Theses supervised by Doç. Dr. Özlen Konu Karakayalı
23 theses · İhsan Doğramacı Bilkent University
SOX2 odak noktasında: SOX2 gen kopya sayısı varyosyonunun TCGA pancancer kümelerinde TP53 mutasyonu ile ilişkisi ve shiny uygulaması kullanılarak de novo SOX2 sentezi için kodon optimize tasarım
Recombinant proteins are crucial for diverse research applications such as biosensors and cancer studies. Proteins are engineered through de novo gene synthesis methods. Numerous tools and databases have emerged to facilitate the design of recombinant proteins, starting from the design of the gene sequence. De novo DNA synthesis enables the synthesis of custom-designed sequences, allowing codon optimization to enhance expression yield in heterologous systems. In cancer research, recombinant expression of proteins involved in tumorigenesis-related signaling pathways is employed for functional studies, potentially revealing new therapeutic targets. A notable example is the pivotal role of SOX2 expression in the formation of cancer stem cells (CSCs) across various cancer types. Previous studies highlight SOX2 expression functionally overlaps with TP53 expression on the PI3K/AKT signaling pathway. This association may stem from the p53-MDM2 interaction. This thesis investigates the association between SOX2 copy number gain and TP53 mutations within TCGA PanCancer cohorts. Fisher's exact test results reveal varying association, dependent on tissue type and specific driver mutations within each cancer type. The findings suggest the potential therapeutic relevance of SOX2 in cancer research. Furthermore, the thesis employs an in-silico approach to design de novo SOX2 synthesis, utilizing a novel shiny app that integrates codon optimization and primer design functionalities. The app enables simultaneous codon optimization for multiple expression systems and offers distance analysis through hierarchical clustering. Codon optimization feature provides control over the rate of replacement value for codon substitution which validated through a case study involving human insulin. Finally, app design set of overlapping primers with synchronized melting temperature to be used in PCR assembly for de novo SOX2 gene synthesis. Keywords: Recombinant Protein, Cancer, TCGA-PANCAN, SOX2, TP53, Association Analysis, Codon Optimization, PCR Assembly, Primer Design
CAP-RNAseq: Gen esansiyelliğine ve mRNA ve protein düzeyleri arasindaki uyuma dayalı RNA-seq veri kümeleme, annotasyon ve önceliklendirme için çevrimiçi bir platform
In recent years, there has been a remarkable growth in the application of RNA-seq in both clinical and molecular biology research contexts. The analysis and interpretation of these RNA-seq data demands a good knowledge of bioinformatics. Many different applications are available to perform the analysis, but more comprehensive applications are needed, especially for researchers without coding experience. Therefore, I developed an all-in-one novel RNA-seq analysis tool, CAP-RNAseq (http://konulabapps.bilkent.edu.tr:3838/CAPRNAseq/), which provide valuable analysis for co-expression cluster prioritization and annotation. CAP-RNAseq in particular performs clustering of the genes based on their expression patterns, annotates mirror clusters that display inverse patterns with a network-based visualizations before prioritization of clusters and/or genes based on "gene essentiality", protein levels and the degree of congruence between mRNA and protein levels of genes. Furthermore, for illustration of the use of CAP-RNAseq in this thesis, I reanalyzed a number of published RNA-seq datasets and identified novel pathways modulated by NTRK2 overexpression (GSE136868) in neural stem cells and also showed significance of the essential genes/pathways in senescent cell clearance focusing on NTRK2 (fibroblast; GSE190998) and THBD (Huh7, GSE228941) siRNA models. In addition, I analyzed our lab's novel RNA-seq data obtained from breast cancer cell lines in CAP-RNAseq; and the findings revealed a) the complex associations between steroid hormones; Drospirenone, Aldosterone, and Estrogen in hormone positive T47D and mineralocorticoid receptor-overexpressing MCF-7 cells; and b) significant differences in essential and non-essential gene expression of the isogenic MCF7 cells overexpressing wildtype or mutant TP53. I also studied a public breast cancer dataset (GSE201085) demonstrating CAP-RNAseq's ability to identify novel breast cancer markers exhibiting high mRNA-protein level correlations. In conclusion, this thesis not only demonstrates the use and power of CAP-RNAseq as a tool to identify essential genes and pathways by analyzing RNA-seq data, but also provides new insights into the roles of essential genes in glioma, senescence and breast cancer.
