YB-1 Expression Research Articles

IDENTIFYING YB1 AND NPM1 AS PART OF THE MUTANT HISTONE 3 PROTEIN INTERACTOME IN H3 MUTATED PHGG

Abstract AIMS Paediatric high-grade gliomas (pHGG) are the leading cause of childhood cancer deaths, accounting for over 40% of deaths in the UK. pHGG tumours harbour histone H3 mutations, defining 50% of pHGG cases and the major mutations are H3.3K27M and H3.3G34R/V. Our previous proteomic research on H3 intercatomes, identified unique and common interacting binding partners of H3-G34R and H3-G34V mutant proteins relative to wild type H3 protein. Among these binding proteins we have focused on YB-1 and Nucleophosmin 1 (NPM1). YB-1, is multifunctional transcription factor and a well- characterised oncogenic protein. NPM1 is a molecular chaperone and frequently overexpressed in cancers. Our aim is to analyse the role of YB-1 and NPM1 in H3 mutated pHGG, in order to identify targeted therapeutic options for these difficult to treat tumours. METHOD H3 mutated glioma, H3 wild type pHGG and control cell lines were cultured and then subjected to comparative immunofluorescence microscopy, and western blot analyses to investigate YB-1 and NPM1 expression. RESULTS Results showed statistically significant altered protein expression and localisation of YB-1 and NPM1 in H3 mutated pHGG in comparison to wild type and control cells. YB-1 expression was increased in H3 mutated cells and NPM1 showed increased cytoplasmic localization in H3 mutated cells in comparison to H3 WT glioma and control cells. CONCLUSION Results of the study support that YB-1 and NPM1 are important interacting proteins in H3G34-mutated pHGGs and can be explored as potential therapeutic targets in H3 mutated pHGG.

HGG-25. IDENTIFYING YB1 AND NPM1 AS PART OF THE MUTANT HISTONE 3 PROTEIN INTERACTOME IN H3 MUTATED PHGG

Abstract Paediatric high-grade gliomas (pHGG) are the leading cause of childhood cancer deaths, accounting for over 40% of deaths in the UK. pHGG tumours harbour histone H3 mutations, defining 50% of pHGG cases. Studies show prominent mutations as being H3.3K27M and H3.3G34R/V. Our previous proteomic research on H3 intercatomes, identified unique and common interacting binding partners of H3-G34R and H3-G34V mutant proteins relative to wild type H3. Among these proteins we have focused on YB-1 and Nucleophosmin 1 (NPM1). YB-1, is multifunctional transcription factor and a well-characterised oncogenic protein. NPM1 is a molecular chaperone and frequently overexpressed in cancers. Our aim is to analyse the role of YB-1 and NPM1 in H3 mutated pHGG, in order to identify targeted therapeutic options for these difficult to treat tumours. H3 mutated glioma cell lines, H3 wild type pHGG cells and control cells were cultured and then subjected to comparative immunofluorescence microscopy, and western blot analyses to investigate YB-1 and NPM1 expression. Results showed statistically significant altered protein expression and localisation of YB-1 and NPM1 in H3 mutated pHGG in comparison to wild type and control cells. YB-1 expression was increased in H3 mutated cells and NPM1 showed increased cytoplasmic localization in H3 mutated cells in comparison to H3 WT glioma and control cells. Results of the study support that YB-1 and NPM1 are important interacting proteins in H3G34-mutated pHGGs and can be explored as potential therapeutic targets in H3 mutated pHGG.

