Dept. of Molecular Biology and Genetics - Ph.D. / Sc.D.
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Item Open Access Development and preclinical characterization of meningococcal outer membrane vesicle vaccine(Bilkent University, 2024-03) Özsürekci, YaseminInvasive meningococcal disease (IMD) is caused by Neisseria meningitidis, with the main serogroups responsible for the disease being A, B, C, W, X and Y. To date, several vaccines targeting N.meningitidis have been developed albeit with a short-lived protection. Given that MenW and MenB are the most common causes of IMD in Europe, Turkey, and Middle East, we aimed to develop an outer membrane vesicle (OMV) based bivalent vaccine as the heterologous antigen source. Herein, we compared the immunogenicity, and breadth of serum bactericidal assays (SBA) based protective coverage of OMV vaccine to X serotype with existing commercial meningococcal conjugate and polysaccharide (PS) vaccines in a murine model. BALB/c mice were immunized with preclinical batches of the W+B OMV vaccine, either adjuvanted with Alum, CpG ODN or their combinations and compared with a MenACYW conjugate vaccine (NimenrixTM, Pfizer) and a MenB OMV-based vaccine (Bexsero®, GSK). The immune responses were assessed through ELISA and SBA. Antibody responses and SBA titers were significantly higher in the W+B OMV vaccine when adjuvanted with Alum or CpG ODN, as compared to the control groups. Moreover, the SBA titers were not only significantly higher than those achieved with available conjugated ACYW vaccines but also on par with the 4CMenB vaccines. In conclusion, the W+B OMV vaccine demonstrated the capacity to elicit robust antibody responses, surpassing or matching the levels induced by licensed meningococcal vaccines. Consequently, the W+B OMV vaccine could potentially serve as a viable alternative or supplement to existing meningococcal vaccines.Item Embargo Identification of actionable drug targets in triple-negative breast cancer(Bilkent University, 2022-09) Tokat, Ünal MetinTriple-negative breast cancer (TNBC) is the most aggressive subtype of the breast cancer, representing 15-20% of all cases. Due to a lack of clinically available/approved targeted therapies, TNBC patients are still in desperate need of chemotherapy. Although TNBC patients initially have relatively high rates of response to chemotherapy, de novo and acquired resistance constitute a major problem, leading to worsened patient outcomes. Considering the initial high response rates and broad availability and affordability of the chemotherapy agents, it is of great utility to explore the molecular mechanisms driving or contributing to de novo and acquired chemoresistance. Therefore, I developed 9 acquired resistant cell lines and 3 acquired resistant xenografts and harnessed 4 de novo TNBC chemoresistance models to identify the resistance-driving factors for each chemotherapy class/agent. Following characterization of these models in comparison to their parental counterparts at protein level, I determined few candidate proteins involved in chemoresistance. By combining experimental and bioinformatic approaches, I showed translational potential of i) p38 and JNK MAPK, and PI3K/AKT inhibitors, and ER stress inducers in de novo and acquired resistance against antimetabolite chemotherapy agents such as gemcitabine and fluorouracil; ii) dual EGFR/HER2, EGFR, SRC and CDK1 inhibitors against an antimicrotubule agent, paclitaxel; iii) FAK and dual PI3K/mTOR inhibitors against an alkylating chemotherapy agent, cisplatin, and iv) FAK, SRC and PI3K/AKT inhibitors against a topoisomerase II inhibitor, doxorubicin. I further observed that molecular underpinnings of de novo and acquired resistance were largely overlapping albeit not the same. We have also investigated cross chemotherapy resistance patterns in each acquired cell line model, which can guide second- or third-line treatment selection. As a result, I present a rational approach to exploit chemotherapy-small molecule inhibitor combinations for previously untreated or treated TNBC patients. In other words, using low-dose chemotherapy and targeted therapy combinations we provide promising preclinical in vitro data for future in vivo testing.Item Open Access The development of prophylactic and therapeutic vaccine using cell derived extracellular vesicles(Bilkent University, 2022-08) Yıldırım, MuzafferThe primary aim of this thesis is to extend the breadth of in vivo application of externally loaded cell-line derived and tumor derived exosomes as a prophylactic and therapeutic carrier against cancer treatment. Exosomes with a size between 30 to 150 nm are small extracellular vesicles secreted by all types of mammalian cells. They mediate a novel mode of intercellular communication through their bioactive cargos such as lipids, nucleic acids, metabolites, and proteins, which can be delivered to the target cells. Exosomes have successfully served as immunotherapeutic nanocarriers in cancer treatment using their natural delivery capabilities. Furthermore, they are attractive as a delivery system because of their stability in circulation, biocompatibility, and low toxicity. In the first part of this thesis, we used exosomes as a nanocarrier system to develop cancer vaccines in a therapeutic murine melanoma cancer treatment. We show that lyophilization of exosomes together with the CpG ODN, model antigen OVA and lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution is successfully accomplished. We analyzed the effect of the lyophilization on a cell line-derived exosomes and we characterized the exosomes by using bead-based technique via flow cytometry, qNano, Scaning electron microscopy, and western blotting. We showed that lyophilization does not harm exosomes’ vesicular integrity and fundamental biological features. Furthermore, we tested the biodistribution and activating capacity of encapsulated exosomes in mouse PEC, mesenteric lymph node, and spleen cells. We found out that loaded exosomes are mostly taken up by antigen-presenting cells. Also, we showed that loading CpG ODN into exosomes significantly improves APC activation markers in macrophages, B cells, and DCs and induced significantly higher IFNγ production from mouse mLN and splenocytes. Finally, we tested the therapeutic utility of the CpG ODN, OVA and αGC encapsulating exosome in the B16F10-OVA melanoma tumor-bearing mice. We found out that therapeutic vaccination with triple (CpG ODN, OVA, and αGC) ligand encapsulating exosomes suppressed the progression of established melanoma tumors in mice. Moreover, our triple ligand loaded exosomes triggered Th-1 biased anti-IgG OVA immunity and converted immune cells in tumor microenvironment to the tumor-suppressing phenotype. In the second part of this thesis, we used tumor-derived exosomes (TEXs) 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. 