TP53 META: TP53 modülatörleri ve mutasyonlarının meme kanseri odağında gen ifade profillerine etkilerini görselleştiren bir web aracı
Belirli bir tedavi ile ilişkili veri setlerinin derlenmesi ve karşılaştırılması, bilimsel bir soruya ilişkin anlayışımıza katkıda bulunabilir. Transkripsiyon faktör protein 53 (TP53), diğer adıyla "Genomun Gardiyanı", sporadik meme kanserinde en çok mutasyona uğrayan transkripsiyon faktörlerinden biridir. TP53 hedef genlerini derleyen, açıklayan ve listeleyen birçok web aracı bulunsa da, bu gen listeleri önemli derecede farklılık gösterebilmekte ve bu listelerin görselleştirilmesini ve meta analizinin yapılmasını sağlayan bir web aracı bulunmamaktadır. Bu bağlamda, R Shiny tabanlı, TP53 META adında, iki koşullu RNA Dizileme veri setlerinden dört farklı setin eş zamanlı karşılaştırılmasına kadar çıkabilen analizlere ve bu analizlerin görselleştirilmesine olanak sağlayan bir araç geliştirdim. TP53 META, seçilen veri setlerinin normalizasyonunu ve limma veya DESeq2 ile diferansiyel ekspresyon analizini bir arada yapabilmektedir. Dahası, bu araç kullanıcıların kümeleme, gen seti zenginleştirme ve transkripsiyon faktörü-hedef zenginleştirme analizleri yapabilmelerini sağlamakta ve bunun yanı sıra ilgili tekli veya kombinasyon tedavilerine ilişkin anlayışımızı güçlendirebilecek hastalık-gen ve tedavi-gen bipartit yapı ağlarının görselleştirmesini yapabilmektedir. TP53 META'nın modülleri transkriptomun yalnızca belirli bir alt kümesine, örneğin TP53 proteininin özelleşmiş hedef genlerinden DREAM hedef genleri listesine, sınırlı tutularak da kullanılabilmektedir. Bir diğer yandan TP53 META, mevcut meta-analiz istatistiksel metotlarından yararlanarak meta-p değeri hesaplamaktadır ve çoğunluğu TP53 ifadesinin; siRNA tedavileri, nutlin ve doxorubicin gibi indükleyiciler ile tedavileri, CHRNA5 deplesyonu ve stabil homozigot veya heterozigot mutasyonlar üzerine modüle olmuş olduğu meme kanseri hücre hatlarına ait verilerden oluşan hem genel kullanıma açık hem de Konu Lab'a ait veri setlerini içermektedir. Genel kullanıma açık veri setleri GEO veri tabanından alınarak, Konu Lab'a ait veri setleri de fastq okuma dosyalarından ham sayma verilerine çevrilerek TP53 META web aracına entegre edilmiştir ve akabindeki hipotezler bu web-aracının işlevselliğini gösterebilmek için test edilmiştir: 1) Literatürde TP53 indükleyicisi olarak bilinen CHRNA5'in deplesyonu MCF7 hücre hattında DREAM hedef genlerinin ilerleyen kaybına yabanıl TP53 varlığında mı yoksa heterozigot TP53 mutasyonları varlığında mı yol açmaktadır?; 2) TP53 indükleyicileri TP53 hedef genlerini yabanıl ve mutant kanser hücre hatlarında eş seviyelerde mi indüklemektedir? Bu tezde aynı zamanda mevcut ve özgün metotları genlerin iki tedavi, örneğin tekli doxorubicin tedavisi ile CHRNA5 deplesyonu durumundaki doxorubicin tedavisi, etkisindeki sinerjistik veya antagonistik ilişkilerinin saptanabilmesi için kullandım. Çıkan sonuçlar MCF7 hücrelerinde yabanıl homozigot TP53 yüksek ifadesinin, heterozigot mutant (R175H, R273H mutasyonlarını içeren bir kopyalı) TP53'ün ifade edildiği koşula kıyasla DREAM hedef genlerini daha çok azalttığını göstermektedir. CHRNA5 deplesyonunun doxorubicin tedavisi ile kombinasyonunda TP53 hedef genlerinin daha çok etkilendiği de gösterilmiştir.
Metabolik disfonksiyon ilişkili yağlı karaciğer hastalığı (MASLD) için çok türlü scrna-seq atlasının geliştirilmesi
Metabolic dysfunction-associated steatotic liver disease (MASLD) affects a significant portion of the human population, potentially leading to severe secondary conditions. Despite extensive research on patients and animal models to understand the disease's initiation and progression, and the development of various therapeutic strategies, current non-invasive techniques remain inefficient, and only one drug has been approved. This underscores the critical need to elucidate the cellular mechanisms driving MASLD to develop new diagnostic and therapeutic approaches. Single-cell RNA sequencing (scRNA-seq) enables the investigation of transcriptomic profiles at the cellular level. Various studies have utilized scRNA-seq to explore MASLD in patient samples and animal models. Although there are numerous publicly available scRNA-seq datasets from these studies, no comprehensive tool exists to explore and analyse them collectively. In this study, publicly available scRNA-seq datasets related to MASLD were collected and reanalysed, adhering to best practices suggested in the literature, and a web tool was developed for their visualisation and custom downstream analyses. Thirteen datasets obtained from the livers of human MASLD spectrum patients, and three datasets different animal models, two mouse and one zebrafish, were uniformly preprocessed. The two human datasets containing cirrhotic samples and two fibrotic datasets, one from mouse and another from zebrafish models, were integrated to reveal the conserved mechanisms of liver fibrosis among human patients and animal models. A web tool was developed in R Shiny to enable visualizations, cell cluster selection, differential expression analysis and gene set enrichment of genes and gene sets. The utility of the app was demonstrated by conducting a case study with the integrated dataset. The results included an endothelial cluster that had cells from each of the four datasets, and differential gene expression analysis on the integrated dataset provided significantly modulated known, e.g., SYNPO and TIMD4 liver fibrosis-associated genes across the three species. As a result, the custom analysis workflow provided in this thesis can allow the detection of conserved markers in different MASDL stages by further analysing this and other integrated datasets in the future.