Abstract 5728: YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors

Abstract Metastatic breast cancer (BC) is the 2nd leading cause of death in women in the US, annually accounting for more than 43,000 deaths and 281,000 new cases of invasive BC. Amongst genetically distinct BC subtypes, those classified as being “triple-negative” (TNBC) are especially devastating due to their highly metastatic behavior, their propensity to recur rapidly, and their low response to standard-of-care therapies. In fact, the acquisition of chemoresistant phenotypes represents the main cause of disease recurrence, metastasis, and death in TNBC patients. Currently, the molecular mechanisms that regulate TNBC progression and metastasis remain unknown, as does the way these metastatic tumors acquire resistance to standard-of-care therapies. We recently established YB1 as a novel driver of these deadly TNBC activities, doing so by stimulating the cancer stem cell phenotype, and by disrupting normal cell cycle progression, therefore promoting therapy resistance and metastasis of TNBC tumors. YB1 is a multifunctional protein that acts as a transcription factor of cancer stem cell genes Nanog, Oct and Sox. Moreover, aberrant activation of YB1 contributes to the metastatic progression of several cancers, including TNBC. Our investigations revealed a major role of YB1 in the regulation of several hallmarks of cancer that drive TNBC tumors progression and metastasis, both in vitro and in preclinical mouse models of TNBC tumors. In extending these discoveries, we now show that aberrant YB1 expression activates oncogenic signaling leading to the dysregulation of cell cycle progression through the regulation of the Cyclin D/CDK4/6 complex signaling and the RB pathway. We also report the identification of a novel small molecule inhibitor, SU056, that specifically targets YB1 and inhibits its oncogenic activity in TNBC cell lines. We further show that the SU056-mediated inhibition of YB1, either as monotherapy or in combination with the CDK4/6 inhibitors, has a significant impact on inhibiting TNBC tumor progression and metastasis. In fact, the SU056-CDK4/6 inhibitors combination showed a synergistic effect when compared to monotherapy, at the same time reducing the toxicity associated with CDK4/6 inhibitors. Mechanistically, our studies revealed that genetic or pharmacologic targeting of YB1 inhibits TNBC tumor progression and metastasis through the regulation cyclin D-CDK4/6-RB pathway and blockade of cell cycle progression. Together, our data support the notion that targeting YB1 represents a potential therapeutic option for the treatment of TNBC, which could be enhanced when combined with standard of care treatment modalities. Citation Format: Wei Wang, Lamyae El Khalki, Neelum Yousaf Zai, Justin Szpendyk, Akram Alkrekshi, Bin Su, Khalid Sossey-Alaoui. YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5728.

Knockdown of DLK4 inhibits non-small cell lung cancer tumor growth by downregulating CKS2.

Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases and is considered as the most common type of cancer. DLX4 was originally identified as a β-globin gene suppressor in red blood cells, which plays critical roles in several types of cancers. However, the role and related mechanism of DLX4 in NSCLC are still unclear. The study aimed to uncover the expression of DLX4 in human NSCLC cells and tissues, reveal its possible role in NSCLC, and investigate the underlying mechanisms. Immunoblot and TCGA database were used to detect the expression of DLX4 in human NSCLC cells and tissues. CCK-8, colony formation, and FCM assays were conducted to detect the effects of DLX4 on the viability and cell cycle of NCI-H2170 and A549 cells. Immunoblot assays were further performed to investigate the possible mechanism underlying DLX4 affecting the growth of NSCLC. We revealed that knockdown of DLX4 inhibited NSCLC cell proliferation. We further revealed that DLX4 knockdown induced the NSCLC cell cycle arrest. Our results further showed that downregulation of DLX4 suppressed YB-1 expression, which further suppressed CKS2 expression, thereby suppressing tumor growth of NSCLC. In conclusion, DLX4 has the potential to serve as a promising drug for NSCLC treatment.

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.