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). We showed that 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 breast cancer model, we found out that administration of 4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regressed tumor growth in 4T1 tumor-bearing mice. As a result, this work implicated that an exosome based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. The last part of this thesis, we studied the therapeutic potential of cell line-derived exosomes loaded with superparamagnetic iron oxide nanoparticles(SPION) and immunostimulatory ligands. We showed that loading SPION enhanced the in vitro delivery of exosomes within immune cells. Also, we found out that spleen cells incubated with exosomes encapsulating with SPION and CpG ODN induced significantly higher levels of IL-12 and IFNγ. Finally, we tested our exosomal vaccine candidate in human hepatocellular carcinoma tumor model in athymic mice. We showed that TLR3 and TLR9 ligands encapsulated with SPION loaded exosomes induced pronounced innate immune activation and regressed tumors and improve survival rate of treated mice.Item Open Access Investigating the targets and biological roles of IRE1’s two enzymatic activities(Bilkent University, 2022-06) Yıldırım, Aslı DilberThe unfolded protein response (UPR) is composed of complex and intricate series of highly regulated pathways geared to resolve the accumulation of misfolded proteins and to maintain cellular homeostasis. Endoplasmic reticulum (ER) is an organelle that regulates the protein folding. Disregulation of the mechanisms underlying ER folding capacity can activate ER-related UPR pathways (UPRER). The inositol-requiring enzyme-1 (IRE1) is an ER stress sensor and proximal regulator of the UPRER. IRE1 harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1’s kinase substrate(s) and their function are unknown. This study was undertaken with the objectives of (i) discovering novel IRE1 kinase targets and (ii) deciphering the pathways regulated by IRE1 during ER stress and related pathophysiological conditions, (iii) investigating the mechanisms underlying how bioactive lipids resolve saturated fatty acid- induced ER stress. For the first part, I found out that phosphorylation of sphingosine 1-phosphate (S1P) lyase (SPL), a S1P degrading enzyme, is induced upon IRE1 kinase activation and this phosphorylation leads to inhibition of SPL’s enzymatic activity. Consequently, SPL inactivation by IRE1 results in the accumulation of cellular S1P levels. Mitochondria is another important organelle and have their own UPR system (UPRmt) to regain organelle proteostasis. However, the link between UPRER and UPRmt is unclear. My experiments further showed that increased S1P levels potentiate UPRmt signaling in an IRE1 kinase activity-dependent manner. As a result of these experiments, we present IRE1 kinase activity as an important regulator of sphingolipid metabolism linking UPRER to UPRmt. I also investigated the function of IRE1 in Kawasaki Disease (KD), a pathophysiological condition leads to coronary artery aneurysm. Upon analysis of publicly available transcrioptome data sets from KD patients, I identified a significant increase in ER stress-related gene signatures that associated with KD progression. The single cell transcriptomics data from Lactobacillus casei cell wall extract (LCWE)- induced murine model of KD further confirmed the activation of ER stress in myeloid cells. Upon confirmation of ER stress activation, I used genetic or small molecule approaches to ablate IRE1 function in order to study IRE1’s role in LCWE-induced KD progression. My data demonstrated significance decrease in caspase-1 induction and IL-1β secretion leading to inhibition of cardiovascular lesion formations that were dependent on IRE1’s RNase activity. The data shows that ER stress plays a causal role in KD progression via IRE1 activity. In last part, I studied how a bioactive lipid molecule, palmitoleate, acts on IRE1 in order to remodel ER membrane and to resolve lipid induced ER stress. My findings show that saturated lipids induce IRE1 oligomerization and activation while the monounsaturated fatty acid palmitoleate, blocks this oligomerization. The promosing results of our mice studies with IRE1 inhibition warrants future investigation to assess IRE1’s therapeutic potential in metabolic and inflammatory human diseases.Item Open Access Characterization of a novel IRE1 substrate pact and interacting miRNAS(Bilkent University, 2022-06) Doğan, Aslı EkinThe double-stranded RNA-dependent protein kinase activator A (PACT) anchors the RNAinduced silencing complex (RISC) to the endoplasmic reticulum (ER)’s membranous platform where RISC nucleation occurs and thus, PACT plays a key role in microRNA (miR)-mediated translational repression. Previous studies have shown that ER stress leads to PACT phosphorylation while simultaneously inducing changes in the expression of many miRs. Here, we demonstrate that PACT is phosphorylated by the ER-resident Inositol-requiring enzyme-1 (IRE1), a bifunctional kinase/endoribonuclease (RNase), both under ER stress and no stress conditions. While the role of IRE1 as a stress-induced RNase driving the unfolded protein response (UPR) is well understood, the function or the target(s) of its kinase activity have remained unexplored. Findings of this thesis show that IRE1- mediated phosphorylation of PACT regulates mature miR-181c levels, which suppresses the expression of key regulators of mitochondrial biogenesis (mitobiogenesis). Phosphorylation by IRE1 causes PACT-mediated suppression of mitobiogenesis and respiration. Partial PACT-deficiency in mice leads to enhanced mitobiogenesis during brown fat activation in cells and mice. Furthermore, cardiopulmonary bypass-induced ischemia/reperfusion injury downregulates PACT protein expression in human hearts while simultaneously inducing mitobiogenesis. Collectively, these findings demonstrate PACTmiR- 181c signaling axis is a key regulator of mitochondrial biogenesis and energetics.Item Open Access Identification of a novel substrate of IRE1 in lipotoxic stress response(Bilkent University, 2022-02) Yıldırım, ZehraFragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is a ribonucleic acid (RNA)-binding protein (RBP) that controls translation of select messenger RNAs (mRNAs). I discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. I show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP-deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. The results presented in my thesis provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggest IRE1 inhibition could be developed as a promising new therapeutic strategy to counteract atherosclerosis.