Glioblastoma tedavisi için kinaz inhibitörleriyle kombine trifluoperazinin yeniden amaçlandırılması: ın vitro ve larval zebra balığı ın vivo modellerinden bulgular
Gliomas are classified into lower-grade gliomas and glioblastomas, with the latter representing the most aggressive and treatment-resistant form, highlighting the urgent need for more effective therapeutic strategies. Recently, the antipsychotic drug trifluoperazine emerged as a potent anticancer drug for multiple cancers, including gliomas. Our previous research identified a potent therapeutic combination of trifluoperazine with sorafenib, a well-established multi-kinase inhibitor with proven efficacy in hepatocellular carcinoma, in Hep3B cell line model. In the present study, the transcriptomic response to trifluoperazine alone or in combination with sorafenib was evaluated in U87-MG glioblastoma cells and revealed that the combinatorial treatment induced a synergistic effect characterized by growth arrest, reduced invasiveness, and transcriptional signatures of metabolic stress and cell fate reprogramming. While trifluoperazine alone promoted a proliferative gene expression profile through the upregulation of essential metabolic and cell cycle-associated genes, its combination with sorafenib counteracted these effects by suppressing oncogenic signaling and amplifying tumor-suppressive pathways. These findings highlight the potential of trifluoperazine as a repurposed agent in kinase-targeted glioblastoma therapy and underscore the benefit of combinatorial strategies to overcome adaptive resistance mechanisms. Although sorafenib treatment in glioblastoma cells provided mechanistic insights into its synergy with trifluoperazine, in vivo exposure to sorafenib caused severe morphological defects in zebrafish larvae over a 72-hour window from 2 to 5 dpf. Moreover, in a previous glial reporter imaging screen using Tg(gfap:GFP) line zebrafish larvae, sorafenib was observed to markedly reduce GFP fluorescence, whereas trifluoperazine alone did not produce any detectable adverse effects in either assay. This in vivo toxicity prompted the search for alternative targeted kinase inhibitors with more favourable safety profiles to combine with trifluoperazine. Other kinase inhibitors that had a growth inhibitory effect on U87-MG as well as A172 cells were identified using MTT assays. A screen of 157 kinase inhibitors resulted in 12 kinase inhibitors with IC50 values less than 5 μM for both cell lines. Among the most promising with full cytotoxic effect were volasertib, INK128, CAY10626, tamatinib, Torin1, bisindolylmaleimide IX, AZD7762, NH125, and BMS345541. Trifluoperazine was also combined with selected kinase inhibitors, and volasertib and INK128 were identified to elicit significant synergism. On the other hand, several kinase inhibitors with significant inhibitory effects alone exhibited significantly reversed impact by the addition of trifluoperazine. The PLK1 inhibitor volasertib was top prioritized due to its highest synergy score in combination with trifluoperazine, and RNA‑seq was performed on U87‑MG cells treated with 2.5 μM volasertib to investigate its translational relevance. The resulting drug‑induced gene signature was evaluated over the TCGA‑GBM cohort, where high volasertib signature scores correlated with significantly improved overall survival, thereby reinforcing the rationale for its repurposing approach in combination with trifluoperazine for glioblastoma therapy. Finally, the early larval toxicity profiles of prioritized single or combinatorial drug treatments were assessed in vivo, and neither induced developmental abnormalities or phenotypic signs of adverse effects. In this context, LDexplore, an R Shiny application was developed to enable multidimensional analysis of zebrafish larval locomotor behaviour under dark:light alternation, incorporating heatmaps and statistical analyses of velocity and acceleration in response to startle stimuli. The app was used to test whether the zebrafish larvae exposed to trifluoperazine alone or in combination with volasertib exhibited different locomotory behaviour and it was found that although trifluoperazine induced a heightened swimming pattern upon stimulus at higher concentrations, the combinatorial treatment was not significantly disruptive to light induced startle response. These findings indicate that the combination of trifluoperazine and volasertib exhibits strong therapeutic potential, with in vivo applicability supported by larval zebrafish assays. Additionally, other kinase inhibitors demonstrating lower toxicity and significant synergistic effects on glioblastoma cell growth were identified, providing insights for further investigation. Keywords: Glioblastoma, Cancer, Drug Screening, Drug Repurposing, Larval Zebrafish Model, Trifluoperazine, Volasertib.
Kolinerjik reseptör nikotinik alfa 5 (CHRNA5) RNAi uygulamasının, meme kanserinde apoptoz, DNA hasarı yanıtı, ilaç duyarlılığı ve hsa-miR-495-3p'nin yüksek ifadesine etkisi
Cholinergic 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. v 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.
Ache mutasyonlu zebrabalığı yavrularında dokuya özgü transkriptomu
Differential expression of specific genes in certain tissues provides information about tissue specificity which might define phenotypes of various tissues. It is possible to understand the tissue-specific effects of knockout or knockdown studies performed on zebrafish embryos, using such genes. In this study, publicly available RNA-seq datasets providing data on 15 of the tissues from 5-9 months old zebrafish were used to estimate tissue specificity for zebrafish genes. Three different normalizations (i.e SD, RPKM, TPM) of 15 tissues were performed to compare; and the results were used to understand whether a given zebrafish mutant has significant enrichments for tissue-specific genes, based on different metrics including Tau, TSI, Hg, Spm, Gini, Counts. Application of these pipelines to the publicly available acetylcholinesterase (ache) mutant vs. healthy zebrafish data (GSE74202) revealed that many retina- muscle-, and liver-specific genes were downregulated in ache mutants. The downregulation of retina and liver specific genes (such as arr3a, rpe65a, rom1b and fabp10a, respectively) were futher confirmed with qPCR on comparative study of ache (+/?) and ache (-/-) 3 days post fertilization (dpf) zebrafish embryos. In addition a pilot experiment testing the effects of constant light and constant dark exposure for 3-5 dpf ache mutant and healthy embryos were performed, suggesting that the expression of retina-specific genes were more prominently affected in 3 dpf mutant embryos regardless of light.
CHRNA5 ifadesi düşürülmüş MCF7 meme kanseri hücre hattı modelinde miR-376 familya mikroRNA'larının etkileri ve ko-kültür rekabet çalışmaları
Cholinergic receptor nicotinic alpha 5 (CHRNA5) is a ligand-gated ion channel and one of the subunits of nicotinic acetylcholine receptors. Role of CHRNA5 in tumorigenesis has been initially shown in the lung tissue in which higher CHRNA5 expression has been significantly correlated with worse prognosis in lung cancer. In addition, our laboratory members recently shown that CHRNA5 depletion in breast cancer cell line MCF7 is antiproliferative (TUBITAK 111T316). In present study, effects of CHRNA5 depletion on miRNA expression profile were investigated and a significant decrease in the expressions of two members of the miR-376 family miRNAs, miR-376a-3p and miR-376c-3p, were identified. To test the effects of these two miRNAs, mimics were used in combination with CHRNA5 depletion on MCF7 cell line model. To investigate the synergism and/or antagonism of miR-376a mimic with CHRNA5 siRNA treatment a microarray study was performed and the signaling pathways involved were identified. Expressions of genes of interest were tested with RT-qPCR for both miRNAs. In addition, the effects of rescue on the cell phenotype and viability were also studied by using phalloidin staining and MTT experiments, respectively. Next a co-culture-based competition assay was developed using MCF7 cell lines expressing different fluorescent molecules to assess competition by both flow cytometer and fluorescent imaging. In summary, the results revealed that combinational treatments of si-CHRNA5 together with the miRNA mimics of two members from miR-376 family revealed enhancement of the antitumor effects. This study has been supported by TUBITAK (grant no. 114S367).
