Y-box Binding Protein-1 Activation Research Articles

Simultaneous Targeting of RSK and AKT Efficiently Inhibits YB-1-Mediated Repair of Ionizing Radiation-Induced DNA Double-Strand Breaks in Breast Cancer Cells

Y-box binding protein 1 (YB-1) overexpression is associated with chemotherapy- and radiation therapy resistance. Ionizing radiation (IR), receptor tyrosine kinase ligands, and mutation in KRAS gene stimulate activation of YB-1. YB-1 accelerates the repair of IR-induced DNA double-strand breaks (DSBs). Ribosomal S6 kinase (RSK) is the main kinase inducing YB-1 phosphorylation. We investigated the impact of RSK targeting on DSB repair and radiosensitivity. The triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468, and Hs 578T, in addition to non-TNBC cell lines MCF7, HBL-100, and SKBR3, were used. MCF-10A cells were included as normal breast epithelial cells. The RSK inhibitor LJI308 was used to investigate the role of RSK activity in S102 phosphorylation of YB-1 and YB-1-associated signaling pathways. The activation status of the underlying pathways was investigated by Western blotting after treatment with pharmacologic inhibitors or transfection with siRNA. The impact of LJI308 on DSB repair and postirradiation cell survival was tested by the γH2AX foci and the standard clonogenic assays, respectively. LJI308 inhibited the phosphorylation of RSK (T359/S363) and YB-1 (S102) after irradiation, treatment with EGF, and in cells expressing a KRAS mutation. LJI308 treatment slightly inhibited DSB repair only in some of the cell lines tested. This was shown to be due to PI3K-dependent stimulation of AKT or constitutive AKT activity mainly in cancer cells but not in normal breast epithelial MCF-10A cells. Simultaneous targeting of AKT and RSK strongly blocked DSB repair in all cancer cell lines, independent of TNBC status or KRAS mutation, with a minor effect in MCF-10A cells. Cotargeting of RSK- and AKT-induced radiation sensitivity in TNBC MDA-MB-231 and non-TNBC MCF7 cells but not in MCF-10A cells. Simultaneous targeting of RSK and AKT might be an efficient approach to block the repair of DSBs after irradiation and to induce radiosensitization of breast cancer cells.

A novel \u03b22-AR/YB-1/\u03b2-catenin axis mediates chronic stress-associated metastasis in hepatocellular carcinoma

β-Adrenergic receptor (β-AR) signalling is strongly associated with tumour progression by the coupling of β-ARs with either a G protein or β-arrestin; however, the related mechanism underlying hepatocellular carcinoma (HCC) metastasis is not clear. Here, we reveal that the transcription factor Y-box binding protein 1 (YB-1) interacts with β2-adrenergic receptor (β2-AR) following stimulation with the agonist isoproterenol (ISO). Clinicopathological analysis demonstrated that β2-AR is significantly correlated with YB-1, which favours the progression of HCC. The binding of YB-1 with β2-AR resulted in YB-1 phosphorylation at serine 102 (S102) via the β-arrestin-1-dependent activation of the PI3K/AKT pathway, followed by the translocation of YB-1 to the nucleus to carry out its tumour-related function. β2-AR-mediated activation of YB-1 facilitated epithelial-to-mesenchymal transition (EMT) and HCC metastasis. The interference of YB-1 expression significantly attenuated liver tumour metastasis induced by chronic stress. Analysis of the transcriptional profile and chromatin immunoprecipitation (ChIP) identified β-catenin as a crucial target of YB-1. Our results unveiled a novel β2-AR-mediated regulatory axis in HCC metastasis that might be helpful for the development of HCC therapeutics.

YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT.

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties.Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149-60. ©2018 AACR.

FOXO3a Expression Regulated by ERK Signaling is Inversely Correlated With Y-Box Binding Protein-1 Expression in Prostate Cancer.