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 Characterization of the fine-scale genetic structure of the Turkish population(Bilkent University, 2022-01) Kars, Meltem EceThe construction of population-based genetic resources plays a pivotal role in the study of human biology and disease. In this study, the fine-scale genetic structure of the Turkish (TR) population was characterized using the whole-exome (WES, n =2,589)andwhole-genome(WGS, n =773)sequencesof3,362unrelatedin-dividuals from Turkey. Significant levels of admixture from Balkan, Caucasus, Middle East, and Europe were detected in the TR subregions, consistent with the history of Anatolia. Results of the population structure analyses showed that the TR and European populations have a closer genetic relationship than previously appreciated. Inbreeding coefficient calculations and runs of homozygosity analysis reflected the unique effects of the high rate of consanguineous marriage on the TR genome. A TR Variome comprising over 40 million variants was constructed using the data generated in this study. Derived allele frequency (DAF) calculations revealed that 28% of TR-WES and 49% of TR-WGS variants in the very rare frequency bins (DAF < 0.005) were not listed in the Genome Aggregation Database. The lists of clinically-relevant variants and human gene knockouts in the TR Variome were also listed in this study, presenting the potential of the TR Variome being an invaluable resource for future disease gene identification studies. Additionally, a reference panel for genotype imputation was generated using TR-WGS data. Since this panel significantly increased imputation accuracy in both TR and neighboring populations, it will probably facilitate genome-wide association studies in these populations. In the second part of the study, the sequencing data of a total of 3,599 unrelated TR individuals were assessed for previously reported pathogenic (RP) variants and predicted pathogenic (PP) variants in Online Inheritance in Men (OMIM) genes associated with a pheno-type. Analyses revealed that no less than 70% of TR people have at least 1 RP variant, and all individuals possess at least one RP and/or PP variant in their genome. Moreover, 25% of individuals carried at least one RP variant in the newborn screening genes. Each individual in the study also had at least a 1 in 17 chance of carrying an RP variant in one of the 73 American College of Medical Genetics recommended actionable genes. MEFV, ABCA4, CYP21A2, PAH,and CFTR displayed the highest cumulative carrier frequencies (CF), consistent with the high prevalence of the phenotypes they are responsible for. By estimating the CF and genetic prevalence in 3,251 OMIM genes using RP and PP variants, this study presents the most comprehensive data so far demonstrating the landscape of genetic disease in the TR population.Item Open Access Characterization of functional and molecular properties of circulating extracellular vesicles of childhood idiopathic nephrotic syndrome patients(Bilkent University, 2021-10) Eroğlu, Fehime KaraNephrotic syndrome (NS) is one of the most common causes of glomerular disease in children and is characterized by the triad of proteinuria, hypoalbuminemia, and edema. The major molecular event in the pathogenesis of NS is the disruption of the glomerular filtration barrier, which is primarily driven by podocyte injury. The most common clinical presentation of NS in children is steroid-sensitive nephrotic syndrome (SSNS), characterized by complete remission within 4 weeks of steroid therapy and no apparent glomerular change in the light microscopic evaluation of kidney biopsies, thereby named as Minimal Change Disease (MCD). Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (SSNS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with SSNS in relapse and remission, 10 healthy controls and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry and western blotting. The major proteins from the plasma EVs were identified via mass spectrometry. A Gene Ontology classification analysis and ingenuity pathway analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and the particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively express proteins which involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared to remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho p38 (p-p38) and decreased levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the nephrotic syndrome phenotype in podocytes in vitro.Item Open Access Multiomics approaches to overcome drug resistance in cancer(Bilkent University, 2021-09) Küçükkaraduman, BarışChemotherapy resistance remains one of the major challenges in cancer treatment. Most of the studies on drug resistance have focused on genetic evolution of cancer cells; however, this focus has shifted to non-genetic and epigenetic mechanisms. There is accumulating evidence that mechanisms of drug resistance are not mutually exclusive but instead coexist within a given cancer to develop resistance and therapy failure. Hence, overcoming resistance requires the comprehension of these complex biological processes. Here, we aimed to characterize drug resistance mechanisms by performing both single omics interrogations and multi-omics integrative analysis. For this purpose, we conducted Gene Set Enrichment Analysis (GSEA), functional enrichment analysis on protein-protein interaction (PPI) networks and miRNA-target networks for interpreting gene and miRNA expression data. To gain further biological insights on resistance mechanisms, we focused on identifying a multi-omics molecular signature that discriminates cancer cells based on their drug response profiles. Collectively, these in silico analyses suggested the epithelial-to mesenchymal transition (EMT) as a mediator of 5-FU/irinotecan resistance in colon cancer and irinotecan/gemcitabine resistance in pancreatic cancer. Drug sensitive cancer cells exhibited a more epithelial phenotype with increased cell proliferation. Multi-omics integration analysis revealed some EMT-related genes such as TGM2 and FOSL1, to contribute differential drug response in cancer cells. On the other hand, response of breast cancer cells to doxorubicin exhibited an opposite profile in which mesenchymal phenotype is sensitive while resistant cells have epithelial phenotype. Secondly, we aimed to induce mesenchymal-to-epithelial transition to overcome EMT-mediated drug resistance. We selected eight natural compounds and two re-purposed agents that have been shown to reverse EMT in various studies. We noted transcriptional changes suggesting a shift towards a more epithelial phenotype in 4 out of the 6 cell lines upon treatment with at least one compound tested. None of the natural compounds or re-purposed agents triggered MET in all cancer cells screened. In addition, compounds with clear or slight MET induction did cause these effects in a specific cell line or only in specific cancer type. We investigated next whether the treatment with natural compounds would result in chemosensitization. MET induction by natural compounds is not uniformly related to increased sensitivity to chemotherapeutics but can result in occasional synergistic or additive effects. Lastly, based on cytotoxic activity of a novel c-Src inhibitor 10a in 15 melanoma cells, we report the identification of a new gene signature that can predict chemosensitivity to 10a. Two distinct phenotypes of cells, defined as sensitive and resistant, were further analyzed to reveal an underlying mechanism for this differential response to 10a. We found that proliferative or mesenchymal features of the cells are associated with distinct sensitivity of 10a. Through a protein−chemical interaction network