissues and paired adjacent normal liver tissues were detected. Then we established HepG2 and Huh7 hepatoma cell lines with knock-down Robo2 by transfection with lentiviral vectors, and examined the occurrence of EMT, proliferation and apoptosis abilities in HCC cells by western blot, flow cytometry, wound healing assay and TUNEL staining. Then we verified the interaction between Robo2 and its target gene by Co-IP and immunofluorescence co-staining, and further explored the mechanism of Robo2 and YB-1 by rescue study. The protein expression level of Robo2 in HCC was considerably higher than that in the normal liver tissues. After successfully constructing hepatoma cells with knock-down Robo2, it was confirmed that down-regulated Robo2 suppressed EMT and proliferation of hepatoma cells, and accelerated the cell apoptosis. High-throughput sequencing and validation experiments verified that YB-1 was the downstream target gene of Robo2, and over-expression of YB-1 could reverse the apoptosis induced by Robo2 down-regulation and its inhibitory effect on EMT and proliferation. Robo2 deficiency inhibits EMT and proliferation of hepatoma cells and augments the cell apoptosis by regulating YB-1, thus inhibits the occurrence of HCC and provides a new strategy for the treatment of HCC.

Modified physiology of burley tobacco plants genetically engineered to express Yb1, a functional EGY enzyme.

Transgenic overexpression of a NtEGY2 gene restores normal green color of burley tobacco plants, but does not increase nitrogen utilization efficiency beyond that exhibited by wild-type individuals. Nitrogen physiology is important in tobacco because of its role in generation of leaf yield and accumulation of nitrogen-containing alkaloids that can react with nitrosating agents in the formation of carcinogenic tobacco-specific nitrosamines. Cultivars of the burley tobacco market class are homozygous for deleterious mutant alleles at the duplicate Yb1 and Yb2 loci which have previously been associated with decreased nitrogen use and utilization efficiency; increased leaf nitrate, total nitrogen, and alkaloid levels; and reduced yields. How mutant alleles at these two loci affect these traits is not well understood. Recent characterization of the Yb1 and Yb2 genes (homologs of Arabidopsis EGY1 gene) enabled overexpression of the wild-type Yb1 allele in yb1yb1yb2yb2 plants to determine if observed unfavorable effects were due to linkage or pleiotropy, and to determine if overexpression could lead to beneficial modifications in any of these traits in transgenic plants relative to naturally-occurring wild-type genotypes. Yb1 overexpression was found to confer an agronomic benefit to yb1yb1yb2yb2 genotypes but no advantage to wild-type genotypes. RNA-Seq was used to carry out a comparative transcriptome analysis of genetically engineered and wild-type nearly isogenic lines (NILs) to gain insight on metabolic pathways affecting carbon and nitrogen metabolism that might be altered as the result of genetic variability at the Yb1 and Yb2 loci. Results indicate that complex changes in the transcriptome of tobacco can be manifested by altered expression of Yb1.

DNAR-14. Y-BOX BINDING PROTEIN 1 REGULATES MEDULLOBLASTOMA TUMOR BIOLOGY AND THERAPEUTIC RESPONSE

Abstract BACKGROUND Medulloblastoma (MB) is the most common pediatric brain tumor. MB can be dividing into 4 genetically distinct subgroups (Sonic Hedgehog (SHH), Wingless (WNT), Group 3, and Group 4). Most patients receive radiation and cisplatin backbone therapy. SHH and Group3 patients demonstrate poor prognosis, especially in cases of MYC amplification or p53 mutation. Given the continued reliance on DNA damage therapies, and our groups previous findings that YB1 regulates IGF2 transcription in SHH MB, we chose to investigate the role of YB1 in the DNA damage response and we performed RIPseq to understand mRNAs regulated by YB1 that may be targeted as novel therapeutics. METHODS Cell lines include primary MBCs derived from NeuroD2-SmoA1 SHH mice, ONS-76 human SHH, Daoy human SHH, D341 and D425 Human Group 3. YB1 overexpression or knockdown followed by radiation and assessment of yH2AX (a marker of DNA de-condensation following damage), Comet assay (a marker of physical damage), and proliferation time courses were used to assess effects of YB1 modulation on radiation response. RIPseq and YB1 knockdown were used to assess the effects of YB1 on PLXND1 levels. Scratch assay and western blotting of EMT markers were used to assess effects of PLXND1 silencing on migration. RESULTS (Radiation Studies) Overexpression of YB1 in primary MBCs followed by allograft into BL6 mice results in decreased overall survival. YB1 silencing followed by radiation results in faster resolution of yH2AX, faster resolution of damage, and lack of pRPA32 accumulation. YB1 silencing sensitizes cells to radiation resulting in substantial decreases in proliferation. (RNA Binding Protein Studies) YB1 binds and positively regulates PLXND1 translation. Silencing PLXND1 results in decreased migration and EMT mark expression. CONCLUSIONS YB1 drives a more error prone non-homologous End-Joining based mechanism of repair and binds and regulates PLXND1 post-transcriptionally. PLXND1 drives a migratory phenotype in SHH MB.