Identification of preclinical implications for novel indole-benzimidazoles and phenothiazines using in vitro cancer cell line and in vivo zebrafish models
Meme kanseri (BC) ve hepatosellüler karsinom (HCC), kayda değer ölüm oranlarına sahip iki önemli sağlık sorunudur. İlaç tedavileri mevcut olmasına rağmen, terapötik başarı sınırlıdır. Düşük biyoyararlanım, yüksek toksisite ve tekrarlayan ilaç direnci nedeniyle yeni tedavi seçenekleri gereklidir. Bu tezde, BC ve HCC'de, sırası ile, yeni indol-benzimidazol ve fenotiyazinlerin anti-kanser etkilerini ve klinik öncesi potansiyellerini test etmek için farklı in vitro, in siliko ve in vivo yaklaşımlar uygulandı. Tezin ilk bileşeninde, in vitro kanser hatları, in vivo zebrabalığı embriyo ve larvaları ve in siliko karşılaştırmalı transkriptomik analizler kullanarak yeni indol-benzimidazol türevlerinin BC hücre hattı toksisitelerini ve östrojen reseptörü (ER) ile ilişkilerini değerlendirdim. İkinci bölümde ise antipsikotik bileşikler olan fenotiyazinler (PTZ), HCC tedavisi için yeniden konumlandırıldılar. Bu nedenle, tek başına veya sorafenib (SFB) ile kombinasyon halinde jenerik PTZ türevleri, in vitro kanser hatları, ardından zebrabalığı gelişim testleri ve embriyonik aşama zenograftları kullanılarak test edildiler. Ek olarak, RNAseq analizleri, ilaçların gen ekspresyon düzeyindeki sinerjistik/antagonistik etkilerini anlamak üzere trifluoperazin (TFP), SFB ve TFP + SFB kombinasyonu ile muamele edilen Hep3B hücreleri üzerinde gerçekleştirildi. Son olarak, yeni PTZ türevlerinin anti-HCC potansiyelleri, in vitro ve in vivo taramalarla araştırıldı. Ayrıca, yeni ve jenerik türevlerin nöral yolaklar üzerindeki etkileri, kolinesteraz testleri ve motor tepki ölçümleri ile değerlendirildi. Tezin bulguları, özgün BC ve HCC tedavilerine istinaden ileri klinik öncesi çalışmaların yürütülmesine potansiyel yol gösterici niteliktedir.
Zebra balığı ksenotransplantasyon çalışmalarını barındıran veri tabanı geliştirilmesi ve ALU-dayanaklı tümör DNA ölçümünün overekspresyon ve mikroçevre örneklendirilmeleri ile zebra balığı ksenograft modellerinde uygulanması
Successful 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. Key words: Zebrafish, Xenograft, Database, ALU repeat, Tumor quantification, Microenvironment, Molecular Modification, Mineralocorticoid Receptor
Meme ve karaciğer kanserlerinde özgün hedefler olarak mineralokortikoid ve glukokortikoid reseptörlerinin incelenmesi
Cell signaling is a complex phenomenon and is maintained through intertwined signal transmissions within and in-between the cells. Anti-cancer therapies are often challenged by this fact due to crosstalk-associated activation of alternative survival routes. Hence, development of new treatment strategies and identification of novel prognostic markers depends on in-depth knowledge on cell signaling routes altered in cancer and possible crosstalk paths. Herein, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) signaling, two closely related members of steroid receptor hormone family, and their possible crosstalk were studied across breast and liver cancer cell lines. In breast cancer cell lines, estrogen responsive and MR expressing T47D was used in order to study possible crosstalk among Estrogen receptor (ER) and MR. MR-GR ligand aldosterone (ALDO) and ER ligand estrogen (E2) administered to breast cancer cells alone and in combination and, MR, ER and GR and their downstream signaling members were studied employing qRT-PCR and Western blot assays. Furthermore, ALDO, E2, ALDO-E2 hormone administrations were also used for cell viability assessments. Our results implied possible interactions of ALDO-E2 signaling at the level of cell viability, and at mRNA levels of progesterone receptor. In liver cancer cell lines, MR and GR was investigated as targets of a novel treatment. Liver cancer subtype hepatocellular carcinoma (HCC) has high mortality rate with limited treatment options. Multi-kinase inhibitor Sorafenib (SFB) with mild effectivity is most known systemic therapy against HCC. To potentiate the effectiveness of SFB and overcome to the crosstalk associated limitations, combinatorial drug treatment approach targeting multiple signaling modalities has been adopted in literature. Previously in our lab, SFB was combined with repurposed anti-psychotic drug TFP as a novel combinatorial treatment against hepatocellular carcinoma (HCC) and liver cancer cell lines. Cellular viability was synergistically reduced by SFB-TFP in HCC cell line Hep3B, while antagonistic effects on viability in SkHep1 was apparent. Herein, two liver cancer cell lines Hep3B and SkHep1, were used in comparison to unravel mechanism of action of SFB-TFP combination at the protein level. Apoptosis, cell cycle, PI3K/AKT/mTOR and MAPK pathways were investigated in addition to MR and GR. Our results revealed several markers indicating success of drug combinations and targeted pathways at protein level which needs to be pursued further.
