Browsing by study line "Genetics and genomics"

Animal coloration is as striking as it is diverse; however, the transcriptional basis of coloration is not deeply understood. Cichlid fishes are a tractable system for studying coloration as they exhibit a wide range of phenotypic diversity while remaining genetically similar. This facilitates the study of genotype-phenotype correlations and the identification of causative genes. RNA sequencing is a powerful approach to investigate the genes which characterize chromatophores. However, RNA-seq results can be plagued by the high abundance of rRNA in cells. This thesis aims to investigate differential gene expression between differently pigmented regions as well as explore the effects of tissue treatments and rRNA depletion on gene expression. Gene sets acquired with polyA selection, riboPOOL probes optimized for zebrafish, and zebrafish probes complemented with newly designed riboPOOL cichlid probes were compared to assess the functionality of these different rRNA depletion strategies. The use of zebrafish probes complemented with newly designed cichlid probes captured the greatest diversity of genes, many transcripts of which were missing from the other gene sets. Furthermore, as experiments such as scRNA-seq rely on a dissociation step, the effect of dissociation on gene expression was examined and found to promote the expression of stress response genes. The results of this upstream optimization were applied in the analysis of differential gene expression between the vertical stripes of the cichlid Pseudotropheus demasoni to better understand the molecular basis of vertical striping in fish. The dark stripes exhibited upregulation of melanic marker genes and the light, iridescent stripes showed an increase in iridophore marker gene expression. These findings were corroborated with cell count data from FACS to link transcriptional profiles and cell type quantifications. Overall, the study provides insight into the transcriptional basis of coloration in cichlid fishes and underscores the importance of optimizing methods drawing meaningful conclusions.

Human induced pluripotent stem cells (iPSCs) are an important in vitro model of disease and development. iPSCs can be differentiated in culture into cell types which are difficult to access from patients, such as neurons. Applying iPSC-derived cellular models to disease studies requires a thorough characterization of the derived cell types, as well as assessing reproducibility across cell lines or differentiation batches. With the aim of providing such a comprehensive molecular characterization at an early stage of cortical neuronal differentiation in vitro, six iPSC lines from four donors were differentiated to cortical neural progenitors using a modification of an established protocol (Shi et al., 2012a). The protocol successfully produced neural progenitors, with over 75% of the differentiated cells aligning with a cortical identity, as confirmed via qPCR and immunocytochemistry of established markers such as PAX6, NES