FOXO3a is a member of the forkhead O transcription factors. FOXO3a induces the factors that contribute to cell cycle arrest and is considered a tumor suppressor in several malignant tumors. Y-box binding protein-1 (YB-1) is a multifunctional protein whose high expression is correlated with poor prognoses in various malignant tumors. In the current study, we investigated the relationship between FOXO3a and YB-1 to validate their functional roles in prostate cancer. Western blotting and cytotoxicity assays were conducted in prostate cancer cells, LNCaP, and 22Rv1 cells. We also evaluated the protein expressions of FOXO3a and YB-1 in human prostate cancer tissues, using radical prostatectomy specimens. Then, we investigated the correlations between protein expressions and clinicopathologic parameters. We found that both FOXO3a and YB-1 proteins were phosphorylated by ERK signaling, resulting in FOXO3a inactivation and YB-1 activation in LNCaP and 22Rv1 cells. Inversely, inhibition of MEK or treatment with metformin activated FOXO3a through inactivation of ERK signaling and suppressed the viability of LNCaP and 22Rv1 cells in a dose-dependent manner. In immunohistochemical analysis, FOXO3a nuclear expression was inversely correlated with YB-1 nuclear expression (P < 0.0001). Furthermore, high FOXO3a nuclear expression was inversely correlated with a higher Gleason grade (P < 0.0001) and higher preoperative PSA (P = 0.0437). These results showed that in prostate cancer, FOXO3a, and YB-1 play inverse reciprocal roles as a tumor-suppressor gene and oncogene, respectively, through their master regulator ERK. Prostate 77:145-153, 2017. © 2016 Wiley Periodicals, Inc.

Phosphorylation-Mediated Interaction of Hepatitis C Virus NS5A and the Cellular YB-1 Controls HCV Propagation and the Early Stage of Viral RNA Replication

About 130-150 million individuals worldwide are chronically infected by hepatitis C virus (HCV); HCV infection has become a leading cause of liver cirrhosis and hepatocellular carcinoma. The HCV life cycle relies on the cooperation of specific viral proteins and various cellular factors. The cellular protein Y-box binding protein 1 (YB-1) has recently been identified as a HCV host cofactor; however, its mechanism has not been fully characterized. The HCV nonstructural protein NS5A plays critical roles in almost every stage of HCV propagation, and has been proposed to control the switch between the defined stages of the HCV life cycle. Moreover, NS5A has recently emerged as a target for the development of novel anti-HCV drugs. Our studies have found that YB-1 not only interacts with NS5A, but also protects NS5A from degradation. Furthermore, both the NS5A/YB-1 interaction and the NS5A-stabilizing activity of YB-1 are dependent on the phosphorylation of YB-1 at serine 102 (S102). Interestingly, the YB-1 S102 site has previously been reported to be phosphorylated by Akt, which is in turn activated by HCV infection. Our study also reveals that DDX3, an YB-1-interacting partner, is another NS5A-binding protein, which plays a different role than YB-1 in HCV RNA replication and infectious virus production. Taken together, the elucidation of YB-1 participation in the HCV life cycle has led to a proposed mechanism of efficient virus propagation via coordination of the different stages of the viral life cycle through controlling stage-wise switches in the viral life cycle. Our finding also provides a novel niche for designing strategies for the development of new anti-HCV drugs by blocking specific virus-host interactions.

Y-Box Binding Protein 1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A To Regulate Viral Propagation.

Replication of hepatitis C virus (HCV) is dependent on virus-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. In this study, we investigated the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate double-stranded RNA (dsRNA) in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of the p58 and p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1-NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs. Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of the HCV life cycle. In this study, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of the p58 and p56 phosphoforms for efficient viral propagation via regulation of cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.

C1QBP negatively regulates the activation of oncoprotein YBX1 in the renal cell carcinoma as revealed by interactomics analysis.

The Y-box-binding protein 1 (YBX1) plays a critical role in tumorigenesis by promoting cell proliferation, overriding cell-cycle check points, and enhancing genomic instability. In this study, the interactome of YBX1 in renal cell carcinoma (RCC) was analyzed by coimmunoprecipitation and mass spectrometry to better understand its function and regulatory mechanism. A total of 129 proteins were identified as potential YBX1 binding partners. The interaction between the complement component 1, q subcomponent binding protein (C1QBP), and YBX1 was further confirmed by immunoprecipitation and Western blotting. Knockdown of C1QBP enhanced the phosphorylation of YBX1and its nuclear translocation, indicating that C1QBP negatively regulated YBX1 activation. The clinical significance of these two proteins was analyzed in the tissues from 52 RCC patients by immunohistochemistry. Expression of YBX1 was markedly elevated in the carcinoma tissues, and its nuclear expression was associated with histological T stage and metastasis. Meanwhile, the level of C1QBP in the carcinoma tissues was significantly lower than that in the adjacent healthy tissues, which was negatively correlated with the nuclear localization of YBX1 in the RCC tissues (P = 0.011). These data suggest that C1QBP is a novel regulator of YBX1, and the expression of C1QBP and the nuclear expression of YBX1 could both be used as independent prognostic makers for cancer progression in the RCC patients. The proteomics data have been deposited to the ProteomeXchange with identifier PXD001493.