Tp53, östrojen sinyal yolaği, ve 14q32.31 miRNA grubu'nun CHRNA5 deplesyon ifade profili üzerine etkilerinin belirlenmesi ve syneRgy uygulamasının geliştirilmesi
Cholinergic receptor subunit alpha 5 (CHRNA5) is a ligand-gated ion channel expressed in not only the nervous system but also other tissues. Differential expression and the polymorphisms of CHRNA5 have been associated with addiction, particularly nicotine and various cancer types. The tumor-suppressive properties of CHRNA5 depletion, i.e., decrease in cell proliferation, induction of DNA damage response, and drug sensitivity, have been identified in breast cancer cell lines. This thesis focuses on identifying critical factors modulating or modulated by the knock-down of CHRNA5 in breast cancer cell lines using both wet-lab and bioinformatics approaches. Here I have first found the significant correlation between CHRNA5 and DNA damage response in breast cancer tumor datasets. Moreover, I discovered that the introduction of siTP53 antagonized the actions of siCHRNA5 and reverted the siCHRNA5-mediated cell cycle inhibition and drug sensitivity in the MCF7 breast cancer cell line. Furthermore, siCHRNA5 was found to inhibit the secondary signaling of estrogen/ESR1 in time and dosage-dependent manners. CHRNA5 depletion also downregulated the conserved 14q32.31 miRNA cluster expression. Among those miRNAs, miR495-3p appeared to be the most prominent candidate, exhibiting a similar expression profile with selective estrogen degraders and partially with siCHRNA5. However, the inhibitory effect of the combinatorial treatment with siCHRNA5 and miR495-3p on the secondary targets of estrogen signaling indicated that siCHRNA5 and miR495-3p might target converging pathways, evidenced by the antagonism (rather than addictiveness) between them. In addition, the Shiny-based syneRgy app was developed to analyze the transcriptome-based synergy between treatments and/or genetic modifications. As a case study, syneRgy analysis using novel MDM2 inhibitor and/or temozolomide treated neuroblastoma cell lines revealed that although the tumor-suppressor effect of combination therapy was more than each individual treatment, it was less than additive. syneRgy was applied to understand the combinatorial treatment of siCHRNA5 with siTP53 as well as siCHRNA5 with miR495-3p mimic and enhanced our understanding of the TFs that might have a role in the crosstalk. To our knowledge, syneRgy is the first-ever online tool to perform statistical synergy analysis using RNAseq count or logFC transcriptomic data and synreg, i.e. our novel methodology allowing for statistical tests of TF target enrichment using regression models.
Gen, im ve yolak saptanmasında sağkalım analizi ve uygulamaları
Cancer literature makes use of survival analyses focused on gene expression based on univariable or multivariable regression. However, there is still a need to understand whether a) incorporating exon or isoform information on expression would improve estimation of survival in cancer patients; and b) applying multivariable regression to gene sets would allow to obtain cancer-specific independent gene signatures in cancer. Differential usage of individual exons, as well as transcripts, are phenomena common to cancerous tissue when compared to normal tissue. The glioblastoma, GBM; liver cancer LIHC; stomach adenocarcinoma, STAD; and breast carcinoma, BRCA datasets from The Cancer Genome Atlas (TCGA) were investigated to identify individual exons and transcripts with transcriptome-wide impact and significance on survival. Aggregation analyses of exons revealed the important genes for survival in each dataset, including GNA12 in STAD, AKAP13 in LIHC and RBMXL1 and CARS1 in BRCA. GSEA was applied on gene sets formed from the exon-based analysis, revealing distinct enrichment profiles for each dataset as well as overlaps for certain GO terms and KEGG pathways. In the second focus of this thesis, multivariable analyses on gene sets whose expressions were obtained from UCSC Xena were used to create two Shiny applications: one for dataset-specific analyses and one for analyses across TCGA-PANCAN. The dataset specific SmulTCan application incorporates Cox regression analyses with expressions of input genes of the user's choice. The SmulTCan application contains additional model validation, best subset selection and prognostic analyses. The ClusterHR application performs clustering analyses with Cox regression results, while it can also be used for bicluster identification and comparison. The axon-guidance ligand-receptor gene sets Slit-Robo, netrins-receptors and Semas-receptors were used for demonstrating the apps. Several hazard ratio signatures and best subsets that can differentiate between prognostic outcomes have been identified from the input gene sets, as well as ligand-receptor pairs with prognostic significance.
BRCA:Cohort-vs-TCGA: meme kanseri kohortlarının mutasyon profillerinin kapsamlı incelenmesi ve TCGA-BRCA ile kıyaslanması için bir web aracı
Comparisons of mutational landscapes between independent breast cancer (BRCA) cohorts in a comprehensive and statistical manner using online tools are likely to help advance our understanding of the diversity of somatic interactions and mutational signatures. In this thesis I have developed a webtool called BRCA:Cohort-vs-TCGA, which makes the mutational landscape comparison between a breast cancer cohort and TCGA-BRCA cohort accessible to the general public. BRCA:Cohort-vs-TCGA app contains modules to conduct mutational signature identification and comparison, differentially mutated gene and pathway identification and comparison, driver gene identification, and somatic interaction identification and comparison. Among the applications generated for similar purposes in literature, BRCA:Cohort-vs-TCGA provides advantages because of its comparative, statistical, and signature related features. When BRCA:Cohort-vs-TCGA was applied to compare the Gustave Roussy Institute (IGR) metastatic breast cancer cohort with the TCGA-BRCA cohort, a unique to IGR mutational signature related to SBS17b, a reactive oxygen species associated signature, which was missed by the original analysis of the authors, was identified. Interestingly, contribution of SBS17b was significantly higher in TNBC samples when compared to HER+ and HR+ samples. Differentially mutated gene analysis identified four genes, namely, ESR1, FSIP2, ARMCX4 and MALRD4, of which the latter two were unique to re-analysis by BRCA:Cohort-vs-TCGA. Differentially mutated pathway analysis on the other hand pointed to the differential mutation of DNA damage related pathways, NRF2 pathway and WNT pathway. The influence of the hypermutator samples in Fisher's exact test based analyses is well documented, as a result of many spurious mutations these samples contain. Re-analysis of the IGR cohort after removal of the hypermutator samples showed ARMCX4 was no longer differentially mutated anymore. Re-analysis of the differentially mutated pathways resulted in identification of three pathways, namely Mismatch Repair - Core, LIN37 independent P53 Targets, and U5, all of which had an odds ratio of less than one. These results, however, could be biased by the presence of hypermutators in the TCGA-BRCA cohort. In the second case study, I have demonstrated how the extracted somatic interactions for a user selected gene, i.e., CHRNA5 and TP53, can be used to prioritize and filter genes. This was done by merging the somatic interactions with the differential expression profiles of MCF7 cells treated with siRNA against CHRNA5 and TP53. Accordingly, BRCA:Cohort-vs-TCGA can help annotate/enrich other high throughput data with somatic interactions of custom genes.