P35.01 YB-1 is a Key Player in Aggressive Behaviour and Chemoresistance in Mesothelioma

Malignant pleural mesothelioma (MPM) is characterised by aggressive growth and frequent resistance to chemotherapy, poor prognosis for patients and limited therapeutic options. To establish potential new therapy targets, a better understanding of the MPM biology underlying these malignant behaviours is crucial. One potential candidate is the multifunctional oncoprotein YB-1, which is often overexpressed in various cancers and associated with aggressiveness, metastasis and poor outcome. YB-1 expression was analysed by qPCR or western blot in cell lines, and in patient material by immunohistochemistry. YB-1 overexpression was achieved by a doxycycline-inducible, retroviral construct, which was stably introduced into MPM cell lines. YB-1 knockdown was performed using YB-1 specific siRNA. For inhibition of YB-1 phosphorylation, the RSK inhibitor BI-D1870 was used. Effects of drug interaction (CI values) were calculated using the CompuSyn software. Cell migration was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. For the zebrafish model, RFP-expressing MPM cells were injected into 48 hour old larvae, imaged after 1 and 2 days and the number of cells in the tail was manually counted. We previously reported that YB-1 is up-regulated in MPM cell lines compared to non-malignant controls. In this study we analysed MPM tissue (n>120) and normal pleura (n=3) specimens for YB-1 expression. While all control samples were negative, tumours showed a heterogeneous YB-1 expression. YB-1 knockdown decreases MPM cell migration and invasion, hence we evaluated the impact of YB-1 overexpression on these phenotypes. MPM cell lines (n=6) which overexpress YB-1 in a doxycycline-inducible manner showed significantly increased cell scattering and migration. Furthermore, YB-1 overexpression lead to significantly higher migratory capacity in vivo using a zebrafish xenograft model. Additionally, when tumour spheroids were co-cultured with endothelial cells, YB-1 overexpression led to a significantly more extensive formation of gaps in the endothelial layer. Finally, since we found that YB-1 knockdown not only decreases cell growth in vitro and in vivo but also decreases the expression of LRP1 and ABCC1, two genes involved in drug resistance, we combined YB-1 knockdown as well as inhibition of YB-1 phosphorylation via an RSK inhibitor with cisplatin chemotherapy. Our data showed highly synergistic combination effects (CI values: 0.1 – 0.5) for several cisplatin doses in combination with either YB-1 siRNA or the RSK inhibitor. In this study we report a deregulated YB-1 expression in MPM tumor specimens compared to non-malignant controls. Our data show an important role of YB-1 in the regulation of cell migration and invasion in vitro and in vivo, which are key characteristics of MPM. Additionally, YB-1 knock down as well as pharmacological inhibition of YB-1 phosphorylation not only reduce MPM cell growth but also sensitise cells to cisplatin chemotherapy. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

YB-1 Oncoprotein Controls PI3K/Akt Pathway by Reducing Pten Protein Level.

YB-1 is a multifunctional protein overexpressed in many types of cancer. It is a crucial oncoprotein that regulates cancer cell progression and proliferation. Ubiquitously expressed in human cells, YB-1 protein functions are strictly dependent on its subcellular localization. In the cytoplasm, where YB-1 is primarily localized, it regulates mRNA translation and stability. However, in response to stress stimuli and activation of PI3K and RSK signaling, YB-1 moves to the nucleus acting as a prosurvival factor. YB-1 is reported to regulate many cellular signaling pathways in different types of malignancies. Furthermore, several observations also suggest that YB-1 is a sensor of oxidative stress and DNA damage. Here we show that YB-1 reduces PTEN intracellular levels thus leading to PI3K/Akt pathway activation. Remarkably, PTEN reduction mediated by YB-1 overexpression can be observed in human immortalized keratinocytes and HEK293T cells and cannot be reversed by proteasome inhibition. Real-time PCR data indicate that YB-1 silencing up-regulates the PTEN mRNA level. Collectively, these observations indicate that YB-1 negatively controls PTEN at the transcript level and its overexpression could confer survival and proliferative advantage to PTEN proficient cancer cells.