IDENTIFYING ROLE OF YB1 AND NPM1 AS PART OF HISTONE 3 MUTANT PROTEIN INTERACTOME, IN H3 MUTATED PHGG

Abstract AIMS Paediatric high-grade gliomas (pHGG) are the leading cause of childhood cancer deaths, accounting for over 40% of deaths in the UK. pHGG tumours harbour histone H3 mutations, defining 50% of pHGG cases. Studies show prominent mutations as being H3.3K27M and H3.3G34R/V. Our previous research has identified unique and common interacting binding partners of H3-G34R and H3-G34V mutant proteins. Among these we have focused on YB-1 and Nucleophosmin (NPM1). YB-1 is multifunctional transcription factor and a well-known oncogenic protein. NPM1 is a molecular chaperone and frequently over- expressed in cancer as well. Our aim is to analyse the role of YB-1 and NPM1 in H3 mutated pHGG, in order to identify targeted therapeutic option for these diffcult to treat tumours. METHOD Further data analyses of mass spectrometric proteomic data were performed. Subsequently, H3 mutated glioma cell lines, and control H3 wild type pHGG cells were cultured and then subjected to proteomic analyses. A comparative immunofluorescence and microscopy, and western blot analyses were performed to investigate the role of YB-1 and NPM-1. RESULTS Results verified the proteomic data and further showed altered protein expression of YB-1 and altered localization of protein NPM1 in H3 mutated pHGG. CONCLUSIONS Results of the study support that YB-1 and NPM1 are important interacting proteins in H3G34-mutated pHGGs and can be explored as therapeutic targets in H3 mutated pHGG.

YB-1 promotes cell proliferation and metastasis by targeting cell-intrinsic PD-1/PD-L1 pathway in breast cancer.

Programmed cell death 1 (PD-1) suppresses T effector functions by inhibiting signaling downstream of the T cell receptor and helping tumor cells escape the immune response. However, the effect and mechanism of cell-intrinsic PD-1 in cancer cells are still unknown. Here, we found that PD-1 is aberrantly upregulated in TNBC patients and cell lines. Cell-intrinsic PD-1 in TNBC cells significantly facilitated tumor growth and metastasis in vitro and in vivo. Further studies indicated that PD-1 effect on TNBC cell growth depends on the cell-intrinsic-PD-1/PD-L1 pathway independent of adaptive immunity. In addition, we further found that the activation of cell-intrinsic PD-1/PD-L1 pathway in TNBC cells is regulated by the gene expression regulator YB-1. Mechanistically, the results of protein degradation analysis, mRNA translationally active analysis, CLICK chemistry and L-azidohomoalanine (AHA) incorporation assays, immunoprecipitation assay and Dual-Luciferase reporter assay showed that YB-1 promotes PD-1 expression through the translational activation pathway. We provide in vitro and in vivo evidence that silencing YB-1 expression in TNBC cells inhibits cell proliferation, tumorigenesis, and metastasis. However, this inhibition can be rescued by simultaneous exogenous expression of PD-1 and PD-L1 proteins. In conclusion, our results identify TNBC cell-intrinsic functions of the PD-1/PD-L1 axis in tumor growth and metastasis; and revealed PD-1/PD-L1 is a critical effector of YB-1-mediated TNBC proliferation and metastasis in vitro and in vivo.

Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters, MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018–2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.

P-gp expression inhibition mediates placental glucocorticoid barrier opening and fetal weight loss