O2-15-2 - Y-Box Binding Protein-1 Activation May Modify the Responses to Endocrine and Her2-Targeted Therapeutics in Breast Cancer

Purpose: YB-1 is a potent oncogene for breast cancer (Bergmann et al. Cancer Res, 2005). Our previous studies have demonstrated that nuclear localization of YB-1 plays pivotal roles in HER2/ErbB2 expression in breast cancer as well as in stomach cancer in vitro and in patients. In our present study, we first asked whether YB-1-induced expression of HER2 gene could be closely coupled with the absence or presence of ERα, and then asked whether YB-1 activation could be biostatistically linked to HER2 and/or ER expression in breast cancer of pre-menopausal and post-menopausal patients.Results: [1] In breast cancer cell lines: YB-1 knockdown by its cognate siRNA resulted in marked reduction of HER2 expression in ERα(+) breast cancer cells, but not in ERα(-) cells; and YB-1 overexpression in nucleus resulted in induction of HER2 expression accompanying by decreased expression of ERα; and ERα overexpression by exogenous transfection of its cDNA induced suppression of YB-1 dependent HER2 expression; and also the functional blockage of ERα by tamoxifen resulted in increasing expression of HER2. [2] In immunohistochemical analyses of clinical samples: In tumors from post-menopausal patients (n = 114), nuclear YB-1 expression was positively associated with HER2 expression, but negatively associated with ERα/PgR expression; however in tumors from pre-menopausal patients (n = 57), nuclear YB-1 expression was not significantly associated with ERα/PgR or HER2 expression.Conclusion and Discussion: Based on our basic and clinical study, YB-1 activation plays an essential role in expression of HER2/ErbB2, depending upon the absence or presence of ERα in breast cancer. The cross-talk of ERα and HER2 through activated YB-1 may thus specify biological characteristics in breast cancers luminal A, luminal B (HER2+), luminal B (HER2-), HER2 disease and triple negative. This study may propose an idea how endocrine and HER2-targeted therapeutics are mutually optimized against breast cancer.

Abstract 3125: Fisetin inhibits p90RSK/YB-1 signaling and downregulates chemoresistance associated P-glycoprotein in A375 melanoma cells

Abstract Malignant melanoma is notoriously difficult to treat due to lack of targeted systemic therapies and emergence of resistance to BRAF inhibitors, currently the treatment of choice for metastatic melanoma. The p90 ribosomal S6 kinase (RSK), an important effector of RAF/ERK signaling promotes melanoma growth and contributes to melanoma chemoresistance by altering response to chemotherapeutic agents. Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor involved in multiple cellular functions including proliferation, migration, and epithelial mesenchymal transition (EMT). Recent studies show that RSK1/2 can activate YB-1 through phosphorylation at its Ser102 residue. We found YB-1 expression to be significantly upregulated in metastatic melanoma cell lines when compared to normal human melanocytes. Treatment of A375 human melanoma cells with the dietary flavonoid fisetin (20-60µM) resulted in inhibition of phosphorylation of YB-1 at Ser102, with subsequent decrease in nuclear translocation and activation. We further studied the effect of fisetin on YB-1 signaling in a 3-D melanoma skin equivalent model, comprising of A375 melanoma cells, cultured with epidermal keratinocytes and dermal fibroblasts that closely simulates the human skin. Studies in the 3-D model showed that fisetin treatment resulted in reduction in melanocytic lesions and tumor progression (p&amp;lt; .01) associated with decreased expression of YB-1, and its downstream targets c-Myc and MTA-1. Fisetin suppressed the expression of EMT markers N-cadherin and Vimentin, with a concomitant increase in E-cadherin in melanoma cells. In addition, a significant decrease in the protein expression of matrix-metalloproteinases -2 and -9 was observed. Next we investigated whether fisetin interfered with RSK signaling involved in YB-1 activation. Immunoblot studies revealed that fisetin inhibited the phosphorylation of p90RSK at the Thr359/Ser363 residue but not at Ser380. Notably, the MDR-1 gene product, P-glycoprotein (P-gp), was markedly decreased in fisetin-treated cells in a dose-dependent manner. Fisetin-mediated increase in the phosphorylation of ERK1/2 at Thr202/Tyr204 residues suggests that diminished P-gp expression is linked to suppression of RSK/YB-1 signaling in fisetin treated cells. Together, our results suggest that fisetin is a potent inhibitor of the p90RSK/YB-1 pathway and may be used to modulate chemosensitivity, which is one of the major obstacles to melanoma treatment. Citation Format: Deeba N. Syed, Rahul K. Lall, Mohammad Imran Khan, Maria Shabbir, Hasan Mukhtar. Fisetin inhibits p90RSK/YB-1 signaling and downregulates chemoresistance associated P-glycoprotein in A375 melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2014-3125