Paralog/miRNA çiftleri ile pancan sağkalım analiz shiny aplikasyonu ve karaciğer kanserinde miRNA sinerjizminin in vitro doğrulanması
Emerging cancer survival tools can predict risk of disease and identify prognostic biomarkers. Multivariable Cox proportional hazards models with mRNA and microRNAs (miRNAs) expression can differentiate survival outcomes. Previous studies showed that genes that belong to the same pathways/families may act independently, and in a cancer-specific manner. In this thesis, cancer-dependent hazard ratios of paralog genes and sense-antisense strands of miRNAs were tested for TCGA PANCAN. The results were presented in a R/Shiny web application that provides gene-by-survival networks. The gene-by-survival network approach also was applied to the plasma membrane-endoplasmic reticulum (PM-ER) calcium channel geneset. Among paralogs, cancer-specific prognostic signatures and functional compartmentalization were observed. Some cancers like UVM, MESO emerged as hub cancers for PM-ER signalling. Further the proposed gene-by-survival network approach has been extended for miRNA-mRNA triplets that may act in synergy in hepatocellular carcinoma (HCC). Next, the effects of synergistic miRNA pairs provided by miRCoop algorithm were tested on cell viability and target gene expression for selected triplets. The results have revealed that the combinatorial miRNA treatments show promising results as RNAi therapeutics yet future studies with different doses and triplets are needed.
Karşılaştırmalı transkriptomik ve diferansiyel gen ekspresyonu için bir shiny uygulamasının geliştirilmesi
RNA sequencing has proven to be an effective technique for divulging information about the transcriptome in molecular biology research. Compared to microarrays and early methods of cDNA sequencing, high-throughput RNA sequencing has better resolution, low background noise, a higher range to quantify gene expression, and relatively lower cost. The development of sequencing technique has led to the development of tools for analyzing the high volume of data that is generated. Statistical methods for normalizing, filtering, performing exploratory and differential analysis, and other functional analyses based on RNA sequencing count data have made RNA sequencing one of the most popular techniques in genomics. To help facilitate the use of such statistical tools, web applications developed in R using the shiny package offer an advantageous environment where researchers can use a graphical interface to give inputs and instructions to the underlying server-side libraries that analyze and generate results in tables and plots. This thesis presents a new tool that has been developed for exploratory analysis, data normalization and filtering, differential gene expression analysis (DGEA), correlation analysis, principal component analysis, and functional analysis such as over-representation analysis and gene set enrichment analysis. When compared to other available applications, this new application offers the ability to run multiple methods for DGEA and compare results between them, leading to the output of gene sets that are discovered as DEGs in multiple tests. Here I present the features of this application in detail where I aim to improve upon the applications that are available in the literature. An example dataset from our lab was also investigated by this RNA-seq tool leading to a better understanding of Mineralocorticoid Receptor (MR) signaling in breast cancer.
Zebra balığı glioma ksenograft modelleri: Enjeksiyon yöntemlerinin in vivo araştırılması ve bir streamlit aplikasyonunun geliştirilmesi
Glioblastoma (GBM) is one of the most aggressive and lethal forms of primary brain cancer, posing significant challenges to effective treatment and patient outcomes. Despite extensive research efforts, our understanding of GBM biology and the development of novel therapeutic strategies remains limited. Zebrafish xenograft models have emerged as a promising tool in cancer research, offering unique advantages in studying GBM. This thesis explores the utility of zebrafish xenograft models in advancing our understanding of GBM. Due to their genetic and physiological similarities to humans, zebrafish provide an excellent platform for studying GBM pathogenesis, tumor progression, and drug screening. Their transparency during early development allows for real-time visualization of tumor growth, invasion, and response to treatments. Moreover, zebrafish models enable rapid and cost-effective high-throughput drug screening, accelerating the identification of potential GBM therapeutics. In this thesis, I focused on creating an application named ZenofishDb Glioma, which is a more evolved and focused version of our previous database called ZenofishDb. ZenofishDb Glioma has been created using Python Streamlit, and comprises only the glioma studies and uses Natural Language Processing (NLP) to classify better, and effectively find and summarize information about zebrafish glioma xenograft models. In addition, after searching ZenofishDb Glioma, I decided to investigate an experimental protocol for injection of glioblastoma cells in the zebrafish model to test effects of injected cell numbers. Using MGG-119-GFP cells and Casper zebrafish, I injected different numbers of cells at different locations and stages, i.e., blastula and 2 days post fertilization, and observed there were significant differences between groups at 5 dpf using multiple quantification strategies. In conclusion, ZenofishDb Glioma can help design effective xenotransplantation strategies and make comparisons to understand how different experimental parameters affect the outcome of zebrafish glioma xenograft models.