analysis, we identified that three histone deacetylase inhibitors, valproic acid, entinostat, and trichostatin A, were predicted to synergize with 10a. The synergizing effect of valproic acid was validated in vitro. We also aimed to define a minimal number of genes that could be used as biomarkers of 10a sensitivity. We show that the expression level of four genes can be used to predict drug sensitivity against 10a.Item Open Access BO-264: Highly potent TACC3 inhibitor as a novel anticancer drug candidate(Bilkent University, 2021-01) Akbulut, ÖzgeBreast cancer has been consistently ranked to be the most common cancer and the second leading cause of cancer-related death in women worldwide for many years. Despite better understanding of tumor biology and the availability of plethora of anti-cancer therapeutics utilizing different strategies, complete response and/or long-term survival is achieved in only a small fraction of patients with aggressive disease. Since microtubule re-organization is an important step during cell division, drugs that interfere with this process have been a major focus of cancer research. Although anti-microtubule agents are widely used in clinic, cytotoxicity to non-tumorigenic cells and drug resistance are still the main obstacles. Therefore, development of alternative target molecules that selectively and efficiently target cancer cells, but restore normal cells are needed. Transforming acidic coiled-coil containing protein 3 (TACC3) is an important TACC family member, having both mitosis-related roles e.g. regulation of centrosomes and microtubule stability and interphase-related roles e.g. regulation of gene expression and cell migration. Being overexpressed in a broad spectrum of cancers and correlation of its expression with disease progression make TACC3 a highly attractive therapeutic target. Although the oncogenic role of TACC3 has been established albeit mostly in in vitro settings, there is currently no TACC3 inhibitor being tested in clinics. Therefore, by combining rational drug design and screening, we aimed to identify and characterize a novel TACC3 inhibitor hit molecule with high potency and minimum toxicity in in vitro and in vivo systems that is amenable for future drug development. BO-264 was identified as a novel inhibitor targeting TACC3 by direct binding validated by using several biochemical methods, including drug affinity responsive target stability, cellular thermal shift assay, and isothermal titration calorimetry. Compared to two other available TACC3 inhibitors, it showed superior inhibition of mitotic progression and cell viability, especially in aggressive basal and HER2+ breast cancer cell lines. Notably, BO-264 had remarkable cytotoxicity effect on several cancer cell lines in NCI-60 human cancer cell line panel (≥ 90% have less than 1 µM GI50 value) and inhibited the proliferation of FGFR3-TACC3 fusion protein-harboring cells, an oncogenic driver in several malignancies. Importantly, BO-264 did not cause any cytotoxicity to non-cancerous cell lines. Noteworthy, its oral administration significantly suppressed tumor growth in both breast and colon cancer syngeneic and xenograft models, and prolonged survival with no major toxicity. Finally, TACC3 expression level has been identified as a strong independent prognostic factor in breast cancer. Collectively, our preclinical findings suggest that BO-264 is a potent and non-toxic anti-cancer agent targeting TACC3 in breast and colon cancer and can be developed further to obtain better drug-like properties.Item Open Access Development of a specialized zebrafish xenotransplantation database and establishment of ALU-based tumor DNA quantification method in zebrafish: focus on models of overexpression and microenvironment(Bilkent University, 2020-09) Targen, SeniyeSuccessful xenotransplantation of human cancer cells into zebrafish host marked a new era in cancer research enabling high throughput in vivo screens. Zebrafish xenotransplantation literature continues to rapidly accumulate, and this necessitates the development of an interactive database for accommodating the collective data for fined-tune search, visualization and statistical representation purposes. Herein, I have introduced an interactive database, ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), housing manually curated details on molecularly-modified cell transplantations, PDXs, stem cell and cancer stem cell transplantation studies as well as transplantation studies bearing modified host details. The database projects collected data in a table format via various attributes and provides graphical representation of the curated details as well as statistical analyses yielding information on incorporated numbers and frequencies of selected attributes; hence can be used for reviews and designing new experiments. Zebrafish PDX studies are separately conceptualized and displayed through ZenoFishDb v1.1. Development of the ZenoFishDb v1.1 leads to a better understanding of tumor analysis methods such as assessment of proliferation and/or tumor growth in xenotransplantation studies and further marks the need for development of novel methods for precise quantification of tumor size. In the light of these findings, I have helped establish a novel qRT-PCRbased proliferation assessment method for xenografts in zebrafish, adapted from previous mouse xenotransplantation studies. Herein, the use and precision of ALU repeat-based quantification of transplanted liver cancer cells in genotyped zebrafish ache mutants and wildtype siblings was shown exemplifying microenvironment as an important factor for tumor growth. I further demonstrated the power of ALU repeatbased quantification in Mineralocorticoid Receptor (MR) overexpressing breast cancer cells (MCF7) injected to the transparent casper zebrafish as a case study. First, I demonstrated that MR expression and signaling was important in breast cancer biology and prognosis based on in silico TCGA and custom RNA sequencing as well as other in vitro and ex vivo assays. I further showed that results obtained from ALU repeatbased quantification of tumor growth in MR-overexpressing MCF7 cells paralleled fluorescent image-based intensity measurements while the former being relatively less time-consuming and more high-throughput. In this study, accurate quantification of MR overexpression in xenografts was also successfully performed by a cDNA-specific primer pair; and the rate of tumor growth based on image analysis, did not correlate with the amount of MR DNA in casper fish xenografts. However, MCF7 cells overexpressing MR exhibited lower cell viability in vitro although some of these effects were due to empty vector (EV) integration. Accordingly, tumor size in xenografts of naïve, EV- and MR-transfected MCF7 cells injected into pigmented AB larvae were quantified for ALU-repeats yet no significant difference was observed due to high within-group variability in vivo. Future studies are needed to assess the role of varying the volume and placement of injected cells along with the amount of MR gene transfected on tumor growth in