Inhibition of RSK/YB-1 signaling enhances the anti-cancer effect of enzalutamide in prostate cancer

Previously, we have shown that Y-box binding protein-1 (YB-1) regulates androgen receptor (AR) expression and contributes to castration resistance. However, the mechanism of YB-1 activation remains unknown. In this study, we aimed to elucidate the mechanism and role of YB-1 activation in relation to castration resistance as well as enzalutamide resistance, with a view to developing a novel therapeutic concept for castration-resistant prostate cancer (CRPC) treatment. The expression and phosphorylation levels of ribosomal S6 kinase 1 (RSK1), YB-1 and AR were examined by quantitative PCR and Western blotting using prostate cancer cells. In addition, the effects of YB-1 inhibition using specific siRNA and small molecule inhibitor SL0101 on AR expression as well as combination treatment with enzalutamide and SL0101 were examined. We found that androgen deprivation, as well as treatment with the next-generation anti-androgen enzalutamide, induced RSK1 and YB-1 activation followed by AR induction, which could be reversed by YB-1 shutdown and RSK inhibitor SL0101. SL0101 and enzalutamide exerted a synergistic tumor-suppressive effect on cell proliferation in androgen-dependent prostate cancer LNCaP cells, as well as castration-resistant C4-2 cells. Furthermore, the phosphorylation levels of RSK1 and YB-1 were elevated in castration- and enzalutamide-resistant cells, compared with their parental cells. Taken together, these findings indicate that RSK1/YB-1 signaling contributes to castration as well as enzalutamide resistance, and that the therapeutic targeting of RSK1/YB-1 signaling would be a promising novel therapy against prostate cancer, especially CRPC when combined with enzalutamide.

Targeting ribosomal S6 kinases/Y-box binding protein-1 signaling improves cellular sensitivity to taxane in prostate cancer.

Taxanes are the only cytotoxic chemotherapeutic agents proved to prolong the survival in patients with castration-resistant prostate cancer. However, because of intrinsic and acquired resistances to taxanes, their therapeutical efficiencies are modest, bringing only a few months of survival benefit. Y-box binding protein-1 (YB-1) promotes cancer cell resistance to various anticancer treatments, including taxanes. Here, we aimed to elucidate the mechanism of taxane resistance by YB-1 and examined overcoming resistance by targeting YB-1 signaling. Gene and protein expression levels were evaluated by quantitative real-time polymerase chain reaction and Western blot analysis, respectively. We evaluated the sensitivity of prostate cancer cells to taxanes using cytotoxicity assays. Natural taxane paclitaxel from Taxus brevifolia activated the Raf-1/extracellular signal-regulated kinase (ERK) pathway, leading to an activation of ribosomal S6 kinases (RSK)/YB-1 signaling. Activated Raf-1/ERK pathway was blunted by YB-1 knockdown in prostate cancer cells, indicating regulation between Raf-1/ERK signaling and YB-1. In addition, ERK or RSK was activated in taxane-resistant prostate cancer cells, resulting in YB-1 activation. YB-1 knockdown as well as RSK inhibition using RSK-specific siRNA or the small molecule inhibitor SL0101 successfully blocked activation of YB-1, leading to suppression of prostate cancer growth and sensitization to paclitaxel. Taken together, these findings indicate that RSK/YB-1 signaling contributes to taxane resistance, and implicate the therapeutics targeting RSK/YB-1 signaling such as RSK inhibitor as a promising novel therapy against prostate cancer, especially in combination with taxane.