BackgroundPrenatal adverse environments can cause fetal intrauterine growth retardation (IUGR) and higher susceptibility to multiple diseases after birth, related to multi-organ development programming changes mediated by intrauterine overexposure to maternal glucocorticoids. As a glucocorticoid barrier, P-glycoprotein (P-gp) is highly expressed in placental syncytiotrophoblasts; however, the effect of P-gp on the occurrence of IUGR remains unclear.MethodsHuman placenta and fetal cord blood samples of IUGR fetuses were collected, and the related indexes were detected. Pregnant Wistar rats were administered with 30 mg/kg·d (low dose) and 120 mg/kg·d (high dose) caffeine from gestational day (GD) 9 to 20 to construct the rat IUGR model. Pregnant mice were administered with caffeine (120 mg/kg·d) separately or combined with sodium ferulate (50 mg/kg·d) from gestational day GD 9 to 18 to confirm the intervention target on fetal weight loss caused by prenatal caffeine exposure (PCE). The fetal serum/placental corticosterone level, placental P-gp expression, and related indicator changes were analyzed. In vitro, primary human trophoblasts and BeWo cells were used to confirm the effect of caffeine on P-gp and its mechanism.ResultsThe placental P-gp expression was significantly reduced, but the umbilical cord blood cortisol level was increased in clinical samples of the IUGR neonates, which were positively and negatively correlated with the neonatal birth weight, respectively. Meanwhile, in the PCE-induced IUGR rat model, the placental P-gp expression of IUGR rats was decreased while the corticosterone levels of the placentas/fetal blood were increased, which were positively and negatively correlated with the decreased placental/fetal weights, respectively. Combined with the PCE-induced IUGR rat model, in vitro caffeine-treated placental trophoblasts, we confirmed that caffeine decreased the histone acetylation and expression of P-gp via RYR/JNK/YB-1/P300 pathway, which inhibited placental and fetal development. We further demonstrated that P-gp inducer sodium ferulate could reverse the inhibitory effect of caffeine on the fetal body/placental weight. Finally, clinical specimens and other animal models of IUGR also confirmed that the JNK/YB-1 pathway is a co-regulatory mechanism of P-gp expression inhibition, among which the expression of YB-1 is the most stable. Therefore, we proposed that YB-1 could be used as the potential early warning target for the opening of the placental glucocorticoid barrier, the occurrence of IUGR, and the susceptibility of a variety of diseases.ConclusionsThis study, for the first time, clarified the critical role and epigenetic regulation mechanism of P-gp in mediating the opening mechanism of the placental glucocorticoid barrier, providing a novel idea for exploring the early warning, prevention, and treatment strategies of IUGR.

The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

ABSTRACT The cold-shock protein Y-box-binding protein (YB)-1 regulates the expression of various chemokines and their receptors at the transcriptional level. Expression of the orphan chemokine CXCL14 is repressed by EGF induced signaling. The possible links between EGF-mediated YB-1 and CXCL14 as well as the functions of critical kinase pathways in the progression of prostate cancer have remained unexplored. Here we examined the correlation between YB-1 and CXCL14, and the ERK/AKT/mTOR pathways in prostate cancer. Knockdown of YB-1 decreased cyclinD1 expression with an upregulation of cleaved-PARP in human prostate cancer cells. EGF treatment upregulated phospho-YB-1 expression in a time-dependent manner, while treatment with an ERK inhibitor completely silenced its expression in prostate cancer cells. EGF treatment stimulates CyclinD1 and YB-1 phosphorylation in an ERK-dependent pathway. Positive and negative regulation of YB-1 and CXCL14 was observed after EGF treatment in prostate cancer cells, respectively. EGF rescues cell cycle and apoptosis via the AKT and ERK pathways. Furthermore, YB-1 silencing induces G1 arrest and apoptosis, while knockdown of CXCL14 facilitates cell growth and inhibits apoptosis in prostate cancer cells. YB-1 and CXCL14 were inversely correlated in prostate cancer cells and tissues. A significant association between poor overall survival and High YB-1 expression was observed in human prostate cancer patients. In conclusion, our data reveal the functional relationship between YB-1 and CXCL14 in EGF mediated ERK signaling, and YB-1 expression is a significant prognostic marker to predict prostate cancer.

A reciprocal feedback of miR-548ac/YB-1/Snail induces EndMT of HUVECs during acidity microenvironment