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells.

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5′UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.

Effect and mechanism of YB-1 knockdown on glioma cell growth, migration, and apoptosis.

Y-box binding protein 1 (YB-1) is manifested as its involvement in cell proliferation and differentiation and malignant cell transformation. Overexpression of YB-1 is associated with glioma progression and patient survival. The aim of this study is to investigate the influence of YB-1 knockdown on glioma cell progression and reveal the mechanisms of YB-1 knockdown on glioma cell growth, migration, and apoptosis. It was found that the knockdown of YB-1 decreased the mRNA and protein levels of YB-1 in U251 glioma cells. The knockdown of YB-1 significantly inhibited cell proliferation, colony formation, and migration in vitro and tumor growth in vivo. Proteome and phosphoproteome data revealed that YB-1 is involved in glioma progression through regulating the expression and phosphorylation of major proteins involved in cell cycle, adhesion, and apoptosis. The main regulated proteins included CCNB1, CCNDBP1, CDK2, CDK3, ADGRG1, CDH-2, MMP14, AIFM1, HO-1, and BAX. Furthermore, it was also found that YB-1 knockdown is associated with the hypo-phosphorylation of ErbB, mTOR, HIF-1, cGMP-PKG, and insulin signaling pathways, and proteoglycans in cancer. Our findings indicated that YB-1 plays a key role in glioma progression in multiple ways, including regulating the expression and phosphorylation of major proteins associated with cell cycle, adhesion, and apoptosis.

Inhibition of Y Box Binding Protein 1 Suppresses Cell Growth and Motility in Colorectal Cancer.

Although chemo- or radiotherapy is usually performed in patients with colorectal cancer, the response is highly variable in locally rectal cancer. Therefore, additional studies are needed on predictable markers and the molecular mechanisms of chemo- and radiotherapy. Y box binding protein 1 (YB1) is an oncoprotein that is aberrantly expressed in many cancers, including colorectal cancer. However, to date there are no targeting agents or strategies to inhibit YB1 expression. Here, we investigate the oncogenic function of YB1 in colorectal cancer and methods to control its expression. We observed that YB1 expression level is correlated with colorectal cancer survival rate. Moreover, YB1 overexpression was associated with colorectal cancer lymph node metastasis and invasion. We also found that radiation exposure increased YB1 expression, which led to radioresistant colorectal cancer, mediated through the activation of cancer stem cell marker CD44 and PI3K/AKT/mTOR signaling. This study revealed, by both in vitro and in vivo assays, that depletion of YB1 could reduce cell proliferation and motility in colorectal cancer. We further demonstrated that the PI3K/mTOR inhibitor BEZ235 suppressed YB1 expression and enhanced the cytotoxicity of radiation. In addition, combined treatment with BEZ235 and radiation showed a significant antitumor response in an in vivo mouse xenograft model. Taken together, our results provide evidence that the activation of YB1 is a major factor in radioresistance and suggest that targeting YB1-mediated signaling is a promising therapeutic strategy for colorectal cancer.

YB-1 modulates the drug resistance of glioma cells by activation of MDM2/p53 pathway.

BackgroundY-box-binding protein-1 (YB-1) is aberrantly expressed in a variety of cancers. However, the biological functional role of YB-1 in glioma is not yet clear.MethodsThe expression of MDM2 and YB-1 was analyzed by real time PCR. Overexpression and knockdown of YB-1 in glioma cells were created by transfection of pcDNA-YB-1 and siRNA against YB-1, respectively. Cell viability was performed by CCK8 assay.ResultsOur findings showed that glioma tissues had higher expressions of YB-1 than that in cancer-free tissues in 54 glioma patients, which were also positively correlated with Murine MDM2 expression. Overexpression of YB-1 or MDM2 renders a drug resistance feature in glioma cell exposed to temozolomide (TMZ), by directly targeting p53. Genetic or chemical inhibition of MDM2 significantly blocked YB-1-modulated response of glioma cells to TMZ. Moreover, inhibition of YB-1 or MDM2 reduced glioma cells metastasis and mortality in mice.ConclusionYB-1 facilitates the resistance of glioma cells to TMZ by direct activation of MDM2/p53 signaling and represents a promising molecular target for glioma treatment.

