YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits.

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

Y box binding protein 1 inhibition as a targeted therapy for ovarian cancer.

Y box binding protein 1 (YB-1) is a multifunctional protein associated with tumor progression and the emergence of treatment resistance (TR). Here, we report an azopodophyllotoxin small molecule, SU056, that potently inhibits tumor growth and progression via YB-1 inhibition. This YB-1 inhibitor inhibits cell proliferation, resistance to apoptosis in ovarian cancer (OC) cells, and arrests in the G1 phase. Inhibitor treatment leads to enrichment of proteins associated with apoptosis and RNA degradation pathways while downregulating spliceosome pathway. In vivo, SU056 independently restrains OC progression and exerts a synergistic effect with paclitaxel to further reduce disease progression with no observable liver toxicity. Moreover, in vitro mechanistic studies showed delayed disease progression via inhibition of drug efflux and multidrug resistance 1, and significantly lower neurotoxicity as compared with etoposide. These data suggest that YB-1 inhibition may be an effective strategy to reduce OC progression, antagonize TR, and decrease patient mortality.

Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms.

Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2-ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2.

Förster Resonance Energy Transfer Based Biosensor for Targeting the hNTH1-YB1 Interface as a Potential Anticancer Drug Target.

The Y-box binding protein 1 (YB1) is an established metastatic marker: high expression and nuclear localization of YB1 correlate with tumor aggressiveness, drug resistance, and poor patient survival in various tumors. In the nucleus, YB1 interacts with and regulates the activities of several nuclear proteins, including the DNA glycosylase, human endonuclease III (hNTH1). In the present study, we used Förster resonance energy transfer (FRET) and AlphaLISA technologies to further characterize this interaction and define the minimal regions of hNTH1 and YB1 required for complex formation. This work led us to design an original and cost-effective FRET-based biosensor for the rapid in vitro high-throughput screening for potential inhibitors of the hNTH1-YB1 complex. Two pilot screens were carried out, allowing the selection of several promising compounds exhibiting IC50 values in the low micromolar range. Interestingly, two of these compounds bind to YB1 and sensitize drug-resistant breast tumor cells to the chemotherapeutic agent, cisplatin. Taken together, these findings demonstrate that the hNTH1-YB1 interface is a druggable target for the development of new therapeutic strategies for the treatment of drug-resistant tumors. Moreover, beyond this study, the simple design of our biosensor defines an innovative and efficient strategy for the screening of inhibitors of therapeutically relevant protein-protein interfaces.

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.

Targeting Phosphorylation of Y-Box-Binding Protein YBX1 by TAS0612 and Everolimus in Overcoming Antiestrogen Resistance.

Nuclear expression of Y-box-binding protein (YBX1) is closely correlated with clinical poor outcomes and drug resistance in breast cancer. Nuclear translocation of YBX1 is facilitated by YBX1 phosphorylation at serine 102 by AKT, p70S6K, and p90RSK, and the phosphorylated YBX1 (pYBX1) promotes expression of genes related to drug resistance and cell growth. A forthcoming problem to be addressed is whether targeting the phosphorylation of YBX1 overcomes antiestrogen resistance by progressive breast cancer. Here, we found that increased expression of pYBX1 was accompanied by acquired resistance to antiestrogens, fulvestrant and tamoxifen. Forced expression of YBX1/S102E, a constitutive phosphorylated form, resulted in acquired resistance to fulvestrant. Inversely, YBX1 silencing specifically overcame antiestrogen resistance. Furthermore, treatment with everolimus, an mTORC1 inhibitor, or TAS0612, a novel multikinase inhibitor of AKT, p70S6K, and p90RSK, suppressed YBX1 phosphorylation and overcame antiestrogen resistance in vitro and in vivo IHC analysis revealed that expression of pYBX1 and YBX1 was augmented in patients who experienced recurrence during treatment with adjuvant endocrine therapies. Furthermore, pYBX1 was highly expressed in patients with triple-negative breast cancer compared with other subtypes. TAS0612 also demonstrated antitumor effect against triple-negative breast cancer in vivo Taken together, our findings suggest that pYBX1 represents a potential therapeutic target for treatment of antiestrogen-resistant and progressive breast cancer.