YB-1 expression promotes epithelial-to-mesenchymal transition in prostate cancer that is inhibited by a small molecule fisetin.

Epithelial-to-mesenchymal transition (EMT) plays an important role in prostate cancer (PCa) metastasis. The transcription/translation regulatory Y-box binding protein-1 (YB-1) is known to be associated with cancer metastasis. We observed that YB-1 expression increased with tumor grade and showed an inverse relationship with E-cadherin in a human PCa tissue array. Forced YB-1 expression induced a mesenchymal morphology that was associated with down regulation of epithelial markers. Silencing of YB-1 reversed mesenchymal features and decreased cell proliferation, migration and invasion in PCa cells. YB-1 is activated directly via Akt mediated phosphorylation at Ser102 within the cold shock domain (CSD). We next identified fisetin as an inhibitor of YB-1 activation. Computational docking and molecular dynamics suggested that fisetin binds on the residues from β1 - β4 strands of CSD, hindering Akt's interaction with YB-1. Calculated free binding energy ranged from -11.9845 to -9.6273 kcal/mol. Plasmon Surface Resonance studies showed that fisetin binds to YB-1 with an affinity of approximately 35 µM, with both slow association and dissociation. Fisetin also inhibited EGF induced YB-1 phosphorylation and markers of EMT both in vitro and in vivo. Collectively our data suggest that YB-1 induces EMT in PCa and identify fisetin as an inhibitor of its activation.

Self-reinforcing loop of amphiregulin and Y-box binding protein-1 contributes to poor outcomes in ovarian cancer

The Y-box binding protein-1 (YB-1) transcription factor is associated with unfavorable clinical outcomes. However, the mechanisms underlying this association remain to be fully elucidated. We demonstrate that YB-1 phosphorylation, indicative of YB-1 activation, is a powerful marker of outcomes for ovarian cancer patients. In ovarian cancer, YB-1 phosphorylation is induced by activation of the lysophosphatidic acid (LPA) receptor (LPAR) via SRC-dependent transactivation of the epidermal growth factor receptor (EGFR) that is coupled to MAPK/p90 ribosomal S6 kinase (p90RSK), but not phosphatidylinositol 3-kinase (PI3K)/AKT signaling. Activation of the LPAR/SRC/EGFR/MAPK/p90RSK/YB-1 axis leads to production of the EGFR ligand amphiregulin (AREG). AREG induces ongoing YB-1 phosphorylation as well as YB-1-dependent AREG expression, thus constituting an AREG/YB-1 self-reinforcing loop. Disruption of transactivation of the EGFR and the downstream self-reinforcing loop decreases invasiveness of ovarian cancer cells in vitro and limits ovarian cancer growth in xenograft models. These findings established the regulation and significance of YB-1 phosphorylation, therefore further exploration of this signaling axis as a therapeutic avenue in ovarian cancer is warranted.

Abstract 506: Targeting Y-box binding protein-1 (YB-1) overcomes drug resistance in triple-negative breast cancer.