Meme kanserinde mineralokortikoid reseptör ve mineralokortikoid reseptörün alt akışında yer alan hedeflerinin östrojen ve aldosteron tarafından düzenlenmesi
Many women suffer from breast cancer worldwide thus accurate diagnosis of this disease has become an important issue for treatment options and improved clinical outcomes. Members of steroid hormone receptors, are a subfamily of nuclear receptors can serve as biomarkers in molecular classification of breast cancer. One of these, Mineralocorticoid Receptor (MR) takes part in many physiological processes in epithelial tissues including mammary epithelia, yet it is not well studied in the context of breast cancer. In this thesis, we investigated expression patterns of MR together with Glucocorticoid Receptor (GR) across multiple breast cancer cell lines at the protein level. Our study revealed that expressions of MR and GR were modulated in breast cancer as a subtype specific manner. We then enquired regulation of MR and its downstream targets, SGK1, NEDD4-2 and subunits of ENaC i.e., α, β and γ, by estrogen (E2) and aldosterone (ALDO) treatment in breast cancer via qPCR and Western Blotting. We found differential responses in expression of MR and its downstream targets to E2 and ALDO suggesting ER status was an important mediator of MR action. We also overexpressed MR in MCF7 cells and then showed that MR, NEDD4-2, β and γENaC mRNA levels increased in response to ALDO only when MR was overexpressed.
mir-497 meme kanserinde, CHRNA5 ile alakalı, önemli bir regülatör mü?
CHRNA5 is an important ligand-gated receptor with roles in addiction and in cancer. In lung cancer, CHRNA5 dysregulation is well known. There is also expression of CHRNA5 in breast cancer cell lines. microRNAs regulate mRNA expression; and different regulatory microRNAs are involved in different cancer types. microRNAs are thus potential biomarkers to diagnose the diseases (e.g. cancer). However, there is no study testing interactions between microRNAs and CHRNA5 in breast cancer. In the present study, mir-497 was found to be one of the most downregulated microRNAs with testable expression levels upon analysis of expression in the breast cancer cell line MCF7 when exposed to CHRNA5 siRNA. RT-qPCR was performed to test the expression level of mir-497. Validated target genes of mir-497 were found to be significantly related to a list of different KEGG pathways significantly (p value < 0.001) among which there were P53 and PI3K-Akt signalling pathways. Mimic-mir-497 treatment, alone or together with CHRNA5 siRNA, was applied on MCF7 cells to understand the interaction between the miRNA and siRNA under investigation. Selected target genes of mir-497 were tested; the most significantly modulated genes were involved in P53 pathway. The results indicated interactions between mir-497 and CHRNA5 however selected targets were not affected by mimicmir- 497. GSE41079 and GSE41074 public datasets containing mRNA and microRNA expression profiles of liver cancer cells treated with mimic-mir-497. Treatment were used to identify novel targets of mir-497 for future use. Immune system was detected in the second place upon REACTOME analysis of GSE41079 and GSE41074. Using multiple online microRNA-mRNA network tools mir-497 mRNA-miRNA networks were extracted for all or only immune genes. The results from network based analyses helped identify additional targets for later use in our mimic-siRNA system.
Meme kanserinde özgün rnai ve nanopartikül yaklaşımlarının anti proliferatif ve ilaç-hassasiyetine yönelik etkilerinin incelenmesi
Drug resistivity remains a major challenge in treating different cancer types. Among several strategies adapted to increase drug sensitivity in breast cancer cells, in the present thesis I studied an RNAi molecule targeting cholinergic receptor nicotinic alpha 5 subunit (CHRNA5) and a red-emitting oligomer nanoparticle, the two agents which I experimentally identified as negative regulators of cell proliferation. Cholinergic signaling is implicated in several different pathologies including cancer. Nicotinic acetylcholine receptors (nAChRs) are shown to be involved in regulation of cell proliferation, however they are mainly studied as mediators of nicotinic activity. CHRNA5 subunit has been shown to have roles in acetylcholine (ACh) production/stability, drug addiction and susceptibility to lung cancer. Few studies of lung and gastric cancers as well as high throughput RNAi screens show CHRNA5 as a modulator of cell proliferation. In the present study multiple CHRNA5 isoforms were cloned from MCF7 breast cancer cells (ER positive, TP53 positive) as in the case of lung cancer; moreover, a significant antimitotic effect of CHRNA5 RNAi application was demonstrated in MCF7 breast cancer cells. Similar effect of CHRNA5 silencing was only partially observed in BT-20 and MDA-MB-231 cells (ER negative, P53 mutant), yet in a seeding density-dependent manner. For the first time in literature the transcriptomic changes associated with CHRNA5 RNAi in the MCF7 cells were studied by microarrays from which differentially expressed gene lists were used to obtain the affected pathways. Additional assays confirmed the reduction in cell viability, DNA synthesis, G1 growth arrest, and changes in cytoskeleton complementing the microarray studies. Use of camptothecin (CPT) and doxorubicin (DOXO) in the absence or presence of CHRNA5 siRNA in MCF7, led to identification of CHRNA5's role in drug sensitivity. Comparisons between CHRNA5 siRNA and public microarray datasets revealed common genes/networks between topoisomerase (TOPO)/cyclin-dependent kinase (CDK) inhibitors and CHRNA5 depletion profile in MCF7 cells. mRNA-miRNA network analysis of differentially expressed common gene sets between TOPO inhibitors and CHRNA5 RNAi treatment identified potential common regulatory miRNAs. In an independent study the anti-cancer as well as drug sensitivity associated effects of a novel CB7-capped, red-emitting conjugated oligomer nanoparticle (Red-CON) were characterized in MCF7 and MDA-MB-231 cells. Red-CON in its encapsulated form exhibited low toxicity and good efficacy as a drug delivery system. This nanoparticle formulation might serve well for future clinical and less toxic chemotherapeutic regimens. Keywords: Breast cancer, CHRNA5, proliferation, motility, drug resistance, nanoparticle.