Co-targeting CK2α and YBX1 suppresses tumor progression by coordinated inhibition of the PI3K/AKT signaling pathway.

Protein kinase CK2 alpha (CK2α) is involved in the development of multiple malignancies. Overexpression of Y-box binding protein 1 (YBX1) is related to tumor proliferation, drug resistance, and poor prognosis. Studies have demonstrated that both CK2 and YBX1 could regulate the PI3K/AKT pathway. In addition, we predicted that CK2 might be the upstream kinase of YBX1 through the Human Protein Reference Database (HPRD). Herein, we hypothesize that CK2 may interact with YBX1 and they regulate the PI3K/AKT signaling pathway together. Expressions of CK2α and YBX1 in cancer cell lines were evaluated by immunoblotting. The results showed that CK2α could regulate the expression of YBX1 at the transcriptional level, which is dependent on its enzymatic activity. Synergistic effects of PI3K/AKT pathway inactivation could be observed through combined inhibition of CK2α and YBX1, and YBX1 was required for CK2α-induced PI3K/AKT pathway activation. Further results demonstrated that CK2α could interact with YBX1 and PI3K/AKT antagonist decreased cell resistance to doxorubicin induced by co-activation of CK2α and YBX1. These results indicated that combined inhibition of CK2α and YBX1 showed synergistic effects in inactivating the PI3K/AKT signaling pathway and may be one of the mechanisms involved in tumor growth and migration.

Targeting the YB-1/PD-L1 Axis to Enhance Chemotherapy and Antitumor Immunity.

Tumor cells can escape immune destruction in tumor chemoresistance, but the mechanism for this phenomenon remains unclear. Y-box binding protein 1 (YB-1), which is upregulated in chemoresistant tumor cells, plays a role in the acquisition of multidrug resistance. Here, we demonstrate that chemotherapy induced an immunosuppressive microenvironment in the tumor and induced immune evasion through YB-1-mediated programmed death-1 ligand 1 (PD-L1) upregulation. Examination of the YB-1 protein and mRNA showed an increase in YB-1 expression in hepatocellular carcinoma (HCC). High YB-1 expression negatively correlated with the overall survival of HCC patients. YB-1 expression positively correlated with PD-L1, and YB-1 induced PD-L1 expression by binding a PD-L1 promoter motif. YB-1 expression was upregulated in chemoresistant HCC cells, and YB-1 knockdown reversed chemoresistance via T-cell activation in the tumor microenvironment due to blocked PD-L1 expression. We also found that inhibition of the tumor immunosuppressive environment and immune evasion was accompanied by proliferation of functional cytotoxic CD8+ T cells and inhibition of myeloid-derived suppressor cells and regulatory T cells in the tumor environment. Our data indicate that targeting the YB-1 signaling axis, which simultaneously reverses both tumor immune evasion and multidrug resistance, may improve the antitumor response. This finding suggests a treatment modality against tumor chemoresistance.

The YB-1/EZH2/amphiregulin signaling axis mediates LPA-induced breast cancer cell invasion.

Lysophosphatidic acid (LPA) has been known to induce epithelial-mesenchymal transition (EMT) to stimulate cancer cell invasion, and resveratrol (3,5,4'-trans-trihydroxystilbene; REV) suppresses the invasion and metastasis of various cancers. The current study aimed to identify the underlying mechanism by which LPA aggravates breast cancer cell invasion and the reversal of this phenomenon. Immunoblotting and quantitative RT-PCR analysis revealed that LPA induces amphiregulin (AREG) expression. Silencing of Y-box binding protein 1 (YB-1) or enhancer of zeste homolog 2 (EZH2) expression efficiently inhibited LPA-induced AREG expression. In addition, transfection of the cells with YB-1 siRNA abrogated LPA-induced EZH2 and AREG expression, leading to attenuation of breast cancer cell invasion. Furthermore, we observed that both REV and 5-fluorouracil (5-Fu) significantly reduce LPA-induced YB-1 phosphorylation and subsequent breast cancer invasion. Importantly, combined treatment of REV with 5-Fu showed more significant inhibition of LPA-induced breast cancer invasion compared to single treatment. Therefore, our data demonstrate that the YB-1/EZH2 signaling axis mediates LPA-induced AREG expression and breast cancer cell invasion and its inhibition by REV and 5-Fu, providing potential therapeutic targets and inhibition of breast cancer.