vivo.Item Open Access Prediction of prognosis and chemosensitivity in breast cancer(Bilkent University, 2020-09) Akbar, Muhammad WaqasBreast cancer (BC) is responsible for the highest mortality and morbidity out of all the cancers in women which is primarily due to both inter and intra-tumoral molecular heterogeneity. This heterogeneity arises from stemness, epithelial to mesenchymal transition and the type of treatment given to patients. These three biological processes are highly related with each other. Traditional therapy when given to breast cancer patients generally results in the transition of epithelial cells to mesenchymal phenotype. Because treatment targets primarily non-stem cells, it can leave stem cells alive which can later result in a relapse of cancer. In this study we aimed to identify such markers that could classify breast cancer patients into stem/mesenchymal or non-stem/epithelial like phenotypes, to determine how generalized the above stated hypotheses are. We developed a gene list of 15 genes we term as CSC/non-CSC gene list (CNCL) which classifies tumors into stemness and/or EMT based phenotypes and can also classify tumor cells based on their relative sensitivity to treatment with traditional therapeutics such as paclitaxel and doxorubicin. When classified into stem/mesenchymal (CS/M) and non-stem/epithelial (NS/E) phenotypes, we showed that Lapatinib and Midostaurin have a specific growth inhibitory effects on NS/E cells, and CS/M cells, respectively. Surprisingly the CNCL showed prognostic significance only for patients who were treated with paclitaxel in neoadjuvant setting, while it could not prognosticate most other BC cohorts. We argue that this is due to the dynamic plasticity of these tumors, as studied within the third aim of this thesis. Secondly, we aimed to identify chemotherapy biomarkers for paclitaxel, cisplatin and doxorubicin to stratify patients in groups that will or will not benefit from these drugs. Using biomarkers, we selected for this purpose, we performed linear regression analysis using breast cancer cell lines to generate cytotoxicity prediction models which can predict IC50 values for these drugs, based on the expression of two genes in each model. Two models were selected for doxorubicin and cisplatin, and three models were selected for paclitaxel. All models were validated both in silico and in vitro. Thirdly, we aimed to evaluate breast cancer plasticity that occurs upon treatment or when a tumor metastasizes. We noted that some breast tumors not only switch their clinical subtype but also change their molecular subtype upon treatment or metastasis. As breast cancer patient treatment in the routine practice is routed based on breast cancer subtype, it is very important to identify the subtype switches which can be critical for changes in treatment decisions. Additionally, we also identified metastatic biomarkers using large number of cohorts. Lastly, as CNCL genes did not show any prognostic importance in terms of both overall survival and metastasis free survival, we checked if the same is true for melanoma. We used Melanin A (MLANA) and Inhibin (INHBA) genes as the markers for invasive/proliferative, stem/non-stem and mesenchymal/epithelial phenotypes. High INHBA expression, which is epithelial, proliferative and non-stem phenotype biomarker, was associated with poor survival and high MLANA expression, which is mesenchymal, invasive and stem phenotype marker, was associated with good prognosis in melanoma patients. Therefore, these findings in melanoma supported our results in breast cancer.Item Open Access Elucidating immunomodulatory effects of telomeric repeat mimicking synthetic A151 oligodeoxynucleotide on immune cell transcriptome(Bilkent University, 2019-09) Yazar, VolkanRecent evidence revealed that DNA is beyond just the blueprint of life; it is also involved in immunomodulation. Unmethylated Cytosine-phosphate-Guanine (CpG) motifs of prokaryotic DNA stimulate immune response by interacting with Toll-like receptor 9 (TLR9). This interaction is mimicked using synthetic oligodeoxynucleotides (ODN) bearing similar DNA motifs to boost vaccinedriven immune response in human. Conversely, mammalian telomeric ends expressing TTAGGG repeats suppress immune response and contribute to fine-tuning of delicate immune balance. In this respect, suppressive ODN A151 with such G-rich telomeric repeats has proven useful in downregulating immune response; an overly active immune response is just as harmful to the host, as in the case of autoimmune disorders. Both CpG ODN and A151 are currently under preclinical/clinical trials with the aim of averting or medically treating a wide range of conditions from cancer to infectious disease or from autoimmune to autoinflammatory conditions. Contrary to CpG ODN, A151 literature is very limited and its modus operandi at gene level remains more of a mystery. Additionally, the degree, duration and breath of A151-induced alterations in immune transcriptome appear partially understood. Given the medical potential A151 holds for immunosuppressive therapy in human as a “self-molecule”, understanding the underlying molecular mechanisms via which A151 operates is invaluable. Toward this end, we attempted to uncover the unidentified features lying behind A151 ODNs immunosuppressive effects on immune cell transcriptome using a combined analysis approach of microarray data in this thesis. We demonstrated for the first time that A151 ODN deprives the cells energy by ceasing cellular uptake of fundamental molecules into the immune cells after derailing the entire intracellular trafficking. Putting it another way, A151 does not directly act on immune system cells but actually suffocates the cells by messing with intracellular trafficking, thereby blocking cellular uptake of fundamental molecules like glucose and glutamine. As such, immune suppression is just an indirect consequence of this larger cellular chaos. Our results indicated that this phenomenon occurs independent of CpG ODN stimulation of the cells and in a timely manner. Most, if not all, regulators of intracellular trafficking, vesicle signaling, and membrane protein transportation were found downregulated after incubation of cells with A151 at a physiologically relevant concentration, as well, implying full-blown entry to this intracellular turmoil at cellular level. The A151 effect on immune transcriptome was not just restricted to setting off a chaos for intracellular dynamics; novel long non-coding RNAs (lncRNAs) with immunometabolic activities were identified within the scope of this study among elements potentially regulated by A151, such as Lncpint, Malat1 and H2-T10 just to name a few. The involvement of lncRNAs in immune regulation is a well-documented phenomenon. Finally, our data showed that as an epiphenomenon of the intracellular turmoil mentioned above A151 has a deep impact in immune cells on mTOR network, the cardinal network of cellular energetics, growth, proliferation, and survival. A major shift in expression profile of relevant genes, i.e. downregulation of many activators