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Astanehe, Arezoo. "Role of Y-box binding protein-1 (YB-1) in breast cancer." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/36668.
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The Y-box binding protein-1 (YB-1) is a multifunctional protein with roles in transcription, translation, DNA repair, and a recently identified function as an extracellular mitogen. YB-1 is over-expressed in various malignancies including breast carcinoma. Previous work from our laboratory has shown that YB-1 is expressed in approximately 40% of invasive breast carcinomas, and its expression correlates with relapse and poor survival. Further, the oncogenic potential of YB-1 has been demonstrated in breast cancer. In the studies presented in this thesis, we sought to understand the contribution of YB-1 as an oncogenic transcription factor to breast cancer. We focused our studies on the basal-like breast carcinoma (BLBC) and the human epidermal growth factor receptor 2 (HER2) over-expressing breast cancers, as patients with these subtypes suffer the worst prognosis. Using BLBC cell lines, we demonstrated that YB-1 induces expression of MET and PIK3CA to promote anchorage-independent growth and invasion respectively. These studies further identified YB-1 as a potential therapeutic target in BLBC. We then directed our focus to the HER2 over-expressing breast cancers. Although the development of trastuzumab (Herceptin®), a targeted therapy against HER2, has provided a substantial advance in the care of affected patients, resistance remains a prevailing challenge. We identified a novel mechanism by which signalling proteins, mitogen activated protein kinase interacting kinase (MNK) and p90 ribosomal S6 kinase (RSK), interact to increase phosphorylation of YB-1. In turn, phosphorylation of YB-1 promotes its nuclear translocation where it regulates transcription of genes involved in trastuzumab resistance. These results further suggest YB-1 as a therapeutic target to improve outcome for women with trastuzumab refractory disease. As a whole, the studies outlined in this thesis have contributed to our understanding of breast cancer pathogenesis and have identified novel aspects of YB-1 function in BLBC and in HER2 over-expressing breast carcinomas.
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EL-Naggar, Amal Mohammad. "Investigation into the role of Y-box binding protein-1 (YB-1) in childhood sarcomas." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45665.
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Davies, Alastair Henry. "Dissecting the function of Y-box binding protein-1 (YB-1) during the development of breast cancer." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44202.
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The series of events that trigger the transformation of a normal cell into a malignant cell are unclear. One prospective driver of tumourigenesis is Y-box binding protein-1 (YB-1). BLG-YB-1 transgenic mice all form mammary tumours and the protein is expressed in over 40% of breast carcinomas. The studies presented in this thesis aimed to uncover the function of YB-1 during the transformation process. To this end, we conditionally expressed YB-1 in normal human mammary epithelial cells (HMECs). In agreement with tumours from the transgenic mice, genomic instability was manifested in the form of numerical and structural chromosomal abnormalities. To query the mechanism responsible for these phenotypes, we assessed global changes in signal transduction using an antibody microarray. Notably, we identified an increase in LIM kinase (LIMK1/2) activity that acted as a catalyst for cytokinesis failure. Subsequent cell cycle checkpoint slippage, due to cyclin E over-expression, potentiated centrosome amplification leading to mitotic spindle abnormalities. The resulting genomic instability was not stochastic but rather it increased susceptibility to cancer by promoting low-level HER2 amplification, as one example. Deeper interrogation revealed that YB-1 was capable of fully transforming HMECs. Through stabilization and upregulation of the histone acetyltransferase p300, YB-1 reprogrammed HMECs into stem/progenitor-like tumour-initiating cells (TICs). Mechanistically, the relaxation of promoter-centered chromatin permitted YB-1 to bind and transcriptionally regulate the TIC-associated genes BMI1, CD44, and CD49f. These cells formed DCIS-like luminal outgrowths in a three-dimensional model of breast acini. Over time, pressures exerted by YB-1 led to the emergence of cells expressing RSK2 and hTERT that had the capacity to form tumours in vivo. These cells were subtyped as triple-negative breast cancer (TNBC), a particularly aggressive form of the disease that is prone to relapse. We discovered that YB-1 regulates the multidrug resistance transporter ABCG2 to render the CD44⁺/CD49f⁺ TIC subpopulation refractory to traditional chemotherapy. However, these cells were responsive to RSK inhibitors, which prevent the activation of YB-1. As a whole, the studies outlined in this thesis indicate that YB-1 facilitates the genesis of TNBC through epigenetic reprogramming and targeting it has the potential to overcome drug resistance and prevent tumour recurrence.
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Lee, Cathy. "Y-box binding protein-1 (YB-1) is essential for the growth and survival of HER-2 over-expressing breast cancer cells." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/220.
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The human epidermal growth factor receptor (HER-2) is over-expressed in 20-30% of breast carcinomas and is a prognostic marker for poor patient outcome. We previously identified the transcription/translation factor Y-box binding protein-1 (YB-1) to be a novel substrate of AKT which binds to epidermal growth factor receptor (EGFR) and HER-2 promoters once phosphorylated (Wu J et al. 2006). YB-1 is over-expressed in approximately 40% of breast cancers; its expression is strongly correlated with HER-2 and is associated with poor patient survival. In order to gain a deeper understanding of the functional role of YB-1 in HER-2 over-expressing breast cancer, we silenced the expression of this factor in BT474-m1 and MDA-MB-453 cells. The loss of YB-1 inhibited the growth of BT474-m1 and MDA-MB-453 cells in monolayer and/or in soft agar. Consistent with this, we found a decrease in the expression of YB-1 responsive gene egfr and/or her-2 in BT474-m1 and MDA-MB-453 cells, which could begin to explain how growth is promoted by this factor. Furthermore, loss of YB-1 expression induced apoptosis in BT474-m1 cells. Beyond its role in tumor growth, YB-1 is also strongly linked to drug resistance. We therefore addressed whether it could play a part in Herceptin sensitivity. Herceptin is currently being used to treat patients with HER-2 positive breast cancer; however, only 30% of the patients respond to the therapy and many of them develop resistance within the first year of treatment. Therefore, it is of utmost importance to understand the biology of HER-2 over-expressing breast cancer to develop novel therapies that can benefit more patients. First we established that Herceptin inhibited BT474-m1 cell growth in anchorage-independent conditions whereas MDA-MB-453 cells were resistant to this treatment. We subsequently demonstrated that knock-down of YB-1 increased sensitivity of BT474-m1 cells to Herceptin while MDA-MB-453 cells failed to respond to the combination treatment. The mechanism for Herceptin resistance in MDA-MB-453 cells still remains elusive and requires further investigation. Thus far, we conclude that YB-1 is needed for the growth and survival of HER-2 positive BT474-m1 and MDA-MB-453 breast cancer cells by inducing members of the HER family.
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Mahomedy, Tephney Gladness. "Investigation of the Stress-Induced Nuclear Localisation of Retinoblastoma Binding Protein 6 (Rbbp6) and its Role in Ubiquitination Of Y-Box Binding Protein-1 (Yb-1)." University of the Western Cape, 2017. http://hdl.handle.net/11394/5900.
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Magister Scientiae - MSc (Biotechnology)Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa RING finger-containing human protein known to serve as an E3 ubiquitin ligase, and to play a role in ubiquitination and suppression of the tumour suppressor p53. It also regulates the stability of mRNA transcripts by modulating 3'-polyadenylation.
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Habibi, Golareh. "Y-box binding protein-1 (YB-1) is a bio-marker of aggressiveness in breast cancer and is a potential target for therapeutic intervention." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/911.
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Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America.
YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells.
We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer.
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Alidousty, Christina [Verfasser]. "Die Regulation der CCL5-Expression in der Monozytendifferenzierung durch post-translationale Modifikation des Y-Box Binding Protein-1 (YB-1) / Christina Alidousty." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2015. http://d-nb.info/107100820X/34.
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Chan, Man Kid. "The interaction between Y box binding protein 1 and DNA replication proteins." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66802.
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A coordinated response to DNA damage is vital to maintain cellular viability and prevent the onset of disease. In mammalian cells, the intra-S phase checkpoint regulator, ATR (Ataxia-telangiectasia mutated and RAD3-related) kinase coordinates the response to DNA damage to ensure the genome is accurately and completely replicated before the cell enters mitosis. Y box Binding Protein 1 (YB-1), a transcription and translation factor, has previously been implicated in cell proliferation and the development of chemotherapeutic resistance. YB-1 has been linked to a wide variety of cellular stresses, but has not been studied in the context of DNA replication. In this study, we determined that YB-1 associates to both the β-globin replication origin (origin-containing) and the control (origin-lacking) DNA regions. This observation suggested that YB-1 may be involved in DNA replication elongation instead of initiation. By immunoprecipiating YB-1, we identified that PCNA and MCM7 preferentially interact with YB-1 during S phase. The examination of the spatial and temporal dynamics of these interactions by immunofluorescence microscopy, however, did not reveal nuclear colocalization of these proteins. By treating cells with hydroxyurea to stall the replication fork, we re-examined the protein-protein interaction between YB-1 and MCM7 and found that following 8 hours of hydroxyurea treatment, YB-1 and MCM7 exhibited diffuse colocalization in the cell nucleus. This finding may implicate YB-1 in exerting a late-onset response to prolonged replication fork arrest either directly at stalled replication forks or at "dormant" origins bound by MCM complexes. A number of roles for YB-1 can be postulated, such as the requirement of YB-1 in facilitating the resumption of DNA replication, or the activation of additional origins to duplicate the genome in the presence of a replication stress. This finding may in turn account foUne réponse coordonnée lors de dommages à l'ADN est vitale pour le maintien de la viabilité cellulaire et pour éviter l'installation de maladies. Dans les cellules de mammifères, le point de contrôle de la phase S, la protéine kinase ATR (Ataxia-telangiectasia mutated and RAD3-related), coordonne la réponse aux dommages de l'ADN afin d'assurer une réplication complète et fidèle du génome avant l'entrée en mitose. La protéine YB-1 (Y box Binding Protein 1), un facteur de transcription et de traduction, est impliqué dans la prolifération cellulaire et la résistance aux chimiothérapies. YB-1 est également lié à une large variété de stress cellulaires but aucune donnée n'est disponible quant à son rôle dans la réplication de l'ADN. Lors de mon travail de Master, j'ai pu montrer qu'YB-1 s'associe à la fois à l'origine de réplication de la β-globine et dans les régions contrôles de l'ADN. Ce résultat suggère qu'YB-1 pourrait être impliqué dans la phase d'élongation de la réplication de l'ADN plutôt que dans celui de l'initiation. Par immunoprécipitation, j'ai identifié PCNA et MCM7 comme interacteurs préférentiels d'YB-1 en phase S. Par contre, en immunofluorescence, je n'observe pas de colocalisation nucléaire entre ces protéines. Lors du blocage de la fourche de réplication par un traitement à l'hydroxyurée, l'interaction entre YB-1 et MCM7 a été réexaminée et j'ai mis en évidence que 8h après le traitement, ces deux protéines présentent une co-localisation diffuse dans le noyau. Ces données indiquent qu'YB-1 pourrait être impliqué dans une réponse tardive suite à un arrêt prolongé de la fourche de réplication soit directement au point d'arrêt soit au niveau d'origines « dormantes » liées aux complexes MCM. YB-1 peut donc avoir plusieurs rôles tels que l'aide à la reprise de la réplication de l'ADN ou l'activation d'origines de réplicatio
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Khandelwal, Payal. "The N-terminal domain of the Y-box binding protein YB-1 plays a major role in cell proliferation and apoptosis." View the abstract Download the full-text PDF version, 2008. http://etd.utmem.edu/ABSTRACTS/2008-016-Khandelwal-index.html.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008.Title from title page screen (viewed on July, 31, 2008). Research advisor: Ramareddy V. Guntaka, Ph.D. Document formatted into pages (xiii, 111 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 90-111).
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Nashchekin, Dmitri. "A Y-box protein/RNA helicase complex links mRNP assembly on the gene to mRNA translation /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-811-8/.
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Reipas, Kristen May. "Inhibiting p90 ribosomal S6 kinase (RSK)/Y-box binding protein-1 (YB-1) signaling is a novel targeted therapeutic strategy with the ability to overcome drug resistance in triple-negative breast cancer." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44616.
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Despite advances in treating breast cancer, disease recurrence rates remain high and secondary tumors are often refractory to chemotherapy. Currently, the treatment for triple-negative breast cancer (TNBC) relies upon conventional chemotherapeutics as no targeted therapies are 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) and associated with disease relapse. It is activated predominantly by phosphorylation via p90 ribosomal S6 kinase (RSK). Once activated YB-1 up-regulates the tumor-initiating cell (TIC) marker, CD44 and promotes drug resistance. These data suggest that blocking YB-1’s activation via RSK inhibition may suppress growth and attenuate the development of chemoresistance in TNBC.