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

BACKGROUND: Y-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. METHODS: The 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. RESULTS: Our 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. CONCLUSION: YB-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.

Complex consisting of antisense DNA and ß-glucan promotes internalization into cell through Dectin-1 and hybridizes with target mRNA in cytosol.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the ß-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

Fisetin targets YB-1/RSK axis independent of its effect on ERK signaling: insights from in vitro and in vivo melanoma models.

The anti-proliferative activity of dietary flavonoid fisetin has been validated in various cancer models. Establishing its precise mechanism of action has proved somewhat challenging given the multiplicity of its targets. We demonstrated that YB-1 promotes epithelial-to-mesenchymal transition and its inhibition suppressed tumor cell proliferation and invasion. The p90 ribosomal S6 kinase (RSK), an important ERK effector, activates YB-1 to drive melanoma growth. We found that fisetin treatment of monolayer/3-D melanoma cultures resulted in YB-1 dephosphorylation and reduced transcript levels. In parallel, fisetin suppressed mesenchymal markers and matrix-metalloproteinases in melanoma cells. Data from cell-free/cell-based systems indicated that fisetin inhibited RSK activity through binding to the kinase. Affinity studies for RSK isoforms evaluated stronger interaction for RSK2 than RSK1. Competition assays performed to monitor binding responses revealed that YB-1 and RSK2 do not compete, rather binding of fisetin to RSK2 promotes its binding to YB-1. Fisetin suppressed YB-1/RSK signaling independent of its effect on ERK, and reduced MDR1 levels. Comparable efficacy of fisetin and vemurafenib for inhibiting melanoma growth was noted albeit through divergent modulation of ERK. Our studies provide insight into additional modes of regulation through which fisetin interferes with melanoma growth underscoring its potential therapeutic efficacy in disease progression.

The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level.

The integrity of stratified epithelia depends on the ability of progenitor cells to maintain a balance between proliferation and differentiation. While much is known about the transcriptional pathways underlying progenitor cells' behavior in the epidermis, the role of posttranscriptional regulation by mRNA binding proteins-a rate-limiting step in sculpting the proteome-remains poorly understood. Here we report that the RNA binding protein YBX1 (Y-box binding protein-1) is a critical effector of progenitors' function in the epidermis. YBX1 expression is restricted to the cycling keratinocyte progenitors in vivo and its genetic ablation leads to defects in the architecture of the skin. We further demonstrate that YBX1 negatively controls epidermal progenitor senescence by regulating the translation of a senescence-associated subset of cytokine mRNAs via their 3' untranslated regions. Our study establishes YBX1 as a posttranscriptional effector required for maintenance of epidermal homeostasis.

Identification of 2,4-dihydroxy-5-pyrimidinyl imidothiocarbomate as a novel inhibitor to Y box binding protein-1 (YB-1) and its therapeutic actions against breast cancer.