of mTOR signaling along with core mTOR components, was validated on the benchtop after different layers of experimental validation using a wide range of marker genes and functional assays, reflecting A151’s ability to vastly shape dynamics of metabolism in favor of a metabolically inert state in macrophages and in B-cells. This knowledge will expand the breadth of A151 therapy in the clinics.Item Open Access The role of lipid-induced integrated stress response in metaflammation and atherosclerosis(Bilkent University, 2019-07) Onat, Umut İnciChronic inflammation resulting from metabolic overloading of organelles (such as the endoplasmic reticulum (ER) and mitochondria that control cellular homeostasis) is a major cause of metabolic disorders including diabetes, obesity and atherosclerosis. ER is an organelle that plays a critical role in cellular metabolism through biosynthesis of lipids, protein maturation and secretion, and calcium storage. Furthermore, a stressed endoplasmic reticulum maintains cellular homeostasis by initiating a conserved stress response pathway that is known as Unfolded protein response (UPR). UPR is activated in response to diverse stimuli that disrupts ER functions and serves asva pro-survival mechanism to regain ER homeostasis. However, in prolonged or severe ER stress, chronic UPR can promote inflammation and apoptosis. Activated UPR, inflammation and necrosis are observed in and causally associated with atherosclerosis. UPR has three branches, one of which is initiated by the protein kinase RNA (PKR) like ER kinase (PERK) and signals to eukaryotic initiation factor 2a (eIF2a). This signaling arm of the UPR is also part of a larger, translational control pathway known as the integrated stress response (ISR). Activation of ISR has been observed in atherosclerosis and could promote atherosclerosis To study the contribution of ISR to atherogenesis, I took advantage of three small molecule inhibitors that can modulate this pathway. I also used a chemical-genetic approach, known as the Adenosine triphosphate (ATP) analog sensitive kinase (ASKA) technology, to interrupt PERK kinase activity. With these multiple tools, I was able to specifically interfere with ISR signaling at multiple molecular nodes in order to study the role of lipid-induced ISR in inflammation, inflammasome activation and atherosclerosis. I discovered that during lipid-induced ER stress, PERK to Activating transcription factor 4 (ATF4) signaling resulted in transcriptional induction of a mitochondrial protease, Lon protease 1 (LONP1), which degrades PTEN induced putative kinase 1 (PINK1) and blocks Parkin-mediated mitochondria clearance (or mitophagy). This in turn causes an increase in mitochondrial reactive oxygen species (ROS) production, inflammasome activation and pro-inflammatory cytokine secretion such as interleukin-1b (IL-1b) in both mouse and human macrophages. I also discovered that these inhibitors are also effective in reducing hyperlipidemia-induced inflammasome activation in Apolipoprotein E-deficient (Apoe/- ) mice and consequently, in preventing atherosclerosis progression. These results point out that intercepting with ISR signaling in hypercholestrolemia can be considered as a novel therapeutic approach that could be developed against atherosclerosis.Item Open Access TLR agonists on autoimmunity, cancer and M1/M2 macrophage polarization(Bilkent University, 2019-07) Horuluoğlu, Begüm HanMacrophages play an important role in the initiation of immune responses and the maintenance of immune homeostasis. Alterations in their phenotype, function and activation state have been implicated in the pathogenesis of autoimmune and inflammatory diseases. An increased M1:M2 ratio is associated with the development of several autoimmune diseases including Systematic Lupus Erythematosus (SLE), vasculitis and myositis. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This work seeks to investigate the ability of PAM3 to induce M2 macrophage differentiation from patient monocytes and evaluate the therapeutic efficacy of PAM3 in a murine model of lupus. Our findings revealed that patients with indicated autoimmune diseases have a significant increase in monocytes of the inflammatory subtypes coupled with a decrease in non-inflammatory classical monocytes compared to healthy controls. Additionally, in the absence of stimulant patient monocytes differentiated into more M1-like macrophages. Nevertheless, phenotypic analysis of in vitro generated macrophages revealed that, PAM3 stimulation induced M2-like macrophage differentiation without any difference from patient and healthy monocytes. Phenotypic analysis was further supported by the high endocytic abilities and secretion of anti-inflammatory cytokines instead of pro-inflammatory cytokines by PAM3 generated macrophages. Lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Upon PAM3 treatment the increased M1:M2 ratio, which was observed in PBS treated group, was decreased to normal levels. The increase in M2 macrophages was accompanied by decreased autoantibody and inflammatory cytokines along with an increase in anti-inflammatory cytokine production. Moreover, kidney damage was significantly suppressed and M2 macrophages were detected in the kidneys of PAM3 treated group. PAM3 treatment prolonged to survival of NZB/W significantly, at 45 weeks of age %60 of mice were still alive whereas in PBS group only %5 were. Our results indicate that, PAM3 induces immunosuppressive macrophages and thus could represent a novel approach to the therapy of autoimmune diseases. The second part of this thesis focused on enhancing the immunomodulatory effects of TLR9 ligands, CpG ODN upon encapsulation within liposomes as cancer vaccine adjuvants. Although both D and K ODN are strictly dependent on TLR9, K ODN trigger pDCs to mature and secrete TNFα while D ODN stimulate pDC to produce IFNα. When cells are incubated with a mixture of K and D ODN, K masks the activity of D. The use of both K and D ODN would be of benefit when preparing vaccine adjuvants and for immunotherapy. Our data indicate that simultaneous delivery of D ODN loaded into neutral liposomes plus K ODN loaded into cationic liposomes improved rather than masked IFNα production while continuing to support TNFα by PBMCs. Liposomal encapsulation did not alter the subcellular localization of either class of ODN, but internalization studies revealed that cationic liposome encapsulation slows and reduces the uptake of K ODN whereas neutral encapsulation of D increases their uptake by pDCs. The efficiency of K plus D liposome combinations was examined in a murine tumor vaccine model. The liposome combinations induced pronounced Th1-biased anti-OVA immunity and led to a significant reduction of B16-OVA tumors following inoculation. Our findings, demonstrate that the beneficial features of D and K ODN could be obtained simultaneously by appropriate liposomal formulation, further extending the breadth of CpG ODN-dependent immunotherapy.Item Open Access Effects of Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi on apoptosis, DNA damage response, drug sensitivity, and HSA-MIR-495-3P overexpression in breast cancer(Bilkent University, 2018-12) Köker, Şahika CıngırCholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is associated with nicotine addiction and it has an important role in the prognosis of lung cancer. Despite its important cellular functions, its role in breast cancer remains to be elucidated. In this thesis, I aimed to identify the alterations in the important cancer signaling pathways occurring upon CHRNA5 depletion. Drug resistance is one of the major obstacles in breast cancer therapy. Heterogeneous nature of breast cancer necessitates identification of more biomarkers which aid in precise diagnosis and hence development of proper treatment options. In this study, by using more than one cell line which is representative of different subtypes of breast cancer, I showed the alterations occurred in cancer signaling pathways such as cell cycle and apoptosis upon CHRNA5 depletion, which could serve as a novel biomarker in breast cancer subtyping. Depending on mutation status of TP53, which is the gatekeeper protein during G1/S checkpoint, CHRNA5 depletion mostly exerted its effects over decreasing the levels of total CHEK1 and pCHEK1 (S345) which significantly altered the response of MCF7 cells to topoisomerase inhibitors in terms of enhanced drug sensitivity. Increases in apoptotic markers, such as BAX/BCL2 ratio along with increased FAS levels, further confirmed that this sensitization of MCF7 cells upon CHRNA5 depletion might have ended with apoptosis. So far in the literature, there is no study examining the regulation of CHRNA5 by small endogenous molecules such as miRNAs. Due to the predictive binding sites in 3’UTR of CHRNA5 and the importance of participating in tamoxifen resistance in breast cancer; I also examined the interplay between miR-15a family and CHRNA5 in MCF7 cells. I showed significant decrease in CHRNA5 levels upon using miR-15a mimic while demonstrating similar activity of miR-15a family mimics with CHRNA5 depletion using RT-qPCR. Another important implication of CHRNA5 depletion in MCF7 cells was the global change in miRNA expression prolife which was verified with independent microRNA arrays. Based on these in silico results, hsa-miR-495-3p appeared as the most downregulated miRNA which is known as a tumor suppressor miRNA. As stated in the literature, the role of miR-495 differs depending on the tumor type. Therefore, I tried to restore its expression by mimicking along with CHRNA5 depletion. The transcriptomic changes observed with CHRNA5 depletion was boosted with the restoration of miR-495 levels.Item Open Access Effects of PI3K/AKT/MTOR and VEGFR pathway inhibitors on liver cancer stem cells and bioactivities of novel pyrazolic chalcone derivatives on liver cancer(Bilkent University, 2017-12) Kahraman, Deniz CansenHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality, such that it the second most frequent cause of cancer death worldwide. Due to its heterogeneous composition and aggressive behavior, it is resistant to conventional therapies and also Sorafenib and Regorafenib which are FDA-approved multikinase inhibitors targeting pathways involved in angiogenesis and proliferation. The mechanisms behind the acquired resistance to Sorafenib were described as activation of compensatory pathways such as PI3K/Akt/mTOR, JAK-STAT, epithelial to mesenchymal transition (EMT), microenvironment and presence of cancer stem cells. Liver cancer stem cells originate from damaged and transformed hepatic progenitor cells (HPCs) which are found responsible for chemo-resistance, tumor relapse, and metastasis. For this reason, the effects of PI3K/Akt/mTOR inhibitors, Sorafenib and DNA intercalators on the enrichment of LCSCs were investigated. CD133+/EpCAM+ population from HCC cells were analyzed by flow cytometry after treatment with inhibitors, and effective inhibitors against LCSCs were further tested for their potential combinatorial effects together with Sorafenib. It was shown that upon treatment with Sorafenib or DNA intercalators the LCSCs were enriched, whereas Rapamycin (mTOR inhibitor), LY294002 (PI3K inhibitor) were able to inhibit the enrichment of LCSCs and reduced the CD133+/EpCAM+ population ratio. Combination studies revealed that when cells are treated initially with Rapamycin and then with Sorafenib, both the LCSC ratio and the sphere formation capacity of cells were reduced compared to cells treated with Sorafenib alone. To understand the alterations in gene expression induced by the inhibitors, a large panel of genes involved in regulation of cancer pathways were analyzed using Nanostring nCounter Technology. Systematic pathway analysis using Cytoscape Score Flow algorithm application allowed us to identify differential response genes involved in stemness. It was shown that genes involved in regulation of stem cells (Wnt and Notch pathway) were downregulated upon treatment with Rapamycin and DAPT (Notch pathway inhibitor), yet Sorafenib treatment resulted in differential regulation of these pathways, where JAG1 gene was found to be up-regulated. Interestingly, IL-8 expression was upregulated dramatically upon treatment with Sorafenib, but downregulated upon DAPT or Rapamycin treatment. Inhibition of IL-8 signaling resulted in reduction in both LCSC ratio and sphere formation capacity of HCC cells, which could be indicating the role of IL-8 signaling in the conservation of stemness features of LCSCs. For this reason, blockade of IL-8 signaling was suggested to be a promising therapeutic approach for HCC. Another topic in this thesis focuses on the potential of VEGFR2 TKIs and quinoids to inhibit both liver cancer cells and liver cancer stem cells. VEGFR TKIs such as Sorafenib, are widely studied for the treatment of many cancers, yet as mentioned above, there are many clinical studies providing the evidence that anti-VEGF or anti-VEGFR therapies lead to stable disease, which is then followed by disease progression in different cancer types. In recent years it has also been shown that antiangiogenic agents are increasing cancer stem cell population via generation of tumor hypoxia. Quinoids, on the other hand, are compounds that are selectively active in hypoxic conditions. Thus, the main aim of this study was to evaluate the bioactivities of compounds from each group on liver cancer cells and also to analyze their effects on the enrichment of LCSCs. Our results have shown that VEGFR2 TKIs were cytotoxic at lower concentrations compared to quinoids. However, it was shown that VEGFR2 TKIs are more likely to enrich LCSC population whereas some of the quinoids were able to reduce this ratio. With this information, a new concept called “aggressiveness factor”, which defines the potential of a compound to cause more aggressive cancer, was introduced. In the last part of this thesis, bioactivities of pyrazolic chalcone derivatives on HCC cell lines and their mechanism of action were investigated. Chalcones and pyrazolic structures are well known for their anti-cancer activities. Newly synthesized pyrazolic chalcone