Abstract Despite advances in treating breast cancer, disease recurrence rates remain high and secondary tumors are often resistant to chemotherapy and incurable. Currently, the treatment for triple-negative breast cancer (TNBC) relies upon conventional chemotherapeutics as there are no targeted therapies available. Although these tumors initially respond well, they paradoxically have the highest relapse rates. Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor abundantly expressed in TNBC (∼70% of patients). It is activated predominantly by phosphorylation via p90 ribosomal S6 kinase (RSK). Once activated it up-regulates the tumor-initiating-cell (TIC) marker CD44 and induces a TIC phenotype[1]. Due to their inherent drug-resistance, TICs survive chemotherapy and go on to drive relapse[2]. We recently identified RSK2 is critical to the survival of TNBC[3]. Inhibiting RSK2 blocks activation of YB-1 and induces apoptosis in TNBC, including CD44+/CD24- cells. Interestingly, inducing YB-1 is sufficient for transformation of human mammary epithelial cells into TNBC. Moreover, P-YB-1S102 is associated with poor overall survival (P&amp;lt;0.001) and relapse (P&amp;lt;0.001) in a cohort of 1057 patients with invasive breast cancer. Taken together, these data indicate YB-1 is important both in the genesis and maintenance of TNBC and implicate it as a key mediator of drug-resistance and relapse within this subtype. Residual cells after paclitaxel or epirubicin have increased P-RSKS221/7, P-YB-1S102 and CD44 and retain mammosphere-forming capability. Cells expressing high P-YB-1S102 exclusively proliferate in the presence of these chemotherapies. Inhibiting YB-1 suppresses growth and induces apoptosis in residual cells and in CD44+/CD24–sorted cells. Pre-emptive knockdown of YB-1 prevents induction of CD44 and sensitizes cells to treatment with paclitaxel. Moreover, blocking RSK/YB-1 signaling suppresses growth and induces cell death in primary TNBC cells (x43) derived from a patient that eventually relapsed. To investigate the role of RSK/YB-1 signaling in bona fide drug-resistant cells, we created paclitaxel-resistant SUM149 cells (SUM149-PTXR) which are cultured in 2 nM paclitaxel (PTX); a concentration that kills &amp;gt;90% of paclitaxel-naive SUM149’s (SUM149-DMSO) by 72 hrs. These cells express increased P-RSKS221/7 and P-YB-1S102 without alterations in total protein levels. Despite their resistance to paclitaxel, these cells remain sensitive to RSK/YB-1 signaling inhibition. We are currently comparing the response of TNBC either with or without YB-1 to paclitaxel in an in vivo orthotopic xenograft model. Collectively, these data indicate that inhibiting RSK/YB-1 signaling can overcome drug resistance and potentially reduce relapse in TNBC. 1.To K, Fotovati A et al. (2010) 2.Li X, Lewis MT et al. (2008) 3.Stratford A, Reipas K et al. (2012) Citation Format: Kristen Reipas, Anna Stratford, Arezoo Astanehe, Kaiji Hu, Sandra Dunn. Targeting Y-box binding protein-1 (YB-1) overcomes drug resistance in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2013-506

Luteolin is a novel p90 ribosomal S6 kinase (RSK) inhibitor that suppresses Notch4 signaling by blocking the activation of Y-box binding protein-1 (YB-1)

Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormone receptors and have limited targeted therapies. Recently, we demonstrated that p90 ribosomal S6 kinase (RSK) is essential for TNBC growth and survival indicating it as a target for therapeutic development. RSK phosphorylates Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, highly expressed in TNBC (~70% of cases) and associated with poor prognosis, drug resistance and tumor initiation. YB-1 regulates the tumor-initiating cell markers, CD44 and CD49f however its role in Notch signaling has not been explored. We sought to identify novel chemical entities with RSK inhibitory activity. The Prestwick Chemical Library of 1120 off-patent drugs was screened for RSK inhibitors using both in vitro kinase assays and molecular docking. The lead candidate, luteolin, inhibited RSK1 and RSK2 kinase activity and suppressed growth in TNBC, including TIC-enriched populations. Combining luteolin with paclitaxel increased cell death and unlike chemotherapy alone, did not enrich for CD44(+) cells. Luteolin's efficacy against drug-resistant cells was further indicated in the primary x43 cell line, where it suppressed monolayer growth and mammosphere formation. We next endeavored to understand how the inhibition of RSK/YB-1 signaling by luteolin elicited an effect on TIC-enriched populations. ChIP-on-ChIP experiments in SUM149 cells revealed a 12-fold enrichment of YB-1 binding to the Notch4 promoter. We chose to pursue this because there are several reports indicating that Notch4 maintains cells in an undifferentiated, TIC state. Herein we report that silencing YB-1 with siRNA decreased Notch4 mRNA. Conversely, transient expression of Flag:YB-1(WT) or the constitutively active mutant Flag:YB-1(D102) increased Notch4 mRNA. The levels of Notch4 transcript and the abundance of the Notch4 intracellular domain (N4ICD) correlated with activation of P-RSK(S221/7) and P-YB-1(S102) in a panel of TNBC cell lines. Silencing YB-1 or RSK reduced Notch4 mRNA and this corresponded with loss of N4ICD. Likewise, the RSK inhibitors, luteolin and BI-D1870, suppressed P-YB-1(S102) and thereby reduced Notch4. In conclusion, inhibiting the RSK/YB-1 pathway with luteolin is a novel approach to blocking Notch4 signaling and as such provides a means of inhibiting TICs.