In spite of advances in breast cancer treatment and early diagnosis, drug toxicity, cancer relapse, multidrug resistance and metastasis are the major impediment to the developments of efficient drugs. However, unique druggable targets of cancer cells distinct from the normal cells provide new rationale in cancer treatment. Previous reports clearly emphasize the differential expression and localization of Y box binding protein-1 (YB-1) between normal breast tissues and different stages of breast cancer. Y box binding protein-1 is DNA as well as RNA binding protein involved in transcription and translation regulation of various proteins involved in cancer progression, apoptosis, cell cycle, epithelial to mesenchymal transition (EMT) and drug resistance. Particularly, during doxorubicin (DOX) treatment and cancer relapse conditions, YB-1 expression was very high in breast cancer tissues and localized in to nucleus which further favours DOX efflux and metastasis. Moreover, siRNA mediated silencing of YB-1 reduces breast cancer progression and metastasis. In this rationale, using an array of computational methods, 2,4-dihydroxy-5-pyrimidinyl imidothiocarbomate (DPI) has been screened out as a drug-likeness antagonist to the YB-1for cancer treatment. In this study, we determined that DPI was toxic to breast cancer cell lines as individual drug as well as in combination with DOX. Moreover, immunofluorescence and confocal studies showed that DPI decreases DOX induced YB-1 nuclear translocation and increases DOX accumulation in breast cancer cell line. A G1/G0 phase cell cycle arrest and apoptosis was also induced by DPI. Moreover, DPI modulated YB-1 downstream targets such as p53, caspase-3, CDK-1 which are involved in cell cycle progression and apoptosis. Further, metastatic functional analysis revealed that DPI inhibits cell adhesion, migration, invasion in aggressive metastatic cell line and inhibits angiogenesis in chick embryonic chorioallantoic membrane (CAM) model. Meanwhile, DPI alters the expression of YB-1 downstream targets which are involved in metastasis such as VEGFR, caveolin, E-cadherin, cytokeratins, desmin and vimentin in MDA-MB-231 xenograft in chick embryonic CAM membrane. The results clearly demonstrated that DPI inhibited YB-1 nuclear translocation, thereby exhibited anti-apoptotic, anti-proliferative and anti-metastatic activities and increases the therapeutic potential of commercial breast cancer drug doxorubicin.

YB-1 expression promotes pancreatic cancer metastasis that is inhibited by microRNA-216a.

Pancreatic cancer is one of the most aggressive cancers. The vast majority of patients are diagnosed with advanced, unresectable disease because of early invasive growth and metastatic spread. The aim of this study was to examine YB-1 expression in pancreatic cancer and determine its effects on cell invasion. YB-1 is overexpressed in pancreatic cancer cell lines and patient tissue samples. In patient tissues, high YB-1 levels correlated with perineural invasion. Silencing of YB-1 significantly reduced cell invasion with decreased expression of MMPs in vitro. Furthermore, we found that the expression of YB-1 was suppressed by miR-216a via direct binding to the YB-1 3'-untranslated region. MiR-216a and YB-1 expression levels were inversely correlated in pancreatic cancer cell lines. In addition, ectopic expression of miR-216a inhibited cell invasion in vitro. Taken together, our findings suggest that YB-1 may play an important role in mediating metastatic behaviour and that repression of YB-1 by miR-216a could have a promising therapeutic potential to inhibit tumor metastasis in pancreatic cancer.

Y-box Binding Protein 1 Is Involved in Regulating the G2/M Phase of the Cell Cycle.

BACKGROUND/AIM: The transcription factor Y-box-binding protein 1 (YB1) is overexpressed in many types of human cancers. YB1 regulates the G1 phase of the cell cycle by controlling transcription of G1 regulators. Here, we report that YB1 is also involved in regulating G2/M phase. MATERIALS AND METHODS: YB1-depleted TKO cells were subjected to quantitative reverse transcription-polymerase chain reaction and cell-cycle analysis. RNA immunoprecipitation (RIP)-chip assay was performed using anti-YB1 antibodies. Precipitated RNAs were subjected to microarray analysis. RESULTS: Silencing YB1 inhibited the proliferation of TKO cells, which lost the machinery required for G1 phase arrest. Cell-cycle analysis showed that silencing YB1 caused G2/M phase cell-cycle arrest. RIP-chip assay showed that YB1 associated with mRNA of multiple cell-cycle-related genes, including G2/M phase regulators. CONCLUSION: YB1 positively regulates not only the G1 phase but also G2/M phase by regulating multiple cell-cycle-related genes.

RSK-mediated nuclear accumulation of the cold-shock Y-box protein-1 controls proliferation of T cells and T-ALL blasts.