BackgroundResearches indicated the process of Endothelial-Mesenchymal-Transition (EndMT) of vascular endothelial cells (ECs) was critically involved in the progression of tumor. ECs demonstrated functional and phenotypic heterogeneity when located under different microenvironments. The extracellular pH of tumor tissues was acidic compared to that of normal tissues. However, there was still unclear whether the acidic microenvironment affected the EndMT of vascular ECs.MethodsHuman Umbilical Vein Endothelial Cell (HUVECs) was cultured under the normal or acidic medium to evaluate the alteration of morphology, migration, permeability, and EndMT markers. Microarray assay was adopted to analyze the differential expression of miRNAs in the acidity-treated HUVECs. Gain- and loss- of function experiments were performed to evaluate the functional role of miRNA-548ac on acidity-induced EndMT of HUVECs. Luciferase reporter and Chromatin-immunoprecipitation assays were conducted to assess the downstream pathway of miRNA-548ac in acidity-induced EndMT of HUVECs.ResultsOur results showed that HUVECs demonstrated mesenchymal transition under acidic conditions with the increase of migration, permeability, and expression of α-SMA and Vimentin, but the expression of vascular endothelial cadherin (VE-cadherin) and CD31 were reduced. In addition, the acidity-treated HUVECs remarkably facilitated the transmigration of pancreatic cancer cells. The expression of miRNA-548ac was significantly decreased in the acidity-treated HUVECs. Moreover, overexpression of miR-548ac inhibited the EndMT of HUVECs and consequently impeded the transmigration of pancreatic cancer cells. The miR-548ac inhibited the expression of YB-1 by binding to the 3’UTR of its mRNA, and YB-1 promoted the translation of Snail which was a critical regulator of EndMT. What’s more, Snail transcriptionally inhibited the expression of miR-548ac through binding to the promoter of its host gene.ConclusionsOur data implicated that the acidic microenvironment promoted the EndMT of HUVECs by the miR-548ac/YB-1/Snail axis, which could contribute to the metastasis of pancreatic cancer.

Rhinacanthin-C enhances chemosensitivity of breast cancer cells via the downregulation of P-glycoprotein through inhibition of Akt/NF-kappa B signaling pathway

Introduction: High expression of P-glycoprotein (P-gp) has been linked to multidrug resistance (MDR) and chemotherapeutic failure. Previously, we demonstrated that rhinacanthin-C, a naphthoquinone from Rhinacanthus nasutus, was able to enhance the cytotoxicity of doxorubicin against breast cancer MCF-7 cells via direct P-gp inhibition. In this study, we looked at its effect on P-gp downregulation and the mechanism involved in the resistance of MCF-7 cells to doxorubicin. Methods: Doxorubicin-resistant MCF-7 (MCF-7/DOX) cells were exposed to rhinacanthin-C for 24-48 hours prior to the assessment of their chemosensitivity via MTT assay, P-gp activity via calcein-AM uptake assay, P-gp expression, and signaling via qRT-PCR and western blot analyses. Results: Pretreatment with 1 µM of rhinacanthin-C for 48 hours significantly enhanced cytotoxicity of doxorubicin, as well as camptothecin and etoposide, to MCF-7/DOX cells. In the rhinacanthin-C-treated cells, reduction of MDR1 mRNA and P-gp levels and increased intracellular calcein were observed. Moreover, phosphorylation of Akt, NF-ᴋB and IκB-α, along with YB-1 expression, significantly decreased after 24-hour treatment with rhinacanthin-C. In contrast, the naphthoquinone had no effect on expression levels of ERK1/2 and phosphorylated ERK1/2 under similar conditions. Conclusion: Rhinacanthin-C, at a non-cytotoxic concentration (1 µM), could downregulate P-gp expression in MCF-7/DOX cells via the inhibition of the Akt/NF-ᴋB signaling pathway and YB-1 expression. Long-term exposure to this natural naphthoquinone may increase the chemosensitivity of cancer cells with MDR phenotype.

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.

Elevated mRNA Level of Y-Box Binding Protein 1 Indicates Unfavorable Prognosis Correlated with Macrophage Infiltration and T Cell Exhaustion in Luminal Breast Cancer.