MAPK and PI3K/AKT mediated YB‐1 activation promotes melanoma cell proliferation which is counteracted by an autoregulatory loop

Y-box binding protein 1 (YB-1) is an oncogenic transcription and translation factor and is overexpressed in several types of cancer. Our previous data showed that YB-1 is upregulated and translocated to the nucleus during melanoma progression and that YB-1 is an important transcription factor regulating proliferation, survival, migration, invasion and chemosensitivity of melanoma cells. It has been suggested that YB-1 is activated and translocated to the nucleus after S102-phosphorylation in the DNA binding domain. In this study, we show that activation of YB-1 by S102-phosphorylation and nuclear translocation is increased during melanoma progression using a human tissue microarray with 100 melanocytic lesions. Furthermore, we analysed the mechanisms governing the expression and activity of YB-1 in melanoma cells. We show that the PI3K/AKT and p53 signalling, growth factors and chemotherapeutic agents increase YB-1 promoter activity. This, however, resulted in no or only modest increase in YB-1 protein expression. We show that the MAPK and PI3K/AKT signalling pathways, both activated in melanoma cells, as well as p53 overexpression increase YB-1 S102-phosphorylation, whereas NFκB signalling inhibits phosphorylation. Overexpression of YB-1 in melanoma cells inhibits translation efficiency and by this proliferation and survival of melanoma cells indicating that there is an autoregulatory loop restricting YB-1 protein expression. These data suggest that there is a tightly regulated feedback mechanism regulating YB-1 expression and activation, necessary for proper cell cycle progression of melanoma cells.

MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibition

Trastuzumab (Herceptin) resistance is a major obstacle in the treatment of patients with HER2-positive breast cancers. We recently reported that the transcription factor Y-box binding protein-1 (YB-1) leads to acquisition of resistance to trastuzumab in a phosphorylation-dependent manner that relies on p90 ribosomal S6 kinase (RSK). To explore how this may occur we compared YB-1 target genes between trastuzumab-sensitive cells (BT474) and those with acquired resistance (HR5 and HR6) using genome-wide chromatin immunoprecipitation sequencing (ChIP-sequencing), which identified 1391 genes uniquely bound by YB-1 in the resistant cell lines. We then examined differences in protein expression and phosphorylation between these cell lines using the Kinexus Kinex antibody microarrays. Cross-referencing these two data sets identified the mitogen-activated protein kinase-interacting kinase (MNK) family as potentially being involved in acquired resistance downstream from YB-1. MNK1 and MNK2 were subsequently shown to be overexpressed in the resistant cell lines; however, only the former was a YB-1 target based on ChIP-PCR and small interfering RNA (siRNA) studies. Importantly, loss of MNK1 expression using siRNA enhanced sensitivity to trastuzumab. Further, MNK1 overexpression was sufficient to confer resistance to trastuzumab in cells that were previously sensitive. We then developed a de novo model of acquired resistance by exposing BT474 cells to trastuzumab for 60 days (BT474LT). Similar to the HR5/HR6 cells, the BT474LT cells had elevated MNK1 levels and were dependent on it for survival. In addition, we demonstrated that RSK phosphorylated MNK1, and that this phosphorylation was required for ability of MNK1 to mediate resistance to trastuzumab. Furthermore, inhibition of RSK with the small molecule BI-D1870 repressed the MNK1-mediated trastuzumab resistance. In conclusion, this unbiased integrated approach identified MNK1 as a player in mediating trastuzumab resistance as a consequence of YB-1 activation, and demonstrated RSK inhibition as a means to overcome recalcitrance to trastuzumab.