Deregulated proliferation is key to tumor progression. Although unrestricted proliferation of solid tumor cells correlates with the cold-shock protein Y-box (YB)-binding protein-1 accumulation in the nuclei, little is known about its expression and function in hematopoietic malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). Here we show that YB-1 protein is highly enriched in the nuclei of activated T cells and malignant human T-ALL cell lines but not in resting T cells. YB-1 S102 mutations that either mimic (S102D) or prevent phosphorylation (S102N) led to accumulation of YB-1 in the nucleus of T cells or strictly excluded it, respectively. Inactivation of ribosomal S6 kinase (RSK) was sufficient to abrogate T-cell and T-ALL cell proliferation, suggesting that RSK mediates cell-cycle progression, possibly dependent on YB-1-phosphorylation. Indeed, phosphomimetic YB-1S102D enhanced proliferation implying that S102 phosphorylation is a prerequisite for malignant T-cell proliferation. At initial diagnosis of T-ALL, YB-1 localization was significantly altered in the nuclei of tumor blasts derived from bone marrow or peripheral blood. Our data show deregulated YB-1 in the nucleus as a yet unreported characteristic of T-ALL blasts and may refine strategies to restrict progression of hematopoietic tumors.

Optimal sequence of antisense DNA to silence YB-1 in lung cancer by use of a novel polysaccharide drug delivery system.

Silencing Y-box binding protein 1 (YB-1) can be an excellent target for cancer therapy and many lung cancer cells express the polysaccharide-recognition receptor Dectin-1. We designed a Dectin-1 targeting vehicle delivering YB-1-antisense DNA. First, we selected five optimal antisense DNA sequences to silence YB-1 from among 153 candidates. We chose the sequence closest to the start codon (AS014), and attached dA40 to the 3' end; dA40 promotes complex formation with a ß-(1➝3)-d-glucan called schizophyllan (SPG). The resultant complexes were applied to 12 human-oriented lung cancer cell lines, and cell viability was examined. The cell lines exhibited decreased viability and showed strong affinity to bind SPG, suggesting the AS014/SPG complex entered the cells via the Dectin-1 mediated pathway.

Inhibition of pro­collagen I expression by oxymatrine in hepatic stellate cells is mediated via nuclear translocation of Y­box binding protein 1.

Accumulating evidence indicated that oxymatrine (OMT), an alkaloid compound from the Chinese medicinal herb Sophora flavescens, exhibits activity against hepatic fibrosis. The present study attempted to explore the underlying mechanisms of OMT­mediated inhibition of collagen production. For this, the LX­2 human hepatic stellate cell line was treated with OMT (240, 480 or 960 mg/l) for 3­5 days. The endogenic expression of pro­collagen I was decreased by OMT in a dose­ and time­dependent manner, accompanied with the downregulation of Y­box binding protein 1 (YB­1), a vital transcription factor, particularly on the fourth day of incubation with a high concentration of OMT. To further explore the intracellular changes in YB­1 levels, nuclear/cytoplasmic proteins were extracted separately, and subsequent western blot analysis revealed a significant upregulation of YB­1 in the nucleus in parallel with its downregulation in the cytoplasm, indicating the nuclear translocation of YB­1 induced by OMT treatment. In another experiment, knockdown of YB­1 using small interfering RNA led to elevated mRNA levels of collagen I, thereby reversing the effects of OMT treatment. In conclusion, these present study suggested that the attenuation of pro­collagen I expression caused by OMT was, to a certain extent, mediated via nuclear translocation of YB­1.

Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement.

Upon exposure to stress, tRNAs are enzymatically cleaved, yielding distinct classes of tRNA-derived fragments (tRFs), yielding distinct classes of tRFs. We identify a novel class of tRFs derived from tRNA(Glu), tRNA(Asp), tRNA(Gly), and tRNA(Tyr) that, upon induction, suppress the stability of multiple oncogenic transcripts in breast cancer cells by displacing their 3' untranslated regions (UTRs) from the RNA-binding protein YBX1. This mode of post-transcriptional silencing is sequence specific, as these fragments all share a common motif that matches the YBX1 recognition sequence. Loss-of-function and gain-of-function studies, using anti-sense locked-nucleic acids (LNAs) and synthetic RNA mimetics, respectively, revealed that these fragments suppress growth under serum-starvation, cancer cell invasion, and metastasis by breast cancer cells. Highly metastatic cells evade this tumor-suppressive pathway by attenuating the induction of these tRFs. Our findings reveal a tumor-suppressive role for specific tRNA-derived fragments and describe a molecular mechanism for their action. This transcript displacement-based mechanism may generalize to other tRNA, ribosomal-RNA, and sno-RNA fragments.