Through an unbiased, functional viability screen comparing breast cancer subtypes, we identified RSK2 as a novel target for TNBC. Pharmacological or siRNA inhibition of RSK2 blocks activation of YB-1, which subsequently decreases growth in TNBC cell lines and delays tumor initiation in immunocompromised mice. Contrary to most conventional chemotherapies, inhibiting RSK/YB-1 signaling eliminates the CD44⁺/CD24‾ cell fraction rather than enriching for it. In an effort to identify novel RSK inhibitors, we screened “off-patent” compounds and identified the flavonoid, luteolin, as a RSK inhibitor. We validated that luteolin inhibits RSK in cell-free assays and further demonstrated it blocks the RSK/YB-1/Notch4 signaling pathway. Luteolin phenotypically mirrored the effects of established RSK inhibitor, BI-D1870, and suppressed growth in TNBC (including CD44⁺/CD24‾-sorted cells) providing further support for the use of RSK inhibitors to treat this subtype. Finally, we demonstrate that cells that survive standard-of-care chemotherapeutics (paclitaxel and epirubicin) exhibit elevated RSK/YB-1 signaling. Inhibiting this pathway sensitizes TNBC to chemotherapy and reduces the residual cell burden. Importantly, RSK inhibition also demonstrates efficacy against a multidrug resistant cell line and primary, drug-refractory TNBC. When taken together, our data identify RSK as a promising target for the treatment of TNBC. RSK inhibition has the unique ability to eliminate CD44⁺/CD24‾cells and overcome broad-spectrum chemoresistance by blocking activation of YB-1 and as such holds potential to reduce relapse in this aggressive subtype.
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Budkina, Karina. "The role of an mRNA-binding protein YB-1 in formation of stress granules and translation." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL006.
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Au cours de la vie de l'ARNm dans la cellule, l'ARNm existe en complexe avec des protéines et n'est jamais libre. Dans le cytoplasme, l'ARNm actif est associé aux ribosomes pour former les polyribosomes tandis que les ARNm réprimés s’associent avec certaines protéines de liaison à l'ARN (RBP) pour former des mRNP. Les mRNP réprimés sont généralement isolés dans le cytoplasme mais ils peuvent également être trouvés dans des compartiments appelés granules d’ARN, notamment lors d'un stress cellulaire. Ces granules d’ARN sont des organelles non membranaires contenant principalement de l'ARNm inactif et coexistent avec des polysomes. Selon les conditions environnementales, il y a un changement dans le ratio des ARNms trouvés dans les granules d’ARN ou dans les polysomes. De plus, il existe des différences dans la teneur en ARNm des différents types de ces organelles en fonction de leur localisation et de leurs fonctions. Actuellement, les granules de stress présentent un grand intérêt pour les chercheurs en raison de leur relation avec certaines maladies neurologiques. Les mutations trouvées dans certaines protéines de liaison à l'ARN telles que TDP43 et FUS sont directement liées à certaines maladies neurodégénératives telles que la sclérose latérale amyotrophique (SLA), la démence frontotemporale (FTLD) et la maladie d'Alzheimer (MA). Dans les neurones affectés, TDP-43 et FUS forment des agrégats cytoplasmiques alors que ces protéines se trouvent généralement dans le noyau dans des conditions physiologiques. Comme elles ont également été trouvées dans les granules de stress cytoplasmiques, les granules de stress peuvent servir d'intermédiaires pour la formation d'agrégats de FUS et TDP-43. En outre, FUS et TDP-43 contiennent des régions intrinsèquement désordonnées (IDR) qui contribuent à leur agrégation.La formation de granules de stress est stimulée par l'exposition à différents facteurs internes et / ou externes. Les granules de stress servent de lieu de stabilisation des ARNm et à les maintenir inactifs jusqu'à ce que les facteurs de stress disparaissent. On considère que les structures secondaires de l'ARNm jouent un rôle important dans l'assemblage des granules de stress. De telles structures servent aussi de sites de liaison pour les RBP, qui les stabilisent davantage (par exemple G3BP). La protéine de liaison Y-box 1 (YB-1) a également été identifiée comme un marqueur pour les granules de stress. YB-1 est une protéine de liaison à l'ARN qui accompagne l'ARNm dès sa synthèse dans le noyau jusqu’à sa dégradation dans le cytoplasme. YB-1 contient un domaine de choc froid (CSD) avec deux motifs de reconnaissance d'ARN (RNP-1 et RNP-2), ainsi qu'un domaine CTD non structuré similaire aux IDR. Pour la plupart des protéines impliquées dans la formation des granules de stress, leur activité stimulante de l'IDR dans ce processus a été démontrée. Dans le même temps, il existe quelques controverses concernant le rôle de YB-1 dans l'assemblage des granules de stress. Selon certains modèles, il y a lieu de le considérer comme un régulateur négatif dans la formation des granules de stress. Selon d'autres, YB-1 présente les propriétés d'un agent favorisant de l'assemblage de granules de stress. Par ailleurs, peu de travaux ont n'a été faits pour déchiffrer l'action de la protéine sur la traduction sous stress oxydatif. Ici, notre objectif était de démêler les mécanismes structuraux par lesquels YB-1 peut réguler négativement la formation de granules de stress et de clarifier son influence sur la traduction dans des conditions de stressDuring mRNA life in cell mRNA exists in complex with proteins and is never free. In the cytoplasm, active mRNA is associated with ribosomes to form polyribosomes while repressed mRNAs in association with RNA-binding proteins forms mRNPs. Repressed mRNPs are generally isolated in the cytoplasm but they can also be found in compartments called mRNP granules, notably during cellular stress. Such mRNP granules are non-membrane organelles contains mostly translationally inactive mRNA and coexist with polysomes. Depending on the environmental conditions, there is a change in the ratio of mRNA found in these types of granules or in polysomes. In addition, there are differences in the mRNA content of the different types of such organelles depending on their localization and functions. Currently, stress granules are of great interest to researchers due to their relation to some neurological diseases. Mutations of some RNA-binding proteins such asTDP43 and FUS are directly linked to some neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTLD), and Alzheimer's disease (AD). In the affected neurons, TDP-43 and FUS form cytoplasmic aggregates while these proteins are generally found in the nucleus under physiological conditions. As they were also found in cytoplasmic stress granules, stress granules may serve as intermediates for the formation of FUS and TDP-43 aggregates. In addition, FUS and TDP-43 contain intrinsically disordered regions (IDRs) which contribute to their aggregation. The formation of stress granules is stimulated by exposure to different internal and/or external factors. Stress granules serve as a place for mRNA stabilization and keeping it inactive until stress factors disappear. It is considered that secondary structures of mRNA play a significant role in the assembly of stress granules. Such structures serve as binding sites for RBPs, which further stabilize them (e.g. G3BP). The Y-box binding protein 1 (YB-1) was also identified as a marker for stress granules. YB-1 is an RNA-binding protein that accompanies mRNA from its synthesis in the nucleus to degradation in the cytoplasm. YB-1 contains a cold shock domain (CSD) with two RNA-recognition motifs (RNP-1 and RNP-2), as well as an unstructured CTD domain similar to IDRs. For most of the proteins involved in the formation of stress granules, their stimulating activity of IDR in this process has been shown. At the same time, there are some controversies regarding the role of YB-1 in the assembly of granules. According to some sources, there is reason to consider it as a negative regulator. According to others, YB-1 exhibits the properties of an inducer during the assembly of stress granules. At the same time, no attempts were made to decipher the mechanism of action of the protein under oxidative stress.Here our aim was to unravel the structural mechanisms by which YB-1 can negatively regulate the formation of stress granules and to clarify its influence on translation in stress conditions
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To, Karen Ka-Yan. "Y-box binding protein-1 induces MET, CD44, and CD49f thereby promoting the capacity for tumour initiation." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/13392.
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Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where its expression correlates with enhanced tumour cell growth and drug resistance. YB-1 is also significantly associated with disease recurrence, suggesting links to tumour-initiating cells (TICs). We recently reported that YB-1 binds to the promoters of genes associated with a stem/progenitor-like phenotype through a chromatin immunoprecipitation (ChIP)-on-chip screen, including genes encoding the MET receptor, CD44, and CD49f. We first confirmed that YB-1 induces oncogene MET by activating its promoter. Further, both YB-1 and MET expression were detected in purified populations of primary human mammary progenitor cells. Similarly, we confirmed that YB-1 binds the gene promoters of reported TIC markers CD44 and the stem cell marker CD49f (α6 integrin) using conventional ChIP. Human breast TICs, reportedly CD44⁺/CD24⁻/low, self-renew, grow in mammospheres, and may evade current drug therapies, leading to increased relapse rates in patients. We observed that P-YB-1S102 co-localized to a CD44High breast cancer cell subpopulation with increased growth capacities. Silencing YB-1 down-regulated MET, CD44 and CD49f, while overexpression of wild-type (YB-1WT) or a constitutively activated form (YB-1S102D) increased MET, CD44 and CD49f transcripts and proteins. Consistent with these findings, the mammary glands of YB-1 transgenic mice had elevated CD44 and CD49f protein with associated hyperplasia. Moreover, the expression of YB-1S102D in SUM 149 cells, enhanced mammosphere formation and growth in soft agar. Conversely, silencing either CD44 or CD49f in these cells reversed YB-1S102D enhanced growth. Consistent with a role for TICs in relapse, Paclitaxel activated YB-1 and this correlated with induced CD44. Further, expression of YB-1WT enhanced mammosphere growth in the presence of Paclitaxel. Taken together, our data suggests that YB-1 induces MET, CD44, and CD49f expression which may be a part of the mechanism used to enhance tumour cell growth, disease relapse, and drug resistance.
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Geiger, Katharina [Verfasser], and Ralf C. [Gutachter] Bargou. "Etablierung eines Vektorsystems zum shRNA-vermittelten Knockdown von Y-box binding protein 1 / Katharina Geiger ; Gutachter: Ralf C. Bargou." Würzburg : Universität Würzburg, 2017. http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149809.
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Tiwari, Aadhya [Verfasser]. "Role of Y-Box Binding Protein-1 mediated cell signaling in proliferation and radiotherapy resistance of breast cancer cells / Aadhya Tiwari." Tübingen : Universitätsbibliothek Tübingen, 2021. http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-937376.
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Dhillon, Jaspreet. "Y-box binding protein-1 is essential for the growth and survival of HER2 over-expressing breast cancers and mediates trastuzumab resistance by inducing CD44." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/24372.
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Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in 40% of all subtypes of invasive breast carcinomas, where its expression is correlated with relapse and poor survival. HER2 amplifications are a frequent genetic abnormality observed in approximately 25% of breast cancers where its over-expression is associated with poor clinical outcome and decreased disease free survival. We recently reported that HER2 over-expressing breast cancers are dependent on YB-1 for growth and survival. In HER2 positive tumours we implicated YB-1 in sustaining cancer cells by its involvement in the STAT3 signalling pathway. The development of trastuzumab, a targeted therapy against HER2, has provided substantial advances in the care and treatment of patients whose tumours over-express HER2. Unfortunately, the development of acquired resistance to trastuzumab remains a prevalent challenge in the treatment of patients whose tumours express HER2. Since YB-1 is also linked to drug resistance in other types of cancer, we addressed its possible role in trastuzumab insensitivity. Employing an in vivo model of acquired resistance, we demonstrated that resistant cell lines have elevated levels of P-YB-1S¹°² and its activating kinase P-RSK and that these levels are sustained following trastuzumab treatment. Further, to demonstrate the importance of YB-1 in mediating drug resistance, the expression of the active mutant YB-1S¹°²D rendered the BT474 cell line insensitive to trastuzumab. Questioning the role of tumour initiating cells (TICs) and their ability to escape cancer therapies, we investigated YB-1’s involvement in inducing the cancer stem cell marker CD44. Notably, the resistant cells expressed more CD44 mRNA and protein compared to BT474 cells, which correlated with increased mammosphere formation. Expression of YB-1S¹°²D in the BT474 cells increased CD44 protein levels, resulting in enhanced mammosphere formation. Further, exposing BT474 cells to trastuzumab selected for a resistant subpopulation enriched for CD44. Conversely, siRNA inhibition of CD44 restored trastuzumab sensitivity in the resistant cell lines. Our findings provide insight on a novel mechanism employed by tumour cells to acquire the ability to escape the effects of trastuzumab and suggest that targeting YB-1 may overcome resistance by eliminating the unresponsive TIC population, rendering the cancer sensitive to therapy.
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Shamsuddin, S. "Biochemical characterization of the interactions between a transcription factor, CTCF and its partners Y-Box binding protein-1, and the large subunit of RNA polymerase II." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269484.
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Breitkopf, Daniel Maximilian [Verfasser], Ute Akademischer Betreuer] Raffetseder, and Ralph [Akademischer Betreuer] [Panstruga. "Notch-3 and a posttranslational modification of Y-box binding protein 1 alter the immune cell balance and the pathophysiology in systemic lupus erythematosus / Daniel Breitkopf ; Ute Raffetseder, Ralph Panstruga." Aachen : Universitätsbibliothek der RWTH Aachen, 2020. http://d-nb.info/1217789650/34.
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Jürchott, Karsten. "Untersuchungen zur subzellulären Lokalisation und zu den Funktionen von YB-1, einem Y-Box-Protein in Säugerzellen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 1999. http://dx.doi.org/10.18452/14569.
Full textAbstract:
YB-1, ein Y-Box-Protein in Säugerzellen, konnte sowohl im Zytoplasma als auch in den Zellkernen von HeLa-Zellen nachgewiesen werden. Es wurde eine Abhängigkeit der intrazellulären Lokalisation von YB-1 vom Verlauf des Zellzyklus beobachtet. In jeder Phase des Zellzyklus war YB-1 im Zytoplasma zu finden. Eine Kernlokalisation von YB- 1 konnte nur in den HeLa-Zellen festgestellt werden, die sich im Übergang von der G1- in die S-Phase oder in der frühen S-Phase des Zellzyklus befanden. Die Abhängigkeit der Lokalisation von YB-1 vom Verlauf des Zellzyklus unterstützt die These, daß YB-1 und andere Y-Box-Proteine an der Regulation der Zellproliferation beteiligt sind. Es wurden verschiedene Proteine identifiziert, die im Zytoplasma von HeLa-Zellen mit YB-1 assoziiert vorkommen. Alle identifizierten Proteine erfüllen Aufgaben im RNA-Metabolismus, was auf eine Beteiligung dieser Proteinkomplexe an der Regulation der mRNA hinweist. Die Interaktion von P32/SF2 (P35) mit YB-1 erwies sich als abhängig vom Zellzyklus, wobei eine maximale Assoziation dieser beiden Proteine beim Übergang der HeLa-Zellen von der G1- in die S-Phase zu beobachten war. In Multidrug-resistenten MCF7/ADR-Zellen konnte eine deutlich verstärkte Interaktion von P32/SF2 mit YB-1 im Vergleich zu den sensitiven MCF7-Zellen festgestellt werden. Im Zytoplasma von HeLa-Zellen konnte YB-1 in Verbindung mit membrangebundenen Polysomen nachgewiesen werden. Eine Assoziation von YB-1 mit freien oder zytoskelettgebundenen Polysomen konnte nicht festgestellt werden. Damit wurde erstmalig gezeigt, daß YB-1 eine Spezifität für eine bestimmte Gruppe von Polysomen besitzt. Die Assoziation mit membrangebundenen Polysomen legte die Vermutung nahe, daß YB-1 an der Translationskontrolle von Polypeptiden beteiligt ist, die am rauhen endoplasmatischen Retikulum synthetisiert werden. Es konnte gezeigt werden, daß YB-1 die Translation von P-Glykoprotein, einem integralen Membranprotein, positiv reguliert. Ein Einfluß auf die Translation der untersuchten sekretorischen Proteine (a-Faktor und Präprolactin) konnte nicht beobachtet werden. Diese Ergebnisse belegen, daß YB-1 ein spezifischer Regulator der Translation bestimmter Membranproteine ist. Am Hand von P-Glykoprotein konnte des weiteren demonstriert werden, daß YB-1 sowohl die Transkription als auch die Translation dieses Proteins positiv reguliert. Die in den Zellkulturen beobachtete Korrelation von YB-1 mit der Expression von P-Glykoprotein konnte auch in primären Mammakarzinomen nachgewiesen werden. Somit ist YB-1 ein entscheidender Faktor bei der Ausbildung einer intrinsischen multiplen Resistenz von Mammakarzinomen gegen die Behandlung mit Chemotherapeutika. Aus diesem Grunde könnte YB-1 einen Ansatzpunkt für die künftige Diagnose und Therapie von Mammakarzinomen und eventuell auch von anderen Tumoren bieten.YB-1, a mammalian Y-box protein was detected in the cytoplasm as well as in the nuclei of HeLa cells. The intracellular localisation of YB-1 depends on the cell cycle. In every part of the cell cycle, YB-1 was found in the cytoplasm. A nuclear localisation of YB-1 was only detectable in the G1- to S-phase transition and in the early S-phase. These observations underline the hypothesis, that Y-box proteins are envolved in the regulation of cell proliferation. Different proteins interacting with YB-1 were identified in the cytoplasm of HeLa cells. All identified poteins are envolved in the RNA metabolism, indicating a role of these protein complexes in the regulation of mRNA. The interaction of P32/SF2 (P35) with YB-1 alternates during the cell cycle with a maximum at the G1- to S-phase transition. A remarkable increase of the association of YB-1 and P32 was observed in the multidrug-resistant MCF7 cells compared with the parental cell line. Furthermore, YB-1 was detected in association with membrane-bound polysomes, suggesting a role of YB-1 at the translational regulation of the synthesis of polypeptides at the rough endoplasmic reticulum. It was shown, that YB-1 stimulate the translation of P-glycoprotein. This influence is specific, beause the translation of a set of control proteins (alpha factor, preprolactin, luciferase) was not effected by YB-1. It was shown, that YB-1 stimulate the expression of P-glycoprotein at the level of transcription as well as at the level of translation. This indicates a central role of YB-1 in the regulation of the biosynthesis of this protein. The correlation of the nuclear expression of YB-1 and the expression of P-glycoprotein was demonstrated in primary breast cancers. Taken together, YB-1 is a important factor for the development of a resistant phenotyp and therefore a possible new target for anti-cancer therapy.
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Jürchott, Karsten. "Untersuchungen zur subzellulären Lokalisation und zu den Funktionen von YB-1, einem Y-Box-Protein in Säugerzellen." [S.l. : s.n.], 1999. http://deposit.ddb.de/cgi-bin/dokserv?idn=961443294.
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Schmidt, Anja. "Das humane Y-Box-Protein YB-1 und seine Bedeutung für die Prognose und den Therapieerfolg beim Mammakarzinom." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=970189966.
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Schmidt, Anja. "Das humane Y-Box-Protein YB-1 und seine Bedeutung für die Prognose und den Therapieerfolg bei Mammakarzinom." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2003. http://dx.doi.org/10.18452/14995.
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Einer der Gründe für das Scheitern derzeitiger Behandlungsmethoden beim Brustkrebs ist die Resistenz gegenüber der angewandten Chemotherapie. Eine große Rolle bei der Entstehung der Multiplen Medikamentenresistenz spielt das MDR1-Gen und sein Genprodukt, das P-Glykoprotein. Das Y-Box-Protein YB-1 reguliert die Expression des MDR1-Gens; eine Überexpression und nukleäre Lokalisation von YB-1 geht im Brustkrebs mit einer gesteigerten P-Glykoprotein Expression einher. In dieser Arbeit wurden Gewebeproben von 83 Brustkrebspatientinnen auf eine YB-1 Überexpression im Tumor und im peritumoralen Epithel untersucht. YB-1 wurde mittels der immunhistochemischen APAAP-Methode an Formalin-fixierten, in Paraffin eingebetteten Brustkrebsgewebeproben nachgewiesen. Die klinische Relevanz der YB-1 Expression wurde untersucht, indem sie mit dem klinischen Verlauf in einem mittleren Beobachtungszeitraum von 61 Monaten und etablierten biologischen Tumorfaktoren wie Lymphknotenstatus, histologisches Grading, Tumorgröße, Hormonrezeptorstatus, uPA und PAI-1 verglichen wurde. In der Kohorte der Patientinnen mit einer postoperativen adjuvanten Chemotherapie zeigte sich eine 5-Jahres-Rezidivrate von 68 % bei einer hohen YB-1 Expression im Tumor und eine Rückfallrate von 39 % bei einer niedrigen YB-1 Expression. Unter Beachtung auch der YB-1 Expression im peritumoralen Epithel konnte ein noch größerer Unterschied hinsichtlich der 5-Jahres-Rezidivrate festgestellt werden. Diese betrug bei Patientinnen mit einer hohen YB-1 Expression 66 %, während bei Patientinnen mit einer niedrigen YB-1 Expression im Nachbeobachtungszeitraum kein Rezidiv festgestellt wurde. Bei der Gegenüberstellung der 5-Jahres-Rezidivraten in der Kohorte der Patientinnen ohne Zytostatikatherapie zeigte sich eine Rückfallrate von 30 % bei einer hohen YB-1 Expression und eine Rückfallrate von 0 % bei einer niedrigen YB-1 Expression. Eine hohe YB-1 Expression war demnach in beiden Kohorten mit einer schlechteren klinischen Prognose assoziiert. Das Ergebnis in der Gruppe der Patientinnen ohne postoperative Chemotherapie zeigt, dass YB-1 mit der Tumoraggressivität beim Brustkrebs korreliert. Eine Korrelation zwischen der YB-1 Expression und den etablierten prognostischen Faktoren Lymphknotenstatus, Tumorgröße und histologisches Grading konnte nicht festgestellt werden. Es wurde jedoch eine signifikante negative Korrelation zwischen der YB-1 Expression und dem Hormonrezeptorstatus und eine positive Korrelation zwischen YB-1 und den Faktoren uPA und PAI-1 gefunden. In dieser Arbeit wurde gezeigt, dass YB-1 eine klinische Relevanz besitzt mit Hinblick sowohl auf eine prognostische als auch eine prädiktive Bedeutung bei der Identifikation von Hoch-Risiko-Patientinnen im Brustkrebs in Ab- und Anwesenheit einer postoperativen Chemotherapie.Intrinsic or acquired resistance to chemotherapy is one of the reasons for failure of current treatment regimens in breast cancer patients. P-glycoprotein and its gene mdr1 plays a major role in the development of a multi-drug resistant tumor phenotype. The Y-box protein YB-1 regulates the expression of mdr1. In human breast cancer, overexpression and nuclear localization is associated with upregulation of P-glycoprotein. In this study, tissues of 83 breast cancer patients have been analyzed with regard to YB-1 overexpression in tumor tissue and in surrounding benign breast epithelial cells. YB-1 has been detached by the immunohistochemical APAAP-method using formalin-fixed, paraffin-embedded breast cancer tissues. Clinical relevance of YB-1 expression was analyzed by comparing it with clinical outcome after a median follow-up of 61 months and with tumor biological factors lymph-node status, tumor size, histological grading, hormone-receptor status and the factors uPA and PAI-1. In patients who received postoperative chemotherapy, the 5-year-relapse rate was 68% in patients with high YB-1 expression in tumor cells and 39% in patients with low expression. With regard to YB-1 expression in surrounding benign breast epithelial cells, the 5-year-relapse rate was 66% in patients with high YB-1 expression whereas in patients with low expression no relapse has been observed so far. YB-1 thus indicates clinical drug resistance in breast cancer. In patients who received no chemotherapy, the 5-year-relapse rate was 30% in patients with high YB-1 expression whereas in patients with low YB-1 expression no relapse occurred. YB-1 thus correlates with breast cancer aggressiveness. In both groups high YB-1 expression was associated with poor clinical outcome. A correlation between YB-1 and tumor biological factors lymph-node status, tumor size and histological grading has not been found. But a significant negative correlation has been observed between YB-1 and hormone-receptor status and a positive correlation between YB-1 and uPA and PAI-1. This dissertation could show the clinical relevance of YB-1 with regard to a prognostic and predictive significance by identifying a high-risk group of breast cancer patients both in presence and absence of postoperative chemotherapy.
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Kantola, Angeline R. "Nuclear magnetic resonance studies of the xUBF Box 1 DNA binding domain /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9228.
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Martin, Daniel. "X-Box Binding Protein-1 is important in maintenance of endothelial integrity and migration." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/xbox-binding-protein1-is-important-in-maintenance-of-endothelial-integrity-and-migration(8390a526-312b-4586-ac18-5f9add13e180).html.
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Background - Sustained activation of spliced x-box binding protein-1 (XBP1), an endoplasmic reticulum stress response transcription factor, results in the development of atherosclerosis in apoE -/- mice. Histone deacetylases (HDACs) play a crucial role in transcriptional regulation through modulation of chromatin structure. In particular, HDAC3 is involved in maintaining endothelial integrity. HDAC3 and XBP1 are similarly expressed in the bifurcation regions of aorta. Unspliced XBP1 (XBPlu) is expressed in a similar manner in endothelial cells. In the present study we investigated the role of XBPlu and XBPls in the maintenance of endothelial integrity and endothelial cell migration. Furthermore, the crosstalk between HDACS and XBP1 signalling pathways was examined. Methods and Results - Our study demonstrated that disturbed flow upregulated HDACS and XBPlu protein production through the VEGF receptor 2 / PI-3-kinase pathway. Knockdown of XBP1 by shRNA lentiviral transfection ablated disturbed flow-induced HDAC3 upregulation. Similarly to HDACS, overexpression of XBPlu by adenoviral gene transfer increased Akt phosphorylation at serine 473 and haem oxygenase 1 gene transcription, which showed a protective role in hydrogen peroxide-induced apoptosis of endothelial cells. Co-immunoprecipitation assays demonstrated that HDACS physically associates with XBPlu and Akt. The use of truncated HDACS constructs demonstrated that XBP1 binds to the central section of HDACS, which is predicted to contain a nuclear export signal. Furthermore, we identified a role for XBP1 in mediating endothelial cell migration. Overexpression of both XBPlu and XBPls enhanced the ability of endothelial cells to migrate. Ablation of XBP1 expression and XBP1 splicing, through knockdown of IRE la expression, reduced endothelial cell migration; however this was without a corresponding decrease in eNOS expression and NO production. Conclusions - These results suggest that XBPlu protects endothelial cells from oxidative stress, including that produced by disturbed flow. The interaction with HDAC3 could be crucial for this effect. The ratio between XBPlu and XBPls is also crucial for correct endothelial cell migration. Modulation of this balance and the interaction with HDAC3 may provide novel therapeutic strategies in vascular disease whilst maintaining endothelial integrity.
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Luce, Ryan A. "Investigations of DNA adducts of adriamycin and molecular interactions between DNA and xUBF Box 1 /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8608.
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Ye, Dewei, and 叶得伟. "Toll-like receptor-4 mediates obesity-induced nonalcoholic steatohepatitis through activation of X-box binding protein-1 in mice." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B47752919.
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Background and objectives:
Nonalcoholic steatohepatitis (NASH), which is characterized by concurrent
existence of hepatic steatosis and predominantly lobular necroinflammation, represents
the more advanced stage in the spectrum of nonalcoholic fatty liver disease (NAFLD).
NASH exhibits dramatically increased risk of progression to end-stage liver diseases
than simple steatosis. Therefore, the progression of hepatic steatosis to steatohepatitis is
the crucial step in the development of obesity-related NASH. Toll like receptor 4
(TLR4), a master regulator of innate immunity, is the principal receptor for endotoxin,
which is a central mediator of liver inflammation associated with both alcoholic and
nonalcoholic liver disease. However, due to a lack of suitable animal models which
fully recapitulate the natural history of obesity-induced NASH, the precise
pathophysiological function of TLR4 signaling in the development of this disease
remains poorly understood.
The objective of this study is to investigate the role of TLR4 in mediating
inflammatory responses in obesity-induced NASH using both in vivo and ex vivo
approaches, and to unveil cellular and molecular mechanisms responsible for TLR4
actions.
Key findings:
1. To address the role of TLR4 in the pathogenesis of NASH, we crossed ApoEdeficient
mice (ApoE-/-) with TLR4 mutant mice (TLR4-/-) to generate ApoE-/-
/TLR4 wild type mice (ApoE-/-/TLR4-WT) and ApoE-/-/TLR4-/- mice. Noticeably,
when fed with high fat high cholesterol (HFHC) diet, ApoE-/-/TLR4-WT mice
developed the typical pathology of NASH (hepatic steatosis, lobular inflammation,
and hepatocyte ballooning) in the context of obesity and metabolic syndrome,
suggesting HFHC-fed ApoE-/- mice as a suitable animal model for NASH.
2. TLR4 inactivation protected ApoE-/- mice against HFHC diet-induced liver injury,
as indicated by a significant improvement in liver histology, a a marked reduction
in serum ALT activity, a dramatic repression of inflammatory infiltrates, as well as
an obvious decrease in hepatic production of pro-inflammatory cytokines.
3. In ApoE-/-/TLR4-WT mice, TLR4 expression was selectively elevated in Kupffer
cells in response to HFHC diet feeding.
4. The activation of XBP1, a transcription factor involved in endoplasmic reticulum
stress, was markedly elevated in liver of ApoE-/-/TLR4-WT mice fed with HFHC
diet, whereas this change was abrogated in HFHC diet-fed ApoE-/-/TLR4-/- mice.
5. In rat primary Kupffer cells, treatment with anti-oxidants blocked endotoxininduced
activation of XBP1 and NF-κB, leading to decreased cytokine production.
In addition, siRNA-mediated knockdown of XBP1 inhibited NF-κB activation and
cytokine production resulted from the treatment with the TLR4 agonist LPS.
6. In ApoE-/-/TLR4-WT mice, adenovirus-mediated expression of dominant negative
XBP1 had no obvious effect on HFHC diet-induced hepatic steatosis and ROS
production, but markedly decreased lobular inflammation, NF-κB activation,
cytokine production in the liver and significantly reduced serum levels of ALT.
Conclusions:
These findings support the role of TLR4 in Kupffer cells as a key player in
mediating the progression of simple steatosis to NASH, by inducing ROS-dependent
activation of XBP1. In light of the obligatory role of XBP1 in TLR4-induced liver
inflammation and injury, therapeutic interventions that inhibit TLR4/XBP1 activation
may represent a promising strategy for treatment of NASH.published_or_final_version
Medicine
Doctoral
Doctor of Philosophy
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Jürchott, Karsten [Verfasser], Hans-Dieter [Gutachter] Royer, Harald [Gutachter] Saumweber, and Uwe [Gutachter] Heinemann. "Untersuchungen zur subzellulären Lokalisation und zu den Funktionen von YB-1, einem Y-Box-Protein in Säugerzellen / Karsten Jürchott ; Gutachter: Hans-Dieter Royer, Harald Saumweber, Uwe Heinemann." Berlin : Humboldt-Universität zu Berlin, 1999. http://d-nb.info/1207654159/34.
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du, Preez Marlene Geraldine. "Molecular analysis of red colouration in 'Bon Rouge' pear (Pyrus communis L.)." University of the Western Cape, 2018. http://hdl.handle.net/11394/6454.
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Philosophiae Doctor - PhD (Biotechnology)The 'Bon Rouge' pear cultivar was developed from a bud mutation on a 'Bon Chretien' pear tree. The latter is characterised by green fruit skin and leaves, while 'Bon Rouge' is characterised by red leaves and red fruit skin as a result of the production of anthocyanin and other pigments. Branch forming buds on 'Bon Rouge' trees often revert to the parent phenotype producing green leaves and fruit skin. The occurrence of both phenotypes on the same tree presents a unique model to study gene expression associated with anthocyanin production in a similar genetic background under the same set of environmental condition.
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Schmidt, Anja [Verfasser], H. [Gutachter] Höfler, H. [Gutachter] Oettle, and Hans-Dieter [Gutachter] Royer. "Das humane Y-Box-Protein YB-1 und seine Bedeutung für die Prognose und den Therapieerfolg bei Mammakarzinom / Anja Schmidt ; Gutachter: H. Höfler, H. Oettle, Hans-Dieter Royer." Berlin : Humboldt-Universität zu Berlin, 2003. http://d-nb.info/1207662550/34.
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Kisielnicka, Edyta. "SCF-mediated degradation of the two translational regulators, CPB-3 and GLD-1, during oogenesis in C. elegans." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-234186.
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The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-‐transcriptional level. This relies on RNA-‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-‐binding proteins abundance in the context of germ cell development.
This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-‐binding proteins, CPB-‐3 and GLD-‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-‐3/CPEB and GLD-‐1/STAR at the pachytene-‐to-‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-‐3 and GLD-‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-‐activated protein kinase, MPK-‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-‐1 signaling may couple RBP-‐mediated regulation of gene expression to progression through meiosis during oogenesis.
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Rodríguez, Solovey Leisa Natacha. "IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs)." Doctoral thesis, Universitat Politècnica de València, 2015. http://hdl.handle.net/10251/58862.
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[EN] ABSTRACT
Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches.
Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling.[ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas. La transducción de la señal del ABA localizada en la membrana plasmática celular juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA.
[CAT] RESUM La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses. Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA.
Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862
TESIS
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Ufer, Christoph. "Untersuchungen zur Expressionsregulation der Phospholipid-Hydroperoxid-Glutathion-Peroxidase." Doctoral thesis, [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=979803632.
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Boukharta, Lars. "Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes." Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-212103.
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Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods. We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target. In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance. Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.
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Yang, Min-Shiou, and 楊旻修. "The role of Y-box binding protein-1 (YB-1) in regulation of DDX3 expression." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/88452208586233895443.
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碩士國立陽明大學
生化暨分子生物研究所
98
Human DDX3 belongs to the DEAD box family which possesses DEAD box motif and ATP-dependent RNA helicase activity. DDX3 plays important roles in many biological functions. For example, DDX3 enhances p21 promoter activity via interacting with Sp1 to repress cell proliferation in liver cancer cell line. However, the regulation of DDX3 expression remains largely unknown. YB-1 (Y-box binding protein 1) is a member of cold-shock domain (CSD) family. YB-1 has nucleic-acid-binding activity and modulates the gene expression through transcriptional regulation. Moreover, the nuclear localization is important for transcription activity of YB-1. It has been shown that AKT could promote nuclear localization of YB-1 by phosphorylating Ser102 of YB-1. In recent studies, YB-1 translocates to nucleus and upregulates many growth related genes expression to enhance proliferation of cancer cells, such as breast cancers, ovarian cancer and lung cancer. In this study, our data showed that DDX3 expression was enhanced in HeLa cells but repressed in HuH-7 cells by YB-1. By using semi-quantitative RT-PCR, real-time quantitative PCR and reporter assay, we demonstrated that YB-1 could modulate DDX3 promoter activity, thereby enhancing or repressing DDX3 expression level in HeLa or HuH-7 cells, respectively. Furthermore, we also found that YB-1 could repress p21 promoter activity in a Sp1-dependent manner in HuH-7 cells, which might result from the inhibition of DDX3 expression and the reduction of DDX3-Sp1 interaction on p21 promoter. In contrast, YB-1 might activate the p21 promoter activity in both Sp1-dependent and Sp1-independent manners in HeLa cells. Consistent with the observation described above, the Ser102 phosphorylation of YB-1 was increased by elevated AKT activity under serum stimulation condition, and DDX3 expression was enhanced in HeLa cells but was repressed in HuH-7 cells. Therefore, the nuclear localization of YB-1 plays an important role in regulation of DDX3 expression. In summary, our findings suggest that YB-1 enhances DDX3 and p21 protein expressions in HeLa cells, but represses them in HuH-7 cells. Therefore, YB-1 protein is a potential regulator of DDX3 protein expression.
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Bettencourt, Maria José Palma. "Y-box binding protein 1 (YB-1) relevance in estrogen receptor-positive (ER+) breast cancer." Master's thesis, 2016. http://hdl.handle.net/10451/23860.
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Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016O cancro da mama é uma doença heterogénea determinada por várias características clínicas e patológicas, incluindo parâmetros histológicos e marcadores moleculares como o recetor de estrogénio (RE), recetor de progesterona (RP) e o recetor 2 do fator de crescimento epidérmico humano (HER2). A classificação de cancro da mama em três principais grupos com interesse clínico reflete essencialmente a classificação molecular: subtipo Luminal, Luminal A (RE+ e/ou RP+, HER2-, Ki67 baixo) e Luminal B (RE+ e/ou RP+, HER2+ ou Ki67 elevado); subtipo com sobre-expressão de HER2 (RE-, PR-, HER2+); e subtipo triplo negativo (RE-, RP- HER2-). Os tumores da mama positivos para o RE são os mais frequentes (cerca de 80%) e são os que apresentam melhor prognóstico. No entanto, cerca de 30% irão progredir com metastização à distância, resultando num aumento da taxa de mortalidade. O cancro da mama RE+ é dependente de estrogénios que ativam o RE, um factor de transcrição importante. Neste contexto, o gene YBX1, que codifica a proteína YB-1 (Y-box binding protein 1), foi recentemente identificado como parte de um preditor de mau prognóstico em cancro da mama dependente de RE. A proteína YB-1 parece estar envolvida em todos os hallmarks do cancro, sendo um indicador de mau prognóstico, recidiva, invasão e resistência a fármacos. Em cancro da mama, a proteína YB-1 encontra-se normalmente sobreexpressa, e tem sido associada com a progressão da doença e com a ausência de RE e RP. Devido à sua capacidade de ligação aos ácidos nucleicos, a proteína YB-1 é um regulador importante de transcrição e tradução de vários genes relacionados com o cancro, como ERBB2, cyclin A/B1, E2F, MDR1, MYC, PIK3CA, e também um componente principal das partículas de ribonucleoproteínas mensageiras, contribuindo assim para a sua estabilização e regulação de tradução. O papel funcional da proteína YB-1 parece ser dependente da sua localização celular, que é estreitamente regulada. Em condições fisiológicas normais a proteína é maioritariamente citoplasmática, embora possa também ser encontrada no núcleo. A translocação nuclear surge, geralmente, em resposta a um stress ou estímulo, e pode ocorrer por clivagem proteolítica ou fosforilação da proteína. A proteína YB-1 é fosforilada no resíduo de serina 102 localizada no cold shock domain, pela cinase p90 ribossomal S6 e pela cinase Akt serina/treonina. Recentemente, utilizando um modelo animal de xenotransplantes ortotópicos de cancro da mama RE+ vs. RE-, com modulação dos níveis de estradiol, o nosso grupo mostrou que o gene YBX1 se encontra sobrexpresso em tumores RE+ que cresceram na presença de 17β-Estradiol (E2), comparativamente a tumores RE+ que cresceram na ausência de E2. Assim, o objetivo deste projeto foi explorar a relevância biológica e clínica da regulação de YB-1 mediada pelo RE em cancro da mama. A nossa hipótese é que as terapias dirigidas ao RE possam afectar o gene YBX1, levando a uma diminuição da sua expressão e que a cessação da hormonoterapia possa desencadear um aumento da sua expressão e consequentemente da proliferação tumoral. Para testar a nossa hipótese usámos uma abordagem translacional, baseada em modelos celulares de cancro da mama e na análise de grupos clínicos relevantes de pacientes com cancro da mama. O estudo do efeito do E2 na expressão do gene YBX1 na linha celular de cancro da mama positiva para RE MCF-7, mostrou que a adição de E2 ao meio de cultura per se não é suficiente para alterar os níveis de expressão de YBX1. De facto, a análise de um painel de 124 genes regulados pelo RE revelou que a sua regulação in vitro difere substancialmente do que foi observado in vivo, o que indica que existirão outros fatores e/ou mecanismos envolvidos, quer sejam intrínsecos da célula ou derivados do hospedeiro, que poderão afetar a relação entre o RE e YBX1. O efeito do E2 nos níveis de YB-1 foi também avaliado ao nível da proteína por Western Blot e imunofluorescência, utilizando anticorpos específicos contra YB-1 total e a forma fosforilada no resíduo de serina 102 (p-YB-1). O estímulo com E2 induziu um aumento da p-YB-1 e da sua localização nuclear. Este efeito mostrou ser dependente do RE, uma vez que foi inibido pelo tamoxifeno e não foi observado na linha celular RE negativa MDA-MB-231. Assim, embora não tenha sido observado um efeito da ativação da via do RE pelo E2 na expressão do gene YBX1, foi detetado um aumento na fosforilação da proteína YB-1, o que sugere que no modelo in vitro, a expressão e atividade de YB-1 pode ser modulada pela via RE ao nível pós-transducional. No contexto clínico, o nosso principal objetivo consistiu em estabelecer uma possível correlação entre a expressão de YB-1 e o cancro da mama RE+. Deste modo, analisámos pela primeira vez a expressão de p-YB-1 em amostras cancro da mama, num conjunto de 60 amostras de tumor primário e 32 metástases emparelhadas. Observou-se uma associação entre níveis elevados de p-YB-1 e tumores RE e RP negativos (P=0,006 e P=0,037, respetivamente). Relativamente aos outcomes clínicos, a expressão de YB-1 e p-YB-1 correlacionou-se com uma diminuição da sobrevivência livre de recidiva (P=0.0442, HR 0.5514 95%CI 0.3088-0.9846 e P=0.0108, HR 0.058 95%CI 0.1230-0.7606 respetivamente), mas não com a sobrevivência global (P=0.2473, HR 0.6097 95%CI 0.3704-1.292 e P=0.0687, HR 0.4789 95%CI 0.2168-1.058). Desta forma, YB-1 e p-YB-1 são importantes biomarcadores de pior prognóstico, nomeadamente para risco de recidiva, especialmente em pacientes com tumores RE-, RP-. Em amostras de metástases, observou-se uma correlação positiva entre a expressão elevada de p-YB-1 e metástases RP negativas (P=0,030), sendo que a marcação de YB-1 não mostrou associação significativa com nenhum dos parâmeros clínicos. Verificámos ainda não existirem níveis diferentes de expressão de ambos os marcadores nos tumores primários e metástases, refletindo uma alteração durante a progressão tumoral (Teste McNemar: P=0.7728 e P=0.0771, respetivamente; Paired t-test: P=0.5754 e P=0.1883, respetivamente). Este estudo consiste n a primeira análise da expressão de YB-1 e p-YB-1 em tumores primários da mama e metástases emparelhadas. Visto que a deteção da proteína YB-1 em tecidos tumorais é um marcador de mau prognóstico, procurámos de seguida avaliar se a deteção de YB-1 no soro de doentes com cancro da mama poderia ser igualmente significante. A deteção de níveis séricos de proteínas é uma técnica minimamente invasiva e de fácil aplicação. Para esta análise foi utilizado um grupo de amostras de soro disponível de doentes com cancro da mama e metástases ósseas. Foi detetada a presença de YB-1 no soro de 22 doentes, correlacionada com a presença de metástases extra-ósseas (P=0.044). A análise multivariada mostrou que os doentes com YB-1 no soro apresentaram uma progressão da doença óssea mais rápida (HR 3.29, 95% CI 1.13 – 9.60, P=0.029), no entanto sem diferenças ao nível da sobrevivência global (HR 2.04, 95% CI 0.86 – 4.87, P=0.108). Este estudo corresponde à primeira análise dos níveis séricos de YB-1 em doentes com cancro da mama. Em suma, os resultados obtidos neste projeto mostram que não só a proteína YB-1, mas também p-YB-1 e YB-1 secretada têm valor de prognóstico em doentes com cancro da mama, reforçando a sua utilidade clínica como fator de prognóstico e possível alvo terapêutico. Este estudo mostrou ainda a regulação in vitro e in vivo de YBX1 não é exclusivamente dependente da presença de E2. No geral, este estudo gerou dados significativos e colocou questões importantes que serão abordadas em projetos futuros.
Estrogen receptor-positive (ER+) tumors are the most frequent breast cancers (BC), and have the better prognosis. Nevertheless, about 30% of patients with ER+ BC will develop distant metastases, with increased mortality rates. ER+ BC is dependent on estrogens that activate ER, an important transcription factor. In this context YBX1, which encodes for Y-box binding protein 1 (YB-1), was recently identified as part of an ER-dependent poor prognosis predictor for ER+ BC. YB-1 is an oncoprotein overexpressed in BC, where it has been associated with disease progression and ER/PR negativity. Recently, using an orthotopic mouse model of ER+ vs. ER- BC, our group showed that YBX1 is overexpressed in ER+ tumors growing under the presence of 17β-Estradiol (E2). Therefore, this project aimed to establish a biological and clinical link between ER and YB-1 expression in BC. Using BC cell lines, we showed that E2 per se did not altered YB-1 expression, at the mRNA or protein level, but induced its phosphorylation. These results need further investigation to address the mechanism behind YB-1/ER connection we observe in the in vivo model. Next, we explored the prognostic value of p-YB-1 expression and secreted YB-1 in the clinical setting. We demonstrated that p-YB-1 is a biomarker of decreased distant metastases-free survival and overall survival, and that secreted YB-1 correlates with faster bone disease progression in patients with BC and bone metastases. In conclusion, the results obtained in this project demonstrate that not only YB-1 but also p-YB-1 and secreted YB-1 have prognostic value in BC patients, reinforcing its clinical utility as prognostic factor and putative target. We also showed that the in vitro and in vivo regulation of YBX1 is not exclusively dependent on the presence of E2. Overall, this work generated significant data and raised important questions to be addressed in future projects.
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Faro, Andrew. "Investigation of the interactions of retinoblastoma binding protein-6 with transcription factors p53 and Y-Box Binding Protein-1." Thesis, 2011. http://hdl.handle.net/11394/3638.
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Philosophiae Doctor - PhDRetinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.
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Lai, Bo-Ying, and 賴柏穎. "Study on the sumoylation of Y-box binding protein 1." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/25685817600443425122.
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碩士國立陽明大學
生化暨分子生物研究所
98
Y-box binding protein 1 (YB-1), a DNA/RNA-binding protein, has been proved to be involved in diverse cellular biological processes, including transcription, translation and DNA repair. However, how the functions of YB-1 are regulated remains unknown. Previous results in our laboratory suggested that YB-1 might serve as a substrate for sumoylation pathway in HEK293T cells. Sumoylation is an important posttranslational modification mechanism, which affects many biological processes. Thus, we are interested to know the molecular mechanism of YB-1 sumoylation and whether sumoylation plays any role in regulation of YB-1 functions. In this study, an in vivo sumoylation assay was performed to confirm the sumoylation of YB-1. We found that YB-1 can be modified by SUMO1, SUMO2 and SUMO3, and it is worthy to note that YB-1 might be preferentially modified by SUMO3. Additionally, three YB-1 protein fragments, amino-acid sequences 1-60, 1-125, and 126-318 were used to map sumoylation sites of YB-1. We observed that sumoylation sites of YB-1 probobly locate in amino-acid sequence from 61 to 324. Nevertheless, YB-1 does not contain consensus sumoylation sequence, ψKXD/E, which mediates the interaction between SUMO conjugating enzyme E2 and target proteins. Accordingly, the exact SUMO acceptor lysine residue of YB-1 might not be characterized by ψKXD/E sequence. By using site-directed mutagenesis, we discovered that the sumoylation status of some YB-1 mutant proteins was reduced, and the subcellular localization of these YB-1 mutant proteins was altered. Thus, our results reveal that mutations at specific amino acid residues affect the sumoylation and subcellular localization of YB-1.
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Huang, Hong-Xuan, and 黃泓軒. "Characterization of Y-box binding protein 1 in Hepatocellular Carcinoma Stem Cells." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/81278407138822858176.
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碩士國立臺灣大學
生化科技學系
102
Hepatocellular Carcinoma (HCC) is the third cause of cancer mortality in the world. The important issues for the treatment of HCC are the high recurrence rate, easy metastasis and drug resistance. Recently, the concept of cancer stem cells (CSCs) provides a new consideration in the cancer therapy including HCC. Cancer stem cells are a subpopulation of cells in the tumor, which have the capability of self-renewal and differentiation and drug resistance in chemotherapy. Previous studies showed that many properties of CSCs, such as high cell mobility, evading immune destruction and reprogramming of energy metabolism, are very different from the original understanding of cancer. As a result, development of the therapy targeting CSCs is one of the novel therapeutic strategies for the cancer in the future. YB-1 is a protein with multiple functions, which has been found associated with many kinds of cancers. YB-1 can increase the expression of stemness marker genes, enhance cell mobility and up-regulate MDR gene expression. Besides, YB-1 is known as a significant regulator during liver development and regeneration. To investigate the regulatory function of YB-1 in CSCs of HCC, I used sphere forming method to enrich CSCs. In the sphere cells, the expression of YB-1 and some pluripotent genes was up-regulated. In addition, the sphere forming ability of YB-1-shRNA knockdown HuH7 HCC cell line was decreased. YB-1 would translocalize to the nucleus of sphere forming cells or side-population cells, and the cancer stem cell population sorted from HCC cell line. These results indicated YB-1 might be involved in the transcriptional regulation in the cancer stem cell-like cells in HCC. Additionally, knock down of YB-1 also down-regulated the stemness, drug resistance and epithelial-mesenchymal transition (EMT) genes expression by qPCR analysis. The detail mechanism would be regulated by Wnt/β-catenin pathway and epigenetic regulation. From these results, YB-1 may play a key role in HCC cancer stem cells.
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Hsieh, Chi Han, and 謝其翰. "Functional impacts of Y-box binding protein 1 on PB2 protein of Influenza A virus." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/97240176180099177211.
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碩士長庚大學
醫學生物技術暨檢驗學系
101
Influenza A virus is a zoonotic pathogen, which has a broad host range that has been able to infect human, birds, pig, and so on. The PB2 627 residue of the influenza A virus is recognized as an important determinant of virulence, pathogenicity and host-range restriction. However, host proteins associated with PB2 627 are yet to be globally investigated and the exactly molecular mechanisms of PB2 627 in determining host range remain unknown. In order to identify cellular proteins that associated with the PB2 protein, FLAG-tagged PB2 627K (human signature) and PB2 627E (avian signature) were generated and their interacting proteins were identified through FLAG-immunoprecipitation and mass spectrometry. The results indicated that the Y-box binding protein 1 (YB-1) belongs to a common interacting protein which had no significant differential binding affinity to PB2 627 K/E proteins. Furthermore, overexpression of YB-1 in A549 cells has been shown to increase the replication of virus, and to increase significantly of minogenome luciferase reporter assay to 58%. In addition, immunofluorescence analysis by confocal microscopy also demonstrated that a strong colocalization between YB-1 and PB2 proteins in the cytoplasm. Our finding suggests that YB-1 is a positive regulator because of increasing replication and the activity of luciferase reporter assay for influenza A virus.
40
Geiger, Katharina. "Etablierung eines Vektorsystems zum shRNA-vermittelten Knockdown von Y-box binding protein 1." Doctoral thesis, 2016. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-149809.
Full textAbstract:
Die Funktion eines Genes zu erforschen, indem man es ausschaltet, und damit seiner Rolle im komplexen Zusammenspiel der einzelnen Prozesse des menschlichen Körpers nachzugehen, stellt heutzutage eines der vielversprechendsten Felder der Gentechnologie dar. Der als RNA-Interferenz bekannte Mechanismus wurde in dieser Arbeit durch den Einsatz von sogenannten short hairpin RNAs (shRNAs), dauerhaft in das Wirtsgenom integrierter Knockdown-Träger, für das Gen bzw. Protein Y box binding Protein (YBX1) angewendet. YBX1, ein Vertreter der Cold-Shock-Proteine, stellt einen zentralen Interakteur lebensnotwendiger Prozesse wie Proliferation, Apoptose und Embryogenese im menschlichen Körper dar. Seine Dysregulation wird jedoch auch in einen Zusammenhang mit Entzündung, Tumorformation und –aufrechterhaltung gebracht, unter anderem auch für das Multiple Myelom. Das Multiple Myelom ist für 1% aller Krebserkrankungen weltweit verantwortlich mit noch immer ungelösten Problemen unzulänglicher Therapie und deletärer Prognose. In der vorliegenden Arbeit wurde die Möglichkeit geschaffen, die Rolle von YBX1 für das Multiple Myelom mit Hilfe einer Maus-Plasmazelllinie in vivo zu untersuchen. Dies geschah durch die Suppression der Genexpression von YBX1 mittels verschiedener gegen YBX1 gerichteter Polymerase II-getriebener short hairpin RNAs (shRNAs). Diese wurden in ein lentivirales Plasmid kloniert. Durch das Vorhandensein von Tetrazyklin induzierbaren Promotoren (Tet-On bzw. Tet-Off) wurde die Möglichkeit geschaffen, einen konditionellen Knockdown von YBX1 zu induzieren. Dies war notwendig, da initiale Arbeiten mit humanen Myelomzelllinien zeigten, dass der Knockdown von YBX1 Apoptose induzieren kann. Mit diesem Konstrukt wurden in HEK293 Zellen lentivirale Partikel hergestellt und damit die murine Plasmozytomzelle MOPC315.BM stabil transduziert. Nach Selektion, Klonierung und Testung (Puromycin-Selektion, RFP-Expression und Western-Blot Analyse) stand ein Zellklon zur Verfügung, der einen induzierbaren YBX1 Knockdown zeigt. Damit gelang die Etablierung eines gegen YBX1 gerichteten Vektorsystems in einer murinen Plasmazellinie in vitro. Mit Hilfe dieser Zelllinien kann nun in weiteren Arbeiten untersucht werden, wie ein YBX1 Knockdown das Tumorwachstum in vivo beinflusstOne of the most dynamic areas in gene technology today is to get an understanding of a gene and its role in the human body via knockout. By using so called short hairpin RNA (shRNA), stably in the host genome integrated knockdown instruments, the mechanism of RNA interference was adopted for the gene and protein YB-1. On the one hand YB-1, a cold-shock-protein, is an important protagonist in life for vital processes such as proliferation, apoptosis and embryogenesis in the human body. On the other hand its dysregulation is associated with inflammation, tumor formation and maintenance, for example in the multiple myeloma. The multiple myeloma is responsible for 1% of all cancer entitities word wide. The therapeutic options are deficient and the prognosis is often unfavourable. In this thesis we had the possibility to analyze the importance of YB-1 for the multiple myeloma by using a multiple myeloma mouse model in vivo. Using different against YB-1 targeted polymerase II driven short hairpin shRNA (shRNA) we suppressed the expression of the gene YB-1. The shRNAs were cloned in a lentiviral plasmid. By using a tetracyclin inducible promotor (tet-on and tet-off) we got the possibility to induce a conditional knockdown of YB-1. This was necessary because the irreversible knockdown of YB-1 can be able to induce apoptosis. Lentiviral particles in HEK293 cells were produced via this construct and MOPC315.BM, a murine plasmocytoma cell, was stably transduced. After selection, cloning and verification (puromycin-selection, RFP-expression and western blot analysis) a cell clon was selected which showed an inducible YB1 knockdown. So we established a vector system targeted against YB-1 in a murine plasmocytoma cell line in vitro. Based on this findings the influence of a YB-1 knockdown on tumor growth in vivo can be investigated in future
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Wang, Wei-Ting, and 王瑋亭. "Study on the role of Y-box binding protein 1 in HCV life cycle." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/rbxa99.
Full textAbstract:
博士國立陽明大學
生化暨分子生物研究所
104
Efficient hepatitis C virus (HCV) propagation relies on the interplay between virus-encoded proteins and numerous cellular cofactors. DDX3 is a well known cellular cofactor of HCV replication. In this study, we explored the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. We identified YB-1 and DDX3 as novel viral nonstructural protein NS5A-interacting proteins. Both YB-1 and DDX3 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, HCV infection severly reduced DDX3 level but have no effect on YB-1 expression. Moreover, while YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, DDX3 palyed a role in stead-state HCV RNA replication. Knockdown of YB-1 in HCV-infected cells inhibited infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, different from the action of DDX3. Notably, YB-1 knockdown severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Moreover, mutations of serine 102 (S102) of YB-1 disrupted 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. Inhibition of the YB-1 S102 phosphorylation by administration of the phosphoinositol 3-kinase (PI3K) inhibitor also reduced the level of ectopically expressed NS5A. While ectopically expression of NS5A enhanced YB-1 S102 phosphorylation which was blocked by the PI3K inhibitor, our results suggest that NS5A may sustain its own level via PI3K/Akt-mediated phosphorylation of YB-1 S102. Collectively, our results support a model in which YB-1 maintains the level and phosphorylation state of NS5A in HCV infection to support HCV RNA replication and infectious virus production, which is possibly regulated by HCV-induced cell signaling. Our finding may provide a new aspect for developing novel anti-HCV drugs.
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Alves, Patrícia Borges 1993. "YB-1 expression in breast cancer: modulation of receptor status and therapeutic implications." Master's thesis, 2019. http://hdl.handle.net/10451/39449.
Full textAbstract:
Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2019O cancro da mama é o segundo cancro mais frequente a nível mundial, sendo o mais incidente entre as mulheres. Apesar dos importantes avanços terapêuticos, o cancro da mama metastático continua a estar associado à maior taxa de mortalidade por cancro entre as mulheres. O cancro da mama é uma doença bastante heterogénea que engloba vários subtipos histológicos e moleculares que influenciam o comportamento, a agressividade tumoral, e consequentemente, o prognóstico clínico. A classificação de cancro da mama com base na expressão molecular do receptor de estrogénio (RE), do receptor de progesterona (RP), do receptor 2 do factor de crescimento (HER2) e do índice proliferativo (Ki-67) tem particular interesse clínico e implicações terapêuticas. Do ponto de vista clínico, existem três subtipos de cancro da mama com maior relevância: o subtipo luminal com dois subgrupos: Luminal A (RE+ e/ou RP+, HER2-, Ki67 baixo) e Luminal B (RE+ e/ou RP+, HER2+ ou Ki67 elevado); o subtipo com sobreexpressão de HER2 ou HER2+ (RE-, PR-, HER2+); e o subtipo triplo negativo (RE-, RP- HER2-). O cancro de mama luminal é o mais comum, correspondendo a cerca de 60% de todos os casos, sendo também o menos agressivo. Uma vez que é caracterizado pela expressão de receptores hormonais e o seu crescimento é hormono-dependente, os doentes com cancro da mama luminal beneficiam de tratamento com hormono-terapia. As opções terapêuticas são várias e incluem o tamoxifeno e o fulvestrant que, por mecanismos diferentes, têm afinidade para o RE, impedindo a ligação do estrogénio e levando ao bloqueio da principal via de sinalização implicada na proliferação das células tumorais. No entanto, cerca de 30% dos casos de cancro de mama luminal torna-se resistente à terapia e os doentes vêm a desenvolver cancro metastático. O cancro metastático é a maior causa de morte por cancro e é considerado o maior desafio quer para os médicos e doentes, quer para a investigação. Assim, é muito importante descobrir novos mecanismos de resistência à terapia, bem como biomarcadores de prognóstico e/ou preditivos de resposta às diversas opções terapêuticas. A YB-1 é uma proteína multifuncional, que pertence à familia de proteinas Y-Box Binding Proteins e que está envolvida em vários processos celulares importantes para o crescimento e o desenvolvimento celular. Geralmente localizada no citoplasma da célula, uma vez activada por fosforilação a YB-1 é translocada para o núcleo onde actua como factor de transcrição e promove a expressão de genes associados ao crescimento, ao ciclo celular e à quimio-resistência. Vários estudos demonstraram o papel oncogénico da YB-1, que foi já associada a todos os hallmarks do cancro. Em relação ao cancro da mama, especificamente, a expressão elevada de YB-1 está associada ao subtipo molecular triplo negativo e a um pior prognóstico dos doentes. Nesta dissertação propusemos estudar a proteína YB-1 em cancro da mama luminal. Neste tipo de cancro, foi já demonstrado in vitro que a expressão aumentada de YB-1 está associada à perda da expressão de RE e resistência à hormono-terapia, e ao aumento da transcrição de HER2. Desta forma, colocámos a hipótese de que a expressão elevada de YB-1 em tumores primários de cancro da mama RE+ pode não só estar associada a um mau prognóstico, mas também à resistência à hormono-terapia e à perda de RE nas metástases. Para abordar esta hipótese, avaliámos retrospectivamente a associação entre YB-1 e p-YB-1 e a sobrevida livre de doença e sobrevida global, usando uma coorte de 80 tumores primários e 51 metástases emparelhadas. Avaliámos ainda uma possível associação com alterações no RE, RP e HER2 nas metástases. Os níveis de proteína foram detectados por imunohistoquímica e as amostras foram avaliadas por dois médicos patologistas independentes. Os resultados mostram que níveis elevados de YB-1 ou a localização nuclear de p-YB-1 estão associados à ausência de RE (p=0,0383 e p=0,0306, respectivamente). Em relação aos outcomes clínicos, a elevada expressão de YB-1 nos tumores, bem como a localização nuclear de YB-1 ou p-YB-1, mostraram estar associadas a menor sobrevivência global (p=0,0437; p=0,0221 e p=0,0163, respectivamente). Embora não tenha sido clara uma associação entre a expressão de YB-1 e as alterações moleculares que se observaram entre os tumores primários e as metástases, 45,8% das metástases com status molecular diferente do tumor primário, eram positivas para YB-1 nuclear, em comparação com 37% sem YB-1 nuclear. Globalmente, a nossa análise de amostras de cancro da mama humano corrobora a importância da YB-1 como biomarcador de mau prognóstico e mostra que esta proteína está implicada na negatividade de RE. Além disso, conseguimos mostrar pela primeira vez que a hormono-terapia adjuvante pode estar implicada na seleção de clones com expressão elevada de YB-1 e que isto pode estar associado com o pior prognóstico de doentes com tumores com elevada expressão de YB-1. Assim, também questionámos se a resistência adquirida à hormono-terapia em cancro da mama RE+ é acompanhada por uma alteração na expressão de YB-1; e sendo assim, se isso afecta a sensibilidade a outras terapias alvo. Para responder a essas questões, seleccionámos in vitro linhas celulares com resistência adquirida a diferentes terapias e avaliámos: a expressão de YB-1, RE, RP, HER2 e Ciclina D1; e a sensibilidade à hormono-terapia, terapia anti-HER2, e inibidores de mTOR e CDK4/6, actualmente usados como agentes únicos ou em combinação, para tratar cancro da mama luminal. Os nossos resultados mostram que a sobreexpressão de YB-1 em células de cancro da mama luminal, MCF-7, leva à diminuição da expressão de receptores hormonais (RE e RP), e aumenta ligeiramente a expressão de HER2. Em consequência, estas células tornam-se menos sensíveis à hormono-terapia (tamoxifeno e fulvestrano) e mais sensíveis à terapia anti-HER2 (lapatinib). De seguida, e para testar se a expressão de YB-1 estaria associada à resistência adquirida, mantivemos quatro linhas celulares de cancro da mama luminal expostas a baixas concentrações de fármacos diferentes por um periodo de cinco meses e avaliámos os níveis de expressão de YB-1, bem como os níveis de expressão dos receptores hormonais RE, RP e HER2. Os nossos resultados mostram que as células MCF-7 resistentes aos fármacos têm uma diminuição na expressão dos receptores hormonais e Ciclina D1, embora não tenham sido detectadas diferenças nos níveis de expressão de YB-1. Em concordância com estes resultados, a linha celular derivada de MCF-7 e resistente ao tamoxifeno apresentou uma menor taxa proliferativa e com menor dependência do estradiol, quando comparada com a linha parental não tratada. No entanto, as linhas celulares derivadas de T47D resistentes ao tamoxifeno, fulvestrano e lapatinib mostram ter um aumento de YBX1, ESR1 e ERBB2, embora nem sempre se traduza num aumento ao nível da proteína. Adicionalmente, avaliámos a resposta das linhas celulares derivadas de MCF-7 e T47D resistentes à hormono-terapia, a três terapias usadas na clínica para cancro da mama luminal metastático: lapatinib (anti-HER2), everolimus (inibidor do mTOR) e palbociclib (inibidor da CDK4/6). Curiosamente, os clones resistentes à hormono-terapia não se tornam mais resistentes às terapias alternativas, havendo até uma tendência para serem mais sensíveis, em especial a linha MCF-7 resistente ao Fulvestrano. Em conclusão, os nossos resultados demonstram que a proteína YB-1 está associada a um pior prognóstico em cancro da mama e que a sua expressão se correlaciona negativamente com os níveis de RE. Assim, esta proteína poderá ser um biomarcador de resposta à terapia em cancro da mama luminal, uma vez que a terapia standard é direccionada para o RE.
Breast cancer (BC) is the most common cancer among women worldwide, and Luminal BC is the most frequent and less aggressive subtype of BC, that affects about 60% of BC new cases. Luminal BC is characterized by the expression of hormone receptors, estrogen receptor (ER) and/or progesterone receptor (PR), and it could be divided in two subgroups depending of HER2 overexpression. Therefore, Luminal BC benefits from hormone-therapy (HT), as tamoxifen and fulvestrant, that blocks the ER-pathway and control the cell growth and development. However, there is a percentage of tumours that became resistant to therapy and develop metastases. Metastatic BC is the biggest challenge, and the major responsible for BC-related deaths. YB-1 is a multifunctional protein that is involved in a variety of cellular processes related with growth and development. Normally, it is localized on cytoplasm and upon activation by phosphorylation, YB-1 translocates to the nucleus. As a transcription factor, YB-1 promotes the transcription of genes related with cell cycle, growth and drug resistance. Therefore, YB-1 has been associated with all hallmarks of cancer in diverse types of cancers, including BC. In this project we aimed to associate YB-1 with prognosis and resistance to HT in luminal BC, using a clinical and in vitro approach. Overall, our analysis in a cohort of 80 primary BC tumours and 51 paired metastases corroborates the importance of YB-1 as a biomarker of poor prognosis and shows that it is associated with ER negativity. Moreover, we provide the first data that shows that adjuvant HT may be implicated in the selection of clones with elevated YB-1 expression, which may be associated with the poor outcome of YB-1High patients. Our in vitro results show that YB-1 overexpression decreases the expression of hormone receptors, and that cells become more resistant to HT and more sensitive to lapatinib (anti-HER2 therapy). Moreover, cells with acquired resistance to tamoxifen, fulvestrant or lapatinib show some alterations in gene transcription, but HT-resistant cells remain sensitive to alternative therapeutics such as lapatinib, everolimus or palbociclib. All things considered, our results suggest that YB-1 could be a predictive biomarker of HT-resistance and tumour aggressiveness, and that it deserves further studies.
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Bello, Ana Rita de Sousa Noia de Mendonça. "The role of YB‐1 in acquired resistance to hormone therapy in breast cancer." Master's thesis, 2019. http://hdl.handle.net/10451/42741.
Full textAbstract:
Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2019O cancro da mama é a neoplasia maligna mais prevalente e com maior mortalidade na mulher. O tratamento da doença metastática permanece um desafio no qual a terapêutica sistémica tem um papel fundamental. A hormonoterapia tem um impacto importante no tratamento de tumores com expressão de receptores hormonais, denominados de Luminais. Infelizmente, uma percentagem significativa de doentes adquire resistência à terapia com progressão da doença. Recentemente, demonstrou-se que a YB-1, uma oncoproteína associada a todos os hallmarks do cancro e à resistência à quimioterapia, está também envolvida na resistência à hormonoterapia. Neste estudo, tivemos como objetivo dar continuidade a um projeto anterior desenvolvido pelo nosso laboratório, cujo propósito foi determinar a associação entre a resistência inata e adquirida à hormonoterapia no cancro da mama luminal e a expressão de YB-1. Neste contexto, expusemos quatro linhas celulares humanas de cancro de mama a diferentes fármacos. Obtivemos clones pós-terapêutica (células released) e avaliámos possíveis alterações no padrão de expressão de YB-1, receptores hormonais e HER2, em células resistentes e released. Os nossos resultados demonstram um aumento na expressão da forma ativada da YB-1, p-YB-1, em células MCF-7 resistentes à hormonoterapia, que se acompanhou de uma maior expressão de HER2. As células released apresentaram um aumento na expressão de YB-1, p-YB-1 e HER2, ocorrendo diminuição da expressão de receptor de estrogénios e de ciclina D1 em células MCF-7 expostas a fulvestrano, sugerindo a emergência de clones mais agressivos, com maior resistência à terapêutica. Nas células HER2+ resistentes ao lapatinib, existe um aumento da expressão de YB-1 e p-YB-1 que é revertido nas células released, com um concomitante aumento de expressão de receptor de estrogénios, possivelmente representando uma sensibilização à hormonoterapia. Este estudo será complementado com ensaios de viabilidade, reavaliação da presença de resistência à hormonoterapia ou lapatinib, bem como a fármacos possivelmente usados numa segunda-linha terapêutica.
Breast cancer (BC) is the most frequent and lethal malignancy among women. Metastatic disease represents a challenge where systemic therapy is crucial. Hormone therapy (HT) has a major role in the treatment of BC with hormone receptor (HR) expression, denominated by luminal BC. Unfortunately, a percentage of patients will develop acquired resistance. Recently, YB-1, a recognized oncoprotein associated with all cancer hallmarks and chemotherapy resistance, was found to be involved in processes related to HT acquired resistance. In this study, we aimed to continue a previous project developed in our lab, which purpose was to determine the association between acquired HT resistance in luminal BC and YB-1 expression. In this context, four human BC cell lines were exposed to different therapies. We derived post-therapy clones (released cells) and assessed possible changes in YB-1, HR and HER2, in both resistant and released cells. Our results show an increase in YB-1’s activated form, p-YB-1, in MCF-7 HT- -resistant cells. When an increase in p-YB-1 was present, it was followed by a rise in HER2. Therapy interruption did not allow phenotype reversion, neither for HT or lapatinib. YB-1 and p-YB-1 expression was raised in released cells, as well as HER2, while ER and cyclin D1 expression decreased in MCF-7 cells exposed to fulvestrant, suggesting the emergence of more aggressive tumor cells, with possible enhanced resistance to therapy. In ER+/HER2+ cells resistant to lapatinib, expression of YB-1 and pYB-1 was increased; this was reversed in released cells, which also showed an increase in HR expression, possibly representing a sensitization to HT. This study must be complemented with cell viability assessments, re-evaluation of therapy resistance and trial of second-line therapies.
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Lin, Chia-Chen, and 林家珍. "Physical and Functional Interaction Between Core-Associated Protein RNA Helicase and Human Y-Box Binding Protein-1." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/45456711019535877607.
Full textAbstract:
碩士國立陽明大學
生物化學研究所
90
Hepatitis C Virus (HCV) is the major cause of transfusion- and community- acquired non-A, non-B hepatitis, which often leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Among components of HCV, the core protein has been reported to affect normal cellular functions and thereby contribute to the pathogenesis of HCV. Our previous study demonstrated that the HCV core protein interacts with a cellular DEAD box RNA helicase, designated CAP-Rf (core-associated protein RNA helicase full-length), and modulates its NTPase/dNTPase activity and transactivation ability of CAP-Rf. To identify cellular factors that interact with CAP-Rf, a yeast two-hybrid screen was performed. Among the CAP-Rf interacting proteins identified, one was a human Y-box binding protein-1 (YB-1). In this study, we focused on both the physical and functional interactions between CAP-Rf and YB-1. The interaction of CAP-Rf and YB-1 was confirmed by glutathione S-transferase pull-down assay. The YB-1-binding site in CAP-Rf was mapped to the region from amino acid residues 536-661, which encompassed its RNA binding and ATP hydrolysis domains. The CAP-Rf interacting domain was located within amino acid residues 126-203 of YB-1, which contained alternating clusters of acidic and basic residues. Moreover, YB-1 markedly enhanced the p53-mediated activation of a synthetic p53-responsive and p21 promoter, while this enhancement was less significant when inclusion of CAP-Rf, suggesting that CAP-Rf interfered with the transactivation of p53/YB-1 complex. Furthermore, CAP-Rf alone elicited a suppressive effect on p21 promoter, but no effect was observed with the synthetic p53-responsive promoter. Besides, CAP-Rf was not associated with the p53-YB-1 complex according to the streptavidin affinity chromatography. These data suggest that CAP-Rf regulates the expression of p21 promoter through a p53-independent pathway. The effect of CAP-Rf, YB-1, and HCV core protein on the activity of MDR1 promoter was also addressed. Both YB-1 and core activated the expression of MDR1, while CAP-Rf alone exhibited a suppressive effect. Coexpression of YB-1/core, YB-1/CAP-Rf, and YB-1/CAP-Rf/core further stimulated the expression of MDR1. These data suggest that HCV core protein, via interacting with CAP-Rf and YB-1, may in part contribute to the multidrug resistant effect to chemotherapy during chronic HCV infection.
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Chang, Yao-Wen, and 張耀文. "Study on the Regulatory Role of Y-box Binding Protein 1 in Mismatch Repair System." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/56277414718525046445.
Full textAbstract:
博士國立陽明大學
生化暨分子生物研究所
102
Y-box binding protein-1 (YB-1) is a DNA/RNA-binding protein that participates in various cellular processes. It has been shown that YB-1 is highly expressed in several types of tumors and associated with tumorigenesis and drug resistance. DNA mismatch repair (MMR) acts as a genome safeguard to ensure the genome fidelity in DNA replication process. MMR also recognizes alkylator-induced O6-meG:T mispairs and consequently triggers the DNA damage response signaling, which ultimately leads to cell cycle arrest and apoptosis. Thus, MMR deficiency is associated with increased risk of the development of genome instability and drug resistance. Previous studies indicated that YB-1 binds to mispaired DNA and interacts with several MMR-related factors. However, its role in the MMR system remains undefined. Here, we found that YB-1 represses MSH6-containing MMR complex formation and reduces the mismatch binding activity of MutSα. Apart from transcriptionally suppressing the expression of key MutSα complex components, MSH6 and MSH2, YB-1 also disrupts interactions among MMR-related factors through interacting with PCNA. In an effort to elucidate how YB-1 exerts this inhibitory effect, we have identified two functional proliferating cell nuclear antigen (PCNA)-interacting protein (PIP)-boxes, which mediate YB-1/PCNA interaction. This interaction is critical for the regulatory role of YB-1 in repressing MutSα mismatch binding activity, preventing association of MutSα with PCNA, disrupting MutSα/PCNA/G/T heteroduplex ternary complex formation and inhibiting in vitro MMR activity. Notably, YB-1-mediated inhibitory effect on 3’ nick-directed MMR activity is more remarkable than 5’ nick-directed MMR activity. Most importantly, YB-1 overexpression is associated with the alteration of microsatellite pattern and the enhancement of spontaneous mutations. Moreover, YB-1-mediated interference with MutSα recruitment to MNNG-modified chromatin might decrease nuclear ATR/ATRIP recruitment and repress MNNG-induced Chk1 phosphorylation. These inhibitory effects on the activation of MMR-dependent damage response signaling might render cells more easily to escape from MNNG-induced G2/M arrest, apoptosis and cytotoxicity. Also, YB-1 overexpression is associated with a higher MNNG-induced mutation frequency. Interestingly, up-regulation of other PIP-box-containing proteins, such as myeloid cell leukemia-1 (Mcl-1) and inhibitor of growth protein 1b (ING1b), has no impact on MMR complex formation, spontaneous mutation frequency, and alkylator-induced mutation accumulation, thus revealing the specific effect of YB-1 on regulating the MMR system. In conclusion, our study demonstrates that YB-1 functions as a PCNA-interacting factor to exert its regulatory role on the MMR process and involves in the induction of genome instability and alkylator resistance, which may partially account for its oncogenic potential and the development of alkylator tolerance in cancer cells.
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"Proteomic analysis of zebrafish folliculogenesis." 2008. http://library.cuhk.edu.hk/record=b5893611.
Full textAbstract:
Lau, Shuk Wa.Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 84-102).
Abstracts in English and Chinese.
Thesis Committee --- p.i
Abstract (in English) --- p.ii
Abstract (in Chinese) --- p.iv
Acknowledgement --- p.v
Table of content --- p.vi
List of figures --- p.ix
Symbols and abbreviations --- p.x
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- Structure of ovarian follicles --- p.1
Chapter 1.2 --- Folliculogenesis and its control --- p.2
Chapter 1.2.1 --- Ovarian follicle growth and development --- p.2
Chapter 1.2.2 --- Follicle recruitment and regulation --- p.4
Chapter 1.2.3 --- Oocyte maturation and ovulation --- p.9
Chapter 1.2.4 --- Intercellular communication between oocytes and somatic cells --- p.10
Chapter 1.3 --- Overview of proteomics --- p.12
Chapter 1.3.1 --- Two-dimensional gel electrophoresis --- p.13
Chapter 1.3.2 --- Mass spectrometry --- p.14
Chapter 1.4 --- Objectives of the study --- p.15
Chapter Chapter 2 --- Proteomic Analysis of Folliculogenesis in Zebrafish Ovary --- p.19
Chapter 2.1 --- Introduction --- p.19
Chapter 2.2 --- Materials and Methods --- p.21
Chapter 2.2.1 --- Animals --- p.21
Chapter 2.2.2 --- Isolation of ovarian follicles --- p.21
Chapter 2.2.3 --- Protein extraction and quantification --- p.22
Chapter 2.2.4 --- Two-dimensional electrophoresis --- p.23
Chapter 2.2.5 --- Staining --- p.24
Chapter 2.2.6 --- In-gel digestion --- p.24
Chapter 2.2.7 --- Mass spectrometry --- p.25
Chapter 2.3 --- Results --- p.25
Chapter 2.3.1 --- Establishment of the protein profiles of different follicle stages --- p.25
Chapter 2.3.2 --- Mass spectrometry analysis on the differentially expressed proteins --- p.26
Chapter 2.4 --- Discussion --- p.27
Chapter Chapter 3 --- Characterization of Y-box Binding Protein 1 (YB-1) in Zebrafish --- p.46
Chapter 3.1 --- Introduction --- p.46
Chapter 3.2 --- Materials and Methods --- p.49
Chapter 3.2.1 --- Animals --- p.49
Chapter 3.2.2 --- Isolation of ovarian follicles --- p.49
Chapter 3.2.3 --- Protein extraction and quantification --- p.49
Chapter 3.2.4 --- SDS polyacrylaminde gel electrophoresis (SDS-PAGE) --- p.50
Chapter 3.2.5 --- Western blot analysis --- p.50
Chapter 3.2.6 --- RNA isolation and reverse transcription --- p.51
Chapter 3.2.7 --- Semi-quantitative RT-PCR quantification of expression --- p.51
Chapter 3.2.8 --- Data analysis --- p.52
Chapter 3.2.9 --- Immunohistochemistry --- p.52
Chapter 3.2.10 --- Cloning of full-length ybl cDNA from zebrafish ovary and construction of recombinant plasmid for expressing ybl --- p.53
Chapter 3.2.11 --- Expression and purification of recombinant zebrafish YB-1 protein --- p.54
Chapter 3.2.12 --- Immunoprecipitation --- p.55
Chapter 3.3 --- Results --- p.58
Chapter 3.3.1 --- Confirmation of the presence of YB-1 --- p.58
Chapter 3.3.2 --- Tissue distribution of YB-1 protein and ybl gene expression in zebrafish --- p.58
Chapter 3.3.3 --- Stage distribution of YB-1 protein and ybl gene expression in ovarian follicles --- p.59
Chapter 3.3.4 --- Localization of YB-1 protein within the ovarian follicle --- p.59
Chapter 3.3.5 --- Degradation of YB-1 in the ovary --- p.60
Chapter 3.3.6 --- Production of recombinant YB-1 (zfYB-1) --- p.60
Chapter 3.3.7 --- Identification of YB-1 -bound partners --- p.60
Chapter 3.4 --- Discussion --- p.61
Chapter Chapter 4 --- General Discussion --- p.77
References --- p.84
47
Chen, Chia-Wei, and 陳家瑋. "CSC-3436 Re-expresses Estrogen Receptor α Through Inhibition of Y-box binding Protein-1 in Triple-Negative Breast Cancer Cells." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/9ymy77.
Full textAbstract:
碩士國立中興大學
生物化學研究所
102
Breast cancer is the most common malignancy among women in Taiwan and its incidence is increasing worldwide. Triple-negative breast cancer (TNBC) accounts for approximately 20% of breast cancer, it is defined by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). However, TNBC is a non-specific receptor expression and is therefore not suitable for use hormone therapy and target therapy, currently there is no specific clinical treatment. Y-box binding protein 1 (YB-1) controls gene expression through both transcriptional and translational mechanisms and is involved in various biological activities such as tumor progression. Expression of HER2 and ER depends upon YB-1 in human breast cancers. In this study, we used (2-PN) CSC-3436, to address a the derivatives hypothesis of that 2-phenylnaphthyridin-4-ones CSC-3436 could inhibit YB-1 expression and re-express ER-α in TNBC cells. The results showed that CSC-3436 decreased YB-1 expression in TNBC cells. CSC-3436 also increased ER-α expression and enhanced the anticancer effect of tamoxifen through inducing apoptosis in TNBC cells. We next found that CSC-3436 inhibited YB-1 expression through decreased Twist expression and Akt signal pathway. Our analysis indicated that Twist and its downstream effector YB-1 provide molecular targets for CSC-3436 synergized with tamoxifen against TNBC cells through the induction of apoptosis.
48
Fedoruk, Matthew Nicholas. "The modulation of androgen action in prostate cancer by exogenous chemicals, efflux transporter P-glycoprotein and Y-box binding protein-1." Thesis, 2005. http://hdl.handle.net/2429/18475.
Full textAbstract:
Prostate cancer is the most frequently diagnosed malignancy and the second leading
cause of cancer deaths in Canada. Human prostate carcinomas are often androgen-dependent
and respond to androgen withdrawal therapy by temporary regression, followed by androgen-
.independent recurrence. These well-established features of prostate cancer strongly suggest
that androgens play a major role in prostate carcinogenesis.
The overall goal of the research described in this thesis is to gain a more comprehensive
understanding of the mechanisms modulating androgen action in prostate cancer cells. This
focused on the influence of exogenous chemicals, the efflux transporter P-glycoprotein and the
multi-functional transcription factor Y-box Binding Protein-1. Epidemiological evidence, based
on agricultural occupational exposures, instigated experimental work that showed pesticides
have the potential to mimic or antagonize hormone action, in many cases through androgen
receptor binding and interfering with transcriptional activity, thus are capable of disrupting the
male hormone-signaling pathway. Furthermore, in prostate cancer cells, androgen
responsiveness is modulated by Pgp activity and expression. The biological consequences of
increased Pgp expression are decreased androgen accumulation and a corresponding
decrease in androgen-regulated transcriptional activity and prostate-specific antigen gene
expression. Experimental evidence further supports the hypothesis that early in prostate cancer
progression, increased YB-1 expression increases Pgp activity, which consequently lowers
androgen levels in prostate tumour cells. Suppression of androgen levels may activate cell
survival pathways and lead to an adaptive survival advantage of androgen-independent prostate
cancer cells following androgen ablation therapy.
Understanding the complex molecular mechanisms by which prostate cancer cells can
control androgen function, and how prostate cancer cells evade apoptotic death, provides a
paradigm to explain the relationship between androgen action and chemotherapeutic
resistance. As a result, the final objective investigated the effects of YB-1 knockdown using
antisense oligonucleotides in vitro, and in vivo after androgen withdrawal in human LNCaP
prostate cancer tumour xenografts, with the aim of suppressing cellular proliferation and
increasing chemosensitivity. Intratumoral injection of 2'-0-(methoxy)ethyl ribose-modified YB-1
ASO and paclitaxel incorporated into a biodegradable, controlled-release formulation in
castrated mice delayed Al progression. These results suggest that YB-1 may be a promising
target for the treatment of prostate cancer based on a strategy of inhibiting cellular proliferation.
Our understanding of the molecular links between androgen action, tumorigenesis, apoptosis,
and drug resistance provides the foundation for a new era of targeted cancer therapy.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
49
Ma, Jui-Wen, and 馬瑞彣. "Aloe-emodin inhibits HER-2 expression through the downregulation of Y-box binding protein-1 in HER-2-overexpressing human breast cancer cells." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/r3876z.
Full textAbstract:
博士國立中興大學
生物化學研究所
106
Breast cancer is the fourth leading cause of death of female in Taiwan, The current treatment of patients with breast cancer target estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor type II (HER-2) receptor type and other tumor markers for personalized medicine. Human epidermal growth factor receptor-2 (HER-2)-positive breast cancer tends to be aggressive, highly metastatic, and drug resistant and spreads rapidly. Studies have indicated that emodin inhibits HER-2 expression. This study compared the HER- 2-inhibitory effects of two compounds extracted from rhubarb roots: aloe-emodin (AE) and rhein. Our results indicated that AE exerted the most potent inhibitory effect on HER-2 expression. Treatment of HER-2-overexpressing breast cancer cells with AE reduced tumor initiation, cell migration, and cell invasion. AE was able to suppress YB-1 expression, further suppressing downstream HER-2 expression. AE suppressed YB-1 expression through the inhibition of Twist in HER-2-overexpressing breast cancer cells. Our data also found that AE inhibited cancer metastasis and cancer stem cells through the inhibition of EMT. Interestingly, AE suppressed YB-1 expression through the downregulation of the intracellular integrin-linked kinase (ILK)/protein kinase B (Akt)/mTOR signaling pathway in HER-2-overexpressing breast cancer cells. In vivo study showed the positive result of antitumor activity of AE in nude mice injected with human HER-2-overexpressing breast cancer cells. These bindings suggest the possible application of AE in the treatment of HER-2-positive breast cancer.
50
Auth, Hak. "The identification of Y-Box binding protein 1 as an osteocalcin stem-loop binding protein and its functional role in the regulation of osteocalcin mRNA stability in rat osteoblast cells (ROS 17/2.8)." 2008. http://www.library.wisc.edu/databases/connect/dissertations.html.
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