P2.06-32 YB-1 - A Key Factor in Mesothelioma Aggressive Growth and Behaviour

Malignant pleural mesothelioma (MPM) is a devastating disease characterized by aggressive growth and local invasion, poor outcome and limited therapeutic options. YB-1 is a multifunctional oncoprotein, which is often up-regulated in cancer and associated with aggressiveness and poor patient outcome. Also, YB-1 is actively secreted by cells upon various stresses. Besides numerous other functions, YB-1 has been described to stimulate cancer cell migration and invasion via regulation of EMT-related factors such as Snail and Twist. Cells were treated with LPS (20 ng/ml) or grown under hypoxic conditions (1% O2, N2 balanced) for 24 hours. YB-1 levels in supernatants were quantified via western blot. Changes in EMT markers were measured by qPCR. Cell migration and cell cycle was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. YB-1 overexpression was achieved by stable transfection with an expression plasmid and confirmed via qPCR and western blot. YB-1 is secreted by MPM cells as well as the immortalised mesothelial cell line Met-5A after exposure to LPS or under hypoxic conditions. When MPM cells as well as primary mesothelial cells derived from pericardial fluid were exposed to soluble YB-1, we observed an upregulation of EMT markers such as SNAIL and TWIST as well as significantly increased migratory capacity. Similar effects were observed in cell lines which overexpress YB-1. YB-1 overexpressing cells migrated at significantly higher speed and covered a larger area. Also, when grown at low density, EMT-like changes in cell morphology as well as scattering was observed. Additionally, while cell divisions occurred at higher frequencies, the duration of the M phase was significantly prolonged. Our data highlight a crucial role of both intracellular and soluble YB-1 in the regulation of migration and invasion, which are key characteristics of MPM. Additionally, YB-1 also interferes with the cell cycle of MPM cells. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

Abstract C13: Direct binding of ERα to YB-1 suppresses HER2 expression in human breast cancer

Abstract [Background] ERα and HER2 are two major biomarkers of therapeutic efficacy in breast cancer patients. Y-box binding protein YB-1 is an oncoprotein involved in breast cancer and a transcription factor for HER2/ErbB2. However, regulatory mechanisms of ERα and HER2 expression by YB-1 are largely unknown. In our present study, we examined how nuclear YB-1 regulates expression of HER2 and ERα in breast cancer cells. [Materials and methods] We established YB-1/Tet-On system in which nuclear YB-1 expression was induced by doxycycline, and examined how YB-1 could modulate expression of HER2 and ERα in breast cancer cells. Furthermore, we established the ERα/Tet-On system, in which ERα expression was markedly induced after treatment with doxycycline. To assess whether the YB-1-HER2-ERα correlation was affected by menopause, we analyzed biopsy samples of 116 premenopausal and 114 postmenopausal patients, who had not received any therapeutic drugs, by IHC. [Results] We first examined whether YB-1 regulates expression of ERα and HER2 by using various human breast cancer cell lines, and observed following findings. (1) Nuclear YB-1 overexpression increased HER2 expression and decreases ERα expression, while HER2 expression was suppressed by estradiol and enhanced by anti-estrogen drugs. (2) ERα binding to YB-1 suppressed YB-1 binding to the HER2 promoter region. Furthermore, binding of YB-1 to ERα was enhanced by estradiol and suppressed by anti-estrogen drugs. (3) YB-1 induced proteasomal degradation of ERα when both interact directly. We have next examined whether expression of HER2, nuclear YB-1, and ERα are significantly associated by IHC analysis of biopsy samples of breast cancers. In breast cancers of postmenopausal, but not premenopausal patients, nuclear YB-1 expression was positively correlated with HER2 expression and negatively correlated with ERα expression. [Conclusions] In our study, we presented a new finding that YB-1 promoted proteasomal degradation of ERα by direct interaction and that YB-1-induced HER2 expression was suppressed by ERα. Furthermore, in breast tumors of postmenopausal patients, nuclear YB-1 expression was positively correlated with HER2 expression and negatively correlated with ERα expression. Therefore, this study could contribute to further development of optimized endocrine- and HER2- targeted therapeutics against breast cancer. Citation Format: Tomohiro Shibata, Kosuke Watari, Hiroto Izumi, Akihiko Kawahara, Satoshi Hattori, Chihiro Fukumitsu, Yuichi Murakami, Ryuji Takahashi, Uhi Toh, Ken-ichi Ito, Maki Tanaka, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Direct binding of ERα to YB-1 suppresses HER2 expression in human breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C13.

Etoposide enhances antitumor efficacy of MDR1-driven oncolytic adenovirus through autoupregulation of the MDR1 promoter activity.

Conditionally replicating adenoviruses (CRAds), or oncolytic adenoviruses, such as E1B55K-deleted adenovirus, are attractive anticancer agents. However, the therapeutic efficacy of E1B55K-deleted adenovirus for refractory solid tumors has been limited. Environmental stress conditions may induce nuclear accumulation of YB-1, which occurs in multidrug-resistant and adenovirus-infected cancer cells. Overexpression and nuclear localization of YB-1 are associated with poor prognosis and tumor recurrence in various cancers. Nuclear YB-1 transactivates the multidrug resistance 1 (MDR1) genes through the Y-box. Here, we developed a novel E1B55K-deleted adenovirus driven by the MDR1 promoter, designed Ad5GS3. We tested the feasibility of using YB-1 to transcriptionally regulate Ad5GS3 replication in cancer cells and thereby to enhance antitumor efficacy. We evaluated synergistic antitumor effects of oncolytic virotherapy in combination with chemotherapy. Our results show that adenovirus E1A induced E2F-1 activity to augment YB-1 expression, which shut down host protein synthesis in cancer cells during adenovirus replication. In cancer cells infected with Ad5WS1, an E1B55K-deleted adenovirus driven by the E1 promoter, E1A enhanced YB-1 expression, and then further phosphorylated Akt, which, in turn, triggered nuclear translocation of YB-1. Ad5GS3 in combination with chemotherapeutic agents facilitated nuclear localization of YB-1 and, in turn, upregulated the MDR1 promoter activity and enhanced Ad5GS3 replication in cancer cells. Thus, E1A, YB-1, and the MDR1 promoter form a positive feedback loop to promote Ad5GS3 replication in cancer cells, and this regulation can be further augmented when chemotherapeutic agents are added. In the in vivo study, Ad5GS3 in combination with etoposide synergistically suppressed tumor growth and prolonged survival in NOD/SCID mice bearing human lung tumor xenografts. More importantly, Ad5GS3 exerted potent oncolytic activity against clinical advanced lung adenocarcinoma, which was associated with elevated levels of nuclear YB-1 and cytoplasmic MDR1 expression in the advanced tumors. Therefore, Ad5GS3 may have therapeutic potential for cancer treatment, especially in combination with chemotherapy. Because YB-1 is expressed in a broad spectrum of cancers, this oncolytic adenovirus may be broadly applicable.

Correlation of Twist and YB-1 up-regulation and epithelial-mesenchymal transition during tumorigenesis and progression of cervical carcinoma

To investigate the clinicopathological significance of Twist and YB-1 up-regulation in cervical cancer, and to correlate the expression of the two genes with E-cadherin, a marker of epithelial-mesenchymal transition (EMT). A total of 202 tissue samples were collected during January 2008 to December 2013, including 50 cases of normal cervical tissues, 100 cases of cervical intraepithelial neoplasia (CIN) and 52 cases of squamous cell carcinoma (SCC). Twist, YB-1 and E-cadherin expression was investigated by MaxVision. Increased expression levels of Twist and YB-1 were found and correlated with the malignant transformation of cervical epithelium, histological progression and metastasis of cervical cancer. In addition, Twist and YB-1 overexpression was also associated with aberrant expression of E-cadherin. Regression analysis revealed that Twist expression was an independent factor for the histological progression of cervical cancer. It is suggested that Twist and YB-1 overexpression is significantly linked to cervical cancer tumorigenesis and progression, likely related to EMT through (YB-1)-Twist-(E-cadherin) pathway. Twist and YB-1 may be markers for determining the metastatic potential of cervical cancer.

Abstract 2366: YB-1 an oncogenic transcription factor regulates macrophage polarization and tumor microenvironment

Abstract Prostate cancer (PCa) is the most common cancer and one of the leading causes of cancer related deaths in men in North America and many western countries. Solid tumors including PCa have been known to be strongly infiltrated by inflammatory leukocytes. Also, significant correlation between increased numbers and/or density of macrophages and poor prognosis has been found in PCa. Based on this, understanding both the recruitment and activation of tumor associated macrophages (TAMs) are considered important targets for therapeutic intervention. We observed that IL-4 (M2 type stimuli) treatment significantly induces YB-1 expression in murine macrophages when compared to LPS (M1 type stimuli). YB-1 is a well-established oncogenic transcription factor known to be expressed by major immune cells like monocytes, T cells and macrophages. However, its expression is found to be relatively lower in differentiated monocytes (i.e. macrophages). Studies have shown an involvement of YB-1 in orchestration of immune responses. In response to proper stimuli like LPS or TGFβ that are associated with inflammatory diseases YB-1 is post-transnationally modified which allows its secretion. This extracellular YB-1 binds to specific unknown receptors on the cell surface of monocytes and promotes activation of different kinases which phosphorylate YB-1 and help in its nuclear translocation. In the nucleus, YB-1 binds to specific gene promoters and activates transcription of target genes like CCL5/RANTES. We over expressed YB-1 in murine macrophages and observed activation of gene network which is representative of M2 phenotype. Macrophages are a fundamental part of the innate defense mechanisms, which can promote specific immunity by inducing T cell recruitment and activation. Despite this, their presence within the tumor microenvironment has been associated with enhanced tumor progression and shown to promote cancer cell growth, angiogenesis and immunosuppression. Further, gain and loss of function studies of YB-1 in murine (RAW264.7) and human (THP-1) macrophages suggested that stable YB-1 knockout in both types of macrophages fails to activate M2 marker genes such as Arg-1, Mrc1, Ass-1, ASL and Pparg. Overexpression of YB-1 in RAW 264.7 macrophages after 16 hours of stimulation with IL-4 (5 ng/ml) significantly decreased the expression of iNOS, an M1 type marker with tumoricidal activity. Our studies provide a novel role of YB-1 in macrophage polarization and provide mechanistic insights as to how YB-1 plays an important role in regulation of tumor microenvironment. Citation Format: Mohammad Imran Khan, Abid Hamid, Vaqar M. Adhami, Hasan Mukhtar. YB-1 an oncogenic transcription factor regulates macrophage polarization and tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2366. doi:10.1158/1538-7445.AM2015-2366

Twist Contributes to Proliferation and Epithelial-to-Mesenchymal Transition–Induced Fibrosis by Regulating YB-1 in Human Peritoneal Mesothelial Cells

Twist is overexpressed in high glucose (HG) damage of human peritoneal mesothelial cells (HPMCs) invitro. Herein, we further identified its precise function related to fibrosis of peritoneal membranes (PMs). The overexpression and activation of Twist and YB-1 (official name, YBX1) and a transformed fibroblastic phenotype of HPMCs were found to be positively related to epithelial-mesenchymal transition progress and PM fibrosis exvivo in 93 patients who underwent continuous ambulatory peritoneal dialysis (PD), and also in HG-induced immortal HPMCs and an animal model of PD. Evidence from chromatin immunoprecipitation and luciferase reporter assays supported that YBX1 is transcriptionally regulated by the direct binding of Twist to E-box. Overexpression of Twist and YB-1 led to an increase in epithelial-mesenchymal transition, proliferation, and cell cycle progress of HPMCs, which might contribute to PM fibrosis. In contrast, the silencing of Twist or YB-1 inhibited HG-induced growth and cell cycle progression of HPMCs; this led to a down-regulation in the expression of cyclin Ds and cyclin-dependent kinases, finally inhibiting PM fibrosis. Twist contributes to PM fibrosis during PD treatment, mainly through regulation of YB-1.

Differential expression of the multidrug resistance 1 (MDR1) protein in prostate cancer cells is independent from anticancer drug treatment and Y box binding protein 1 (YB-1) activity.

The development of a drug-resistant phenotype is the major challenge during treatment of castration-resistant prostate cancer (PC). In solid cancer entities, one of the major contributors to chemoresistance is the multidrug resistance 1 (MDR1) protein. Believed to be involved in the induction of MDR1 expression is the presence of anticancer drugs as well as the Y box binding protein 1 (YB-1). Basal as well as drug-induced expression of MDR1 in established PC cell lines was assessed by Western blotting and mass spectrometry. Subsequently, the influence of YB-1 on MDR1 expression was examined via transient overexpression of YB-1. While LNCaP and PC-3 cells showed no detectable amounts of MDR1, the resistance factor was found to be expressed in 22Rv1 cells. Despite this difference, all three cell lines demonstrated similar growth behavior in the presence of the first-line chemotherapeutic agent docetaxel. Incubation of 22Rv1 cells with docetaxel, cabazitaxel, and abiraterone did not significantly alter MDR1 expression levels. Furthermore, overexpression of the MDR1 controlling factor YB-1 showed no impact on MDR1 expression levels. MDR1 was detectable in the PC cell line 22Rv1. However, this study suggests that MDR1 is of less importance for drug resistance in PC cells than in other types of solid cancer. Furthermore, in contrast to YB-1 properties in other malignancies, MDR1 regulation through YB-1 seems to be unlikely.

Roles of Y box-binding protein 1 in SK-BR-3 breast cancer proliferation

To explore the roles of Y box-binding protein 1 (YB-1) in breast cancer cell proliferation. Twenty cases of surgical removal of breast cancer tissue (diagnosed with invasive ductal carcinoma, stage II, by postoperative paraffin pathology) and normal breast tissues adjacent to carcinoma were collected during June 2013 to August 2013.Quantitative real-time PCR (qRT-PCR) was performed to detect the YB1 mRNA levels. Cultured mammary epithelial cells (HBL-100) and breast cancer cells (MCF7, MDA-MB-231 & SK-BR-3 cells) were harvested and qRT-PCR was performed to detect the YB1 mRNA levels.SK-BR-3 cells were stimulated with various concentrations of PDGF-BB and YB1 expression levels were detected by qRT-PCR. Down-regulation or over-expression of YB1 by si-YB1 or Ad-GFP-YB1 was detected in SK-BR-3 cells. And MTS cell proliferation assay kit was used to detect cell proliferation. YB1 mRNA levels were significantly higher in breast cancer tissues and MDA-MB-231 and SK-BR-3 breast cancer cell lines than that in adjacent normal breast tissues and HBL-100 mammary epithelial cells respectively (P < 0.05).YB1 expression levels increased in PDGF-BB stimulated SK-BR-3 cells in a dose-dependent manner. A down-regulation of endogenous YB1 decreases and an over-expression of exogenous YB1 promotes the proliferation activity in SK-BR-3 cells.

YB-1 promotes transcription of cyclin D1 in human non-small-cell lung cancers.

Cyclin D1, an oncogenic G1 cyclin, and YB-1, a transcription factor involved in cell growth, are both over-expressed in several human cancers. In human lung cancer, the functional association between YB-1 and cyclin D1 has never been elucidated. In this study, we show YB-1 is involved in the transcription of cyclin D1 in human lung cancer. Depletion of endogenous YB-1 by siRNA inhibited progression of G1 phase and down-regulated both the protein and mRNA levels of cyclin D1 in human lung cancer cells. Forced over-expression of YB-1 with a cyclin D1 reporter plasmid increased luciferase activity, and ChIP assay results showed YB-1 bound to the cyclin D1 promoter. Moreover, the amount of YB-1 mRNA positively correlated with cyclin D1 mRNA levels in clinical non-small-cell lung cancer (NSCLC) specimens. Immunohistochemical analysis also indicated YB-1 expression correlated with cyclin D1 expression in NSCLC specimens. In addition, most of the cases expressing both cyclin D1 and CDC6, another molecule controlled by YB-1, had co-existing YB-1 over-expression. Together, our results suggest that aberrant expression of both cyclin D1 and CDC6 by YB-1 over-expression may collaboratively participate in lung carcinogenesis.

YB-1 Is Activated by IL-7 and Is Overexpressed in Pediatric Pre-B Acute Lymphoblastic Leukemia

Abstract 1468 Background:Increased YB-1 expression correlates with poor prognosis, drug resistance and metastasis in several different cancers including B cell lymphoma. Phosphorylation and nuclear localization of YB-1 in response to growth factors leads to increased survival through expression of proteins such as survivin and multidrug resistance protein 1. Until now, its role in leukemia has not been investigated. We hypothesized that YB-1 expression is aberrantly regulated in pediatric pre-B acute lymphoblastic leukemia (pre-B ALL), and that YB-1 may be activated downstream of IL-7. This cytokine facilitates the differentiation and survival of pre-B cells and has been implicated in the drug resistance of pre-B ALL. Methods:YB-1 and IL-7Ra protein expression was investigated by flow cytometry in normal pre-B cells (CD19+CD10+CD20−), and mature B cells (CD19+CD10−CD20+) as well as diagnostic and relapsed pre-B ALL (CD19+CD10+/−). Cell surface and cytoplasmic expression was quantified by mean fluorescent intensity (MFI). Bone marrow from healthy donors was used as a source of normal pre-B cells, while mature B cells were derived from PBMCs; leukemic cells at presentation and relapse were obtained following local IRB approval and informed consent. Activation of YB-1 downstream of IL-7 stimulation (25 ng/ml) was examined in pre-B ALL cell lines or NSG (NOD scid gamma) mice-expanded pre-B ALL by Western blotting using anti-phosphoYB-1(S012). Pre-B ALL cell lines used in these experiments were 697, 380, RCH and RS-4;11. Signaling pathways were investigated by pre-treatment of cells with pharmacological inhibitors followed by Western analyses. For the transient overexpression of YB-1, pEGFP or a pEGFP/YB-1 fusion protein was electroporated into freshly isolated mature B cells (which have a low basal expression of YB-1) and YB-1 and IL-7Ra expression was assessed by flow cytometry after 24 h. Results:While intracellular YB-1 expression was significantly higher in leukemia samples at presentation compared to normal pre-B cells, the highest YB-1 levels were found in relapsed pre-B ALL (see figure, part A). All examined pre-B ALL cell lines had levels comparable to those of the relapse samples. Similarly, surface IL-7Ra (CD127) levels (MFI medians; upper-lower range) were increased in onset (221; 150–286), and relapsed (1840; 651–2030) ALL compared to normal pre-B cells (528; 333–2673). (normal pre-B vs. leukemia at presentation, p<0.001, Mann-Whitney). Overexpression of YB-1-GFP in normal mature B cells resulted in increased expression of IL-7Ra (see figure, part B), suggesting an link between the YB-1 and IL-7 signaling pathways.Activated YB-1 is phosphorylated on S102 and relocated to the nucleus. Addition of IL-7 to pre-B ALL cell lines led to phosphorylation of YB-1 within 30 min. Similar results were shown for patient-derived, NSG mice-expanded pre-B ALL samples. Intracellular immunostaining using Imagestream technology (Amnis) showed that IL-7 treatment of pre-B ALL cell lines increased nuclear YB-1 levels 4-fold.As PI3K and MEK1 are involved in signaling downstream of IL-7, we investigated their role in YB-1 signaling in both pre-B ALL cell lines and NSG-mouse expanded pre-B ALL using pharmacological inhibitors. Western analyses showed that inhibition of PI3K using LY294002 did not prevent IL-7-mediated phosphorylation of YB-1 but the MEK1 inhibitor U0126 did, indicating the involvement of MAPK (see figure, part C). Conclusion:We show that YB-1, which is highly expressed in pediatric pre-B ALL compared to normal pre-B cells, is expressed at even higher levels after relapse. We demonstrate a link between the YB-1 and IL-7 signaling pathways which could offer a novel target for the treatment of refractory leukemia. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NONO and RALY proteins are required for YB-1 oxaliplatin induced resistance in colon adenocarcinoma cell lines

BackgroundYB-1 is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. Recent data have shown that YB-1 is also overexpress in colorectal cancer. In this study, we tested the hypothesis that YB-1 also confers oxaliplatin resistance in colorectal adenocarcinomas.ResultsWe show for the first time that transfection of YB-1 cDNA confers oxaliplatin resistance in two colorectal cancer cell lines (SW480 and HT29 cell lines). Furthermore, we identified by mass spectrometry analyses important YB-1 interactors required for such oxaliplatin resistance in these colorectal cancer cell lines. A tagged YB-1 construct was used to identify proteins interacting directly to YB-1 in such cells. We then focused on proteins that are potentially involved in colorectal cancer progression based on the Oncomine microarray database. Genes encoding for these YB-1 interactors were also examined in the public NCBI comparative genomic hybridization database to determine whether these genes are localized to regions of chromosomes rearranged in colorectal cancer tissues. From these analyses, we obtained a list of proteins interacting with YB-1 and potentially involved in oxaliplatin resistance. Oxaliplatin dose response curves of SW480 and HT29 colorectal cancer cell lines transfected with several siRNAs corresponding to each of these YB-1 interactors were obtained to identify proteins significantly affecting oxaliplatin sensitivity upon gene silencing. Only the depletion of either NONO or RALY sensitized both colorectal cancer cell lines to oxaliplatin. Furthermore, depletion of NONO or RALY sensitized otherwise oxaliplatin resistant overexpressing YB-1 SW480 or HT29 cells.ConclusionThese results suggest knocking down NONO or RALY significant counteracts oxaliplatin resistance in colorectal cancers overexpressing the YB-1 protein.