PurposeThe Y-box binding protein 1 (YBX1) gene encodes the multifunctional protein YB1 that is associated with the dysregulation of numerous cancer-related genes. However, the prognostic value of YBX1 and its correlation with immune cell infiltration in breast cancer (BRCA) remain unclear.MethodsYBX1 expression data in various malignancies were obtained from Oncomine, Tumor Immune Estimation Resource (TIMER), Cancer Cell Line Encyclopedia, UALCAN and cBio Cancer Genomics Portal databases. Survival data were analyzed with Kaplan–Meier plotter. Immune cell infiltration and its association with YBX1 expression level were assessed with TIMER and LinkedOmics. YB1 expression was evaluated by immunohistochemistry and Western blotting, and changes in cancer cell viability and T cell activity following YBX1 knockdown were assessed with an immunocyte–tumor cell co-culture assay.ResultsYBX1 was downregulated in the BRCA cohort, which was closely associated with worse prognosis in the luminal A subtype (overall survival [OS]: hazard ratio [HR] 1.93, 95% confidence interval [CI] 1.22–3.05, P = 0.0042; recurrence-free survival [RFS]: HR 1.85, 95% CI 1.51–2.28, P = 3.1e-9) and luminal B subtype (OS: HR 1.08, 95% CI 0.68–1.70, P = 0.75; RFS: HR 1.29, 95% CI 1.02–1.62, P = 0.03). YBX1 expression was positively correlated with the M2 macrophage infiltration and expression of T cell exhaustion markers such as indoleamine 2,3-dioxygenase 1 (IDO1) (rs = 0.388, P = 4.93e-37) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (rs = 0.321, P = 2.54e-25) in luminal BRCA. Kaplan–Meier analysis revealed a correlation between YBX1 expression, M2 infiltration and survival outcome. Co-culture with macrophages or T cells enhanced the decrease in luminal BRCA cell viability induced by YBX1 knockdown.ConclusionHigh YBX1 mRNA levels predict a poor prognosis in luminal BRCA, which is correlated with M2 macrophage infiltration and T cell exhaustion in the tumor microenvironment. Combining classic therapeutics with immune checkpoint inhibitors and M1 polarization agents may be an effective treatment strategy for luminal BRCA with YBX1 overexpression.

De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

The dual distinct role of telomerase in repression of senescence and myofibroblast differentiation.

Many aging related diseases such as cancer implicate the myofibroblast in disease progression. Furthermore genesis of the myofibroblast is associated with manifestation of cellular senescence of unclear significance. In this study we investigated the role of a common regulator, namely telomerase reverse transcriptase (TERT), in order to evaluate the potential significance of this association between both processes. We analyzed the effects of TERT overexpression or deficiency on expression of CDKN2A and ACTA2 as indicators of senescence and differentiation, respectively. We assess binding of TERT or YB-1, a repressor of both genes, to their promoters. TERT repressed both CDKN2A and ACTA2 expression, and abolished stress-induced expression of both genes. Conversely, TERT deficiency enhanced their expression. Altering CDKN2A expression had no effect on ACTA2 expression. Both TERT and YB-1 were shown to bind the CDKN2A promoter but only YB-1 was shown to bind the ACTA2 promoter. TERT overexpression inhibited CDKN2A promoter activity while stimulating YB-1 expression and activation to repress ACTA2 gene. TERT repressed myofibroblast differentiation and senescence via distinct mechanisms. The latter was associated with TERT binding to the CDKN2A promoter, but not to the ACTA2 promoter, which may require interaction with co-factors such as YB-1.

YB1 regulates miR-205/200b-ZEB1 axis by inhibiting microRNA maturation in hepatocellular carcinoma.