Highlights of This Issue

Epithelial-mesenchymal transition (EMT) has been implicated in the initial stages of metastasis. What remains unclear, however, is how it is maintained in late metastatic cells and whether it also regulates the subsequent dormant state of micrometastases. Tran and colleagues detail a mechanistic link between EMT and cancer dormancy in which Snail1 and Twist1, two key EMT inducers, cooperate with each other to initiate and maintain EMT, respectively, and in so doing Twist1 contributes directly to the cancer dormancy program. Therefore, Twist1 represents a potential therapeutic target to manipulate tumor dormancy for clinical benefits.The transmembrane protein Trop2 has been reported to be overexpressed in carcinomas, but its function remains largely unknown. In an effort to elucidate its role in tumorigenesis, Wang and colleagues generated Trop2−/− animals, which lack an obvious tumor phenotype. However, when mated into an Arf-null background and exposed to carcinogens, Trop2−/− mice develop tumors with both squamous and spindle cell histology. Accordingly, immortalized cells from Trop2−/− Arf−/− mice show hyperactivation of MAPK and Src pathways. Additionally, poorly differentiated head and neck cancers exhibit low Trop2 levels. These results identify a tumor suppressor role and highlight unexpected signaling properties for this protein.In this study, Azad and colleagues present the first report of DNA-dependent protein kinase (DNA-PK) blockade leading to accelerated senescence in irradiated human cancer cells. The study shows that DNAPK inhibition modulates repair of therapeutically induced DNA doublestrand breaks and thus promotes p53-dependent accelerated senescence in irradiated cells. Significantly, this report notes the lack of clinical trials using novel pharmacological inhibitors of DNA-PK in conjunction with ionizing radiation. Thus, these data provide a sound rationale for both preclinical and clinical evaluation of novel DNA-PK inhibitors in combination with ionizing radiation and other anticancer agents that induce doublestrand breaks or inhibit double-strand break repair.Many strategies used to kill cancer cells induce stress-responses that inhibit treatment-induced cell death and promote emergence of a treatment resistant phenotype. Whereas both Y-box binding protein-1 (YB-1) and clusterin are known to confer taxane resistance in cancer, their mechanistic interrelationships are undefined. Shiota and colleagues found that many varied anticancer therapeutic agents including taxanes induce endoplasmic reticulum stress with activation of YB-1. YB-1 was shown to be a critical regulator of stress-induced clusterin transcription and expression, and clusterin was shown to play a dominant downstream mediator of YB-1 in stressinduced cytoprotection and paclitaxel resistance in prostate cancer cells.

Upregulation of mdr1 gene is related to activation of the MAPK/ERK signal transduction pathway and YB-1 nuclear translocation in B-cell lymphoma

Multidrug resistance (MDR) in human B-cell lymphoma constitutes a major obstacle to the effectiveness of chemotherapy. The aim of this study was to investigate the molecular mechanism of MDR in B-cell lymphoma. The B-cell lymphoma MDR sublines were developed by exposing the parental Daudi cells to stepwise increasing concentrations of doxorubicin. Interaction of Y-box binding protein-1 (YB-1) with the Y-box motif of the mdr1 gene promoters was studied by electrophoretic mobility shift assay. The effects of YB-1 on mdr1 promoter activity were examined by luciferase assay. After silencing of YB-1 gene by shRNA, the role of YB-1 nuclear translocation in the formation of induced MDR was examined. Expression of mdr1 and YB-1 was examined further after Daudi cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitor PD98059 for 1 hour. Doxorubicin-resistant sublines was generated from the Daudi cell line by stepwise selection in doxorubicin. We found that acquisition of MDR is associated with enhanced YB-1 nuclear translocation and MAPK/extracellular signal-regulated kinase (ERK) activity. Electrophoretic mobility shift assay revealed that doxorubicin increased binding of YB-1 to the Y-box of mdr1 promoter. Luciferase reporter assays demonstrated that the Y-box region is essential for YB-1 regulation of mdr1 expression. The introduction of exogenous YB-1 shRNA into Daudi cells resulted in decreased levels of the expression of mdr1 gene and P-glycoprotein induced by doxorubicin. When Daudi cells were pretreated with MAPK inhibitor PD98059, the phosphorylation of ERK was effectively inhibited as well as the nuclear translocation of YB-1 and the expression of mdr1 gene. Doxorubicin can increase expression of mdr1/P-glycoprotein through activating MAPK/ERK transduction pathway, then increasing expression of YB-1, inducing YB-1 nuclear translocation, and enhancing DNA-binding activity of YB-1.

Molecular Decoy to the Y-Box Binding Protein-1 Suppresses the Growth of Breast and Prostate Cancer Cells whilst Sparing Normal Cell Viability

The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2, and to a lesser degree PKCα and AKT. Herein, we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1S102 phosphorylation based on molecular docking. In cancer cells, the CPP blocked P-YB-1S102 and down-regulated both HER-2 and EGFR transcript level and protein expression. Further, the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably, the growth of breast (SUM149, MDA-MB-453, AU565) and prostate (PC3, LNCap) cancer cells was inhibited by ∼90% with the CPP. Further, treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast, the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells, primary breast epithelial cells, nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics.