Browsing by Author "Ersan, Pelin Gülizar"
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Item Open Access The role of mediator complex in tamoxifen resistance of ER-positive breast cancer(Bilkent University, 2022-01) Ersan, Pelin GülizarBreast cancer is the most prevalent cancer type and the leading cause of cancer mortality among women worldwide. Estrogen receptor-positive (ER+) breast cancer is the most common clinical subtype with an incidence rate of approximately 80% of all breast cancers. Tamoxifen is a highly effective hormonal therapy for ER-positive breast cancer patients. However, its remarkable success is hampered by de novo or acquired resistance. Despite several advances in therapy options for relapsing patients, tamoxifen resistance is still an urgent clinical problem that needs to be addressed. Therefore, there is a dire need for novel targeted therapies to confer tamoxifen resistance in ER-positive breast cancer. The architecture of Mediator complex links DNA-bound transcription factors to the general transcription machinery RNA polymerase II. Mediator kinase module is dissociable part of the Mediator complex and broadly involved in human cancers. However, the role of kinase module in tamoxifen resistance has not been investigated. In this dissertation, I deciphered the association of Mediator kinase module in tamoxifen resistance both in vitro and in vivo settings. Initially, our gene expression profiling and survival analyses revealed that Mediator subunit 13 (MED13) and cyclin-dependent kinase 8 (CDK8) were significantly higher in tamoxifen-treated patients, and this outcome strongly correlated with worsened patient survival. In vitro inhibition of either MED13 via genetic modulation or CDK8 by highly selective inhibitor, SNX631, significantly reversed tamoxifen resistance. Notably, targeting MED13 or CDK8 resulted in inhibition of HER2/mTOR signaling and triggered apoptosis. Mechanistically, we identified that inhibition of either MED13 or CDK8 combined with tamoxifen treatment reduced ErbB2 mRNA level. We further demonstrated that CDK8 post-transcriptionally controls ErbB2 level via regulating mRNA stability. Moreover, inducible silencing of MED13 in combination with tamoxifen impaired the tumor growth. Similarly, in vivo treatment of SNX631 together with tamoxifen reduced tumor growth in xenografts and prolonged the lifespan in an aggressive transgenic mouse model. These results provided insight into how transcriptional programmers MED13 and CDK8, could contribute to mediating tamoxifen resistance and added new dimension to treatment strategies for ER-positive breast cancer.Item Open Access Systems-level analysis reveals multiple modulators of epithelial-mesenchymal transition and identifies DNAJB4 and CD81 as novel metastasis inducers in breast cancer(American Society for Biochemistry and Molecular Biology, 2019) Üretmen-Kagıalı, Z. C.; Sanal, E.; Karayel, Ö.; Polat, A. N.; Saatci, Ö.; Ersan, Pelin Gülizar; Trappe, K.; Renard, B. Y.; Önder, T. T.; Tunçbağ, N.; Şahin, Özgür; Özlü, N.Epithelial-mesenchymal transition (EMT) is driven by complex signaling events that induce dramatic biochemical and morphological changes whereby epithelial cells are converted into cancer cells. However, the underlying molecular mechanisms remain elusive. Here, we used mass spectrometry based quantitative proteomics approach to systematically analyze the post-translational biochemical changes that drive differentiation of human mammary epithelial (HMLE) cells into mesenchymal. We identified 314 proteins out of more than 6,000 unique proteins and 871 phosphopeptides out of more than 7,000 unique phosphopeptides as differentially regulated. We found that phosphoproteome is more unstable and prone to changes during EMT compared with the proteome and multiple alterations at proteome level are not thoroughly represented by transcriptional data highlighting the necessity of proteome level analysis. We discovered cell state specific signaling pathways, such as Hippo, sphingolipid signaling, and unfolded protein response (UPR) by modeling the networks of regulated proteins and potential kinase-substrate groups. We identified two novel factors for EMT whose expression increased on EMT induction: DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) and cluster of differentiation 81 (CD81). Suppression of DNAJB4 or CD81 in mesenchymal breast cancer cells resulted in decreased cell migration in vitro and led to reduced primary tumor growth, extravasation, and lung metastasis in vivo. Overall, we performed the global proteomic and phosphoproteomic analyses of EMT, identified and validated new mRNA and/or protein level modulators of EMT. This work also provides a unique platform and resource for future studies focusing on metastasis and drug resistance.Item Open Access TLR ligand loaded exosome mediated immunotherapy of established mammary Tumor in mice(Elsevier BV, 2021-11) Yıldırım, Muzaffer; Yıldırım, Tuğçe Canavar; Turay, Nilsu; Bildik, Tuğçe; İbibik, Bilgehan; Evcili, İrem; Ersan, Pelin Gülizar; Tokat, Ünal M.; Sahin, Ö.; Gürsel, İhsanTumor-derived exosomes (TEXs) could be harnessed as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. Engineering TEXs to harbor immunostimulatory molecules still remains a challenge. Previously, we demonstrated that nucleic acid ligand encapsulated liposomes could trigger synergistic strong humoral, and cell mediated immune responses and provokes tumor regression to that of their standalone counterparts. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). Engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic cancer model, administration of4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regress tumor growth in 4T1 tumor-bearing mice. Taken together this work implicated that an exosome-based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. This approach offers a personalized tumor therapy strategy that could be implemented in the clinic.