BackgroundY‐box binding protein 1 (YB1 or YBX1) plays a critical role in tumorigenesis and cancer progression. However, whether YB1 affects malignant transformation by modulating non‐coding RNAs remains largely unknown. This study aimed to investigate the relationship between YB1 and microRNAs and reveal the underlying mechanism by which YB1 impacts on tumor malignancy via miRNAs‐mediated regulatory network.MethodsThe biological functions of YB1 in hepatocellular carcinoma (HCC) cells were investigated by cell proliferation, wound healing, and transwell invasion assays. The miRNAs dysregulated by YB1 were screened by microarray analysis in HCC cell lines. The regulation of YB1 on miR‐205 and miR‐200b was determined by quantitative real‐time PCR, dual‐luciferase reporter assay, RNA immunoprecipitation, and pull‐down assay. The relationships of YB1, DGCR8, Dicer, TUT4, and TUT1 were identified by pull‐down and coimmunoprecipitation experiments. The cellular co‐localization of YB1, DGCR8, and Dicer were detected by immunofluorescent staining. The in vivo effect of YB1 on tumor metastasis was determined by injecting MHCC97H cells transduced with YB1 shRNA or shControl via the tail vein in nude BALB/c mice. The expression levels of epithelial to mesenchymal transition markers were detected by immunoblotting and immunohistochemistry assays.ResultsYB1 promoted HCC cell migration and tumor metastasis by regulating miR‐205/200b‒ZEB1 axis partially in a Snail‐independent manner. YB1 suppressed miR‐205 and miR‐200b maturation by interacting with the microprocessors DGCR8 and Dicer as well as TUT4 and TUT1 via the conserved cold shock domain. Subsequently, the downregulation of miR‐205 and miR‐200b enhanced ZEB1 expression, thus leading to increased cell migration and invasion. Furthermore, statistical analyses on gene expression data from HCC and normal liver tissues showed that YB1 expression was positively associated with ZEB1 expression and remarkably correlated with clinical prognosis.ConclusionThis study reveals a previously undescribed mechanism by which YB1 promotes cancer progression by regulating the miR‐205/200b‒ZEB1 axis in HCC cells. Furthermore, these results highlight that YB1 may play biological functions via miRNAs‐mediated gene regulation, and it can serve as a potential therapeutic target in human cancers.

Trimethylamine N-oxide mediated Y-box binding protein-1 nuclear translocation promotes cell cycle progression by directly downregulating Gadd45a expression in a cellular model of chronic kidney disease

AimsCell cycle arrest plays critical roles in preventing renal tubular epithelial cell (RTEC) injury and maladaptation after the onset of chronic kidney disease (CKD), but the underlying mechanism governing this arrest has not been fully elucidated. This study was designed to determine the underlying role of YB-1 in promoting cell cycle progression and nuclear translocation in HK-2 cells induced by trimethylamine N-oxide (TMAO). Main methodsYB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. Gene expression was analysed using RNA sequencing in HK-2 cells treated with TMAO. Cell cycle progression was analysed via flow cytometry. Luciferase assay and ChIP-PCR were performed to determine the relationship between transcription factor YB-1 and Gadd45a promoter region. Additionally, mice were fed with TMAO to test renal dysfunction and measure the expression of YB-1, GADD45a and CCNA2 in the kidney sections through immunohistochemistry. Key findingsYB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. RNA sequencing analysis showed that the cell cycle checkpoint genes growth arrest and DNA damage (Gadd)45a, Gadd45g, cyclin (Ccn)a2, Ccnb1, Ccne1 and Ccnf were differentially expressed in HK-2 cells after treated with 400 μM TMAO for 30 min. Flow cytometry results demonstrated that cell cycle progression was blocked at the G2/M checkpoint. In animal models, elevated dietary TMAO directly led to progressive renal tubulointerstitial dysfunction and inhibited the expression of YB-1 in kidney. Moreover, YB-1 was determined to regulate Gadd45a expression by directly binding to its promoter region. YB-1 expression was negatively correlated with the expression of Gadd45a and Gadd45g but positively correlated with Ccna2, Ccnb1, Ccne1 and Ccnf in CKD. SignificanceYB-1 may be a reliable molecular target and an effective prognostic biomarker for CKD.

Abstract 5000: YB-1 based oncolytic virotherapy in combination with small molecular inhibitors for treatment of refractory osteosarcoma