derivatives were tested against different cancer cells, and selection based on the IC50 values of compounds was made to analyze their effect on a panel of HCC cells. Results have shown that, compounds 39, 42, 49 and 52 were the most effective derivatives which had anti-proliferative activities in less than 5 μM concentrations. Further investigation of cell cycle progression and cell death mechanisms have revealed that compounds 42 and 52 caused cell cycle arrest at the G2/M phase and induced apoptotic cell death. Also, levels of cell cycle proteins, p21, CDK1, and phospho-CyclinB1 were shown to decrease upon treatment with these compounds.Item Open Access Prediction of prognosis and chemosensitivity in gastrointestinal cancers(Bilkent University, 2017-12) Demirkol, SeçilColon and gastric cancers are the third and fifth cancer types with the poorest survival. Surgery is considered the primary treatment option, which can be curative. However the decision as to whether chemotherapy administration after surgery is needed, is critical for especially stage 2 colon cancer; since a group of patients who receive chemotherapy do not have significantly improved clinical outcome. For this purpose, clinical risk factors are currently evaluated to determine patients with a high risk of progression. However this is not standardized by guidelines yet. Therefore, in this thesis my first aim was to ex vivo validate two novel independent mRNA based biomarkers, ULBP2 and SEMA5A, which have been previously identified in our lab, and to generate a prognostic signature which could stratify colon cancer patients with differential prognostic profiles using the best stratification method. I showed that a 3-group prognostic signature, SU-GIB, based on expression of these two genes is associated with cancer-specific, disease-free, and overall survival independent of clinical confounding factors in colon cancer. I performed in silico analysis in order to understand the biological details of the prognostic distinction, and revealed that patients with poorer prognosis show higher expression of pro-inflammatory cytokines and a more mesenchymal profile. Patients with better clinical outcome exhibit a more epithelial profile with higher levels of phosphorylated EGFR and Shc proteins. Analysis of high-throughput drug cytotoxicity databases also showed that colon cancer cell lines with „Bad‟ SU-GIB signature are more sensitive to a dual PI3K-MTOR inhibitor, BEZ235. In this thesis, my second goal was to define prognostic and molecular sub-groups for gastric cancer and characterize the specific biology of each sub-group. Utilizing an unsupervised approach on publicly available microarray data, I identified 3 biological groups, which harbor differential characteristics related to ECM involvement, EMT, proliferation and cell cycle. Moreover I identified distinct prognostic groups which are related to this molecular classification which can further stratify patients with known pathologic subtypes (diffuse and intestinal). I found that EMT was an important parameter for molecular and prognostic classifications for both types of cancers. I therefore, studied high-throughput drug cytotoxicity databases in order to identify selective compounds with selective growth inhibition on epithelial or mesenchymal cancer cells. I identified EGFR inhibitors as being significantly more effective on epithelial cancer cells regardless of the tissue type. My future plans include the large scale validation of SU-GIB, ex vivo validation of the gastric prognostic signature, and in vitro studies that would demonstrate effectivity of EGFR inhibitors on epithelial cancer cells and combination of EGFR inhibitors with MET inducers.Item Open Access Identifying and targeting coding/non-coding molecular switches regulating drug resistance and metastasis in breast cancer(Bilkent University, 2017-09) Raza, UmarBreast cancer is the second most common cancer and the leading cause of cancer associated deaths in women worldwide. Despite the availability of large number and various types of therapy agents which are effective in limiting tumor burden at initial stages, cancer cells still manage to resist to therapy treatment and exhibit re-growth of existing tumor or metastasize to distant organs. Therefore, there is a dire need to identify underlying molecular mechanisms to enhance therapy response and to block metastasis. In addition to coding genome, non-coding RNAs have also play active role in controlling proliferation, apoptosis, invasion and drug resistance in cancer. Therefore, I aimed to identify novel coding/non-coding molecular switches regulating drug resistance and metastasis in breast cancer. In the first part of this dissertation, I identified miR-644a as a novel tumor suppressor inhibiting both cell survival and epithelial mesenchymal transition (EMT) whereby acting as pleiotropic therapy-sensitizer in breast cancer. Both miR-644a expression and its gene signature are associated with tumor progression and distant metastasis-free survival. Mechanistically, miR-644a directly targets the transcriptional co-repressor C-terminal binding protein 1 (CTBP1) whose knock-outs by the CRISPR-Cas9 system inhibit tumor growth, metastasis, and drug resistance, mimicking the phenotypes induced by miR-644a. Furthermore, miR-644a/CTBP1-mediated upregulation of wild type- or mutant-p53 acts as a ‘molecular switch’ between G1-arrest and apoptosis by inducing p21 or Noxa, respectively. Interestingly, an increase in mutant-p53 by either overexpression of miR- 644a or downregulation of CTBP1 was enough to shift the balance between cell cycle arrest and apoptosis in favor of apoptosis through the upregulation of Noxa. Notably, p53-mutant patients, but not p53-wild type ones, with high CTBP1 level have a shorter survival suggesting that CTBP1 could be a potential prognostic factor for breast cancer patients with p53 mutations. Overall, modulation of the miR-644a/CTBP1/p53 axis may represent a new strategy for overcoming both therapy resistance and metastasis. In the second part of this dissertation, I performed whole transcriptome sequencing with downstream pathway analysis in the chemoresistant triple negative breast cancer (TNBC) tumors we developed in vivo. This suggested a potential role of integrins and hypoxia in chemoresistance. Mechanistically, we showed that our candidate gene is hypoxia-induced and is overexpressed in resistant tumors, and activates integrin subunit alpha 5 (ITGA5). In the meantime, hypoxia-mediated downregulation of a miRNA targeting our candidate gene, leads to further activation of the ITGA5. This culminates in the activation of FAK/Src signaling thereby mediating resistance. Importantly, higher expression of our candidate gene, or lower expression of miRNA was associated with poorer relapse-free survival only in chemotherapy-treated TNBC patients. Finally, inhibition of candidate gene increased the efficacy of chemotherapy in highly aggressive TNBC models in vivo providing pre-clinical evidence for testing inhibitors against our candidate gene to overcome chemoresistance in TNBC patients.