Tür içi ve türler arası transkriptom meta-analizi kullanılarak kanser teşhisi, prognozu ve tedavisi için yeni araçların geliştirilmesi
In the past decades, a considerable number of studies have performed meta-analysis on large data collections to prioritize sets of genes, pathways or types/categories of disease focusing on either differential expression, survival analysis, or co-expression networks. However, not many web applications or databases have been developed from these studies thus findings largely remained restricted to the addressed questions and it was not possible for other researchers to use the collected data for the evaluation of novel hypotheses. In this thesis, transcriptomic meta-analysis strategies have been applied to untangle complexities in multiple aspects of cancer research including treatment, diagnosis, and prognosis. Furthermore, three different web-tools have been developed which are not limited to a single type of meta-analysis. In this context, in addition to interesting cancer related findings, novel methodologies have been proposed and tested in the field of meta-analysis and cancer research. First chapter of the thesis presented a general introduction on the concepts of the thesis. Second chapter focused on a pathway comparison strategy based on meta-analysis that was used to reveal concordant/discordant aspects of rapamycin-mediation on transcriptomes of zebrafish and mouse. Analysis has shown that ribosomal terms were significantly upregulated while proteasome was downregulated in both species. Zebrafish has undergone a whole-genome duplication event; I also found out that rapamycin treatment resulted in largely concordant behavior of duplicated gene pairs. In addition, an online database, CompariZome, was developed to evaluate the duplicate zebrafish gene pairs in multiple GEO datasets in zebrafish in comparison to respective human expression datasets. In the third chapter of this thesis, I focused on identification of correlation between a trio of genes, CDH1, HNF4A, and GRHL3, using Cancer Cell Line Encyclopedia (CCLE) dataset to reveal the significance of association between these genes in different cancers, including breast and other epithelial cancers. The findings indicated correlation within the module and has demonstrated the power of meta-analysis using CCLE dataset. In the fourth chapter of this thesis, I focused on understanding the association of CHRNA5, a subunit of cholinergic receptors, with epithelial-to-mesenchymal transition (EMT) as well as epithelial differentiation, TP53 induction, and estrogen (E2) signaling with respect to breast cancer. Meta-analysis of in-vitro and in-vivo microarray expression datasets showed that CHRNA5, itself, and its positively co-expressed neighbors, were likely secondary targets of E2-signaling; overexpressed in ER- breast cancer patients; and indicators of worse prognosis. Functional annotation revealed that CHRNA5 and its co-expression network was indeed associated with proliferation related pathways. Based on meta-analysis of different cohorts processed in the study, an online database E2S (Estrogen (E2) to Survival) was developed that can facilitate user to query any gene for evaluation of E2-mediated effects, regulation by estrogen receptor (ER), prognostic importance and co-expression network along with functional annotations. In the fifth chapter of this thesis I focused on deciphering the correlation and deregulation between a human parolog pair of genes, i.e., mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) in breast cancer. Meta-analysis of a separate normal/tumor cohort revealed that both genes were downregulated in breast cancer and their expressions were highly positively correlated. However, deregulation analysis predicted that expression of MR and GR was more tightly regulated in normal breast hence its regulation might be lost with the onset of tumorigenesis. Another Shiny database, DualExpBC, was developed to evaluate differential expression of a gene in breast cancer as well as correlation and deregulation of expression between any two input genes in the breast normal/cancer expression cohort. With this thesis, I have developed novel tools and approaches for intra- and inter-species comparative transcriptomics and meta-analysis providing potential diagnostic, prognostic and therapeutic biomarkers.
Acıbadem meme kanseri kohortu RNA-seq verilerini gen imzalari ve klinik/mutasyon verileriyle moleküler alt tiplere göre keşfetmek, betimlemek ve sınıflandırmak için bir web aracı
Transcriptomics-based approaches have revealed the molecular heterogeneity and distinct gene expression patterns across breast cancer subtypes since the early 2000s. This led to the usage of molecular subtypes in clinics and translational research in prognostic assessment, therapeutic efficacy prediction, and retrospective analysis of cohort studies. In this thesis, breast cancer subtypes of Acıbadem Breast Cancer Cohort (ABCC) RNA-seq data were classified with immunohistochemistry (IHC), PAM50, and SCMOD1 as molecular subtype predictors. The results revealed the moderate concordance of the methods across ABCC and selected five other public datasets. In addition, it was shown that the classification of ABCC and TCGA-BRCA RNA-seq data strongly depends on the gene signature selection. Further, a machine learning model trained with TCGA-BRCA RNA-seq data and PAM50 genes as predictors showed moderate results for ABCC and MATADOR due to the imbalanced nature of datasets where feature importance revealed a subset of PAM50 genes as predictors. Additionally, the R-Shiny-based classABCC app was developed to facilitate clustering of ABCC with six gene signatures, molecular subtyping of ABCC, and prediction of subtypes with TCGA-BRCA RNA-seq trained machine learning model.
İlacin yeniden konumlandırılması ve glioblastoma tedavisine yönelik yeni kombinasyonların in vitro ve zebrafish in vivo modeller kullanılarak araştırılması
Glioblastoma is the most common and aggressive brain cancer type with the survival rate less than 2 years after diagnosis. Yet, potent drug treatments used in patients are limited and the field is in need of development of new potential drugs. In this study, repurposing of approved drugs alone or in combination and novel drugs are investigated in terms of inhibition of cell viability, glial fluorescent signals and their effects on behavior in zebrafish larval models. The main aim of this study was to test whether phenothiazines, trifluoperazine and a novel molecule 10, could be repurposed for glioblastoma treatment with lower dosages and more potency when combined with Sorafenib, an approved drug, in glioblastoma cell lines and zebrafish larvae. Those drug combinations were not found as toxic in the dosages studied while acted on glia cells in zebrafish transgenic larval models. Last but not least, behavior and stress response of the wild type and heterozygous mutant ache larvae in comparison with homozygous siblings were tested upon drug administration to assess genotype by drug interactions. Combination treatments exhibited higher efficacies suggesting phenothiazines with sorafenib could have potential in glioblastoma treatment. Genotype specific effects of individual and combination treatments on larval light-dark behavior, stress response and recovery exhibited potential for passage of blood brain barrier by the tested drugs. The established protocols for genotype and drug interactions could be applied to other kinases in combination with phenothiazines.