Abstract This in vitro study evaluates YB-1 based oncolytic virotherapy in combination with molecular target therapy as a novel therapeutic strategy for refractory osteosarcoma. In vitro combination approaches include CDK4/6-, MDM2- and PARP-inhibitors. The treatment strategy was explored in four established osteosarcoma cell lines (143B, MG-63, SJSA, U2OS). Intracellular YB-1 expression was evaluated by immunofluorescence for oncolytic therapy with YB-1 selective adenovirus XVir-N-31. Efficacy of mono- and combination therapy is assessed using cell survival assays. The Chou Talalay method is used to determine synergistic effects between combinational components. Characterization of molecular pathways associated with oncolytic cytotoxicity is done using cell cycle analysis, western blotting, hexon titer test, sqPCR and ELISA. We observe a potent preclinical anti-tumor effect using XVir-N-31 monotherapy on all tested osteosarcoma cell lines. However, most cell lines were resistant towards inhibitor monotherapy in clinically achievable concentrations. In contrast, all dual combinations resulted in significantly enhanced cytotoxicity. These results require reduced numbers of infectious viral particles and are observed at clinically implementable inhibitor concentrations. Increased oncolytic cytotoxicity is associated with changes of tumor cell cycle regulation due to inhibitor treatment and modulation of infectious viral particle formation. In summary, we determine XVir-N-31 as a potent oncolytic agent for the treatment of refractory osteosarcoma. We provide a strong preclinical rationale for the combination of oncolytic adenovirus with molecular target therapy. A phase I clinical trial with XVir-N-31 in combination with molecular target therapy for refractory childhood sarcoma is planned. Citation Format: Charlotte Friederike Middendorf, Sebastian Johannes Schober, Corazón Kordass, Roman Nawroth, Stefan E. Burdach, Per Sonne Holm, Uwe Thiel. YB-1 based oncolytic virotherapy in combination with small molecular inhibitors for treatment of refractory osteosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5000.

BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

BackgroundBRD7 is a tumor suppressor known to inhibit cell proliferation and cell cycle progression and initiate apoptosis in breast cancer. However, the function and underlying molecular events of BRD7 in tumor invasion and metastasis in breast cancer are not fully understood.MethodsBRD7 expression was assessed in two stable cell lines MDA231 and MCF7 with BRD7 overexpression and one stable cell line MDA231 with BRD7 interference using qRT-PCR and western blotting. CCK8 assay was used to examine the proliferation ability of MDA231 and MCF7 cells. Scratch wound healing assay was used to evaluate cell migration in MDA231 and MCF7 cells. Both Matrigel and three-dimensional invasion assays were performed to investigate the cell invasion ability after BRD7 overexpression or silencing or YB1 restoration in MDA231 and MCF7 cells. The potential interacting proteins of BRD7 were screened using co-immunoprecipitation combined with mass spectrometry and verified by co-immunoprecipitation in HEK293T cells. Additionally, we confirmed the specific binding region between BRD7 and YB1 in HEK293T cells by constructing a series of deletion mutants of BRD7 and YB1 respectively. Finally, xenograft and metastatic mouse models using MDA231 cells were established to confirm the effect of BRD7 on tumor growth and metastasis.ResultsHere, the results of a series of assays in vitro indicated that BRD7 has the ability to inhibit the mobility, migration and invasion of breast cancer cells. In addition, YB1 was identified as a novel interacting protein of BRD7, and BRD7 was found to associate with the C-terminus of YB1 via its N-terminus. BRD7 decreases the expression of YB1 through negatively regulating YB1 phosphorylation at Ser102, thereby promoting its proteasomal degradation. Furthermore, gene set enrichment analysis revealed that epithelial-mesenchymal transition (EMT) is the common change occurring with altered expression of either BRD7 or YB1 and that BRD7 represses mesenchymal genes and activates epithelial genes. Moreover, restoring the expression of YB1 antagonized the inhibitory effect of BRD7 on tumorigenicity, EMT, invasiveness and metastasis through a series of in vitro and in vivo experiments. Additionally, BRD7 expression was negatively correlated with the level of YB1 in breast cancer patients. The combination of low BRD7 and high YB1 expression was significantly associated with poor prognosis, distant metastasis and advanced TNM stage.ConclusionsCollectively, these findings uncover that BRD7 blocks tumor growth, migration and metastasis by negatively regulating YB1-induced EMT, providing new insights into the mechanism by which BRD7 contributes to the progression and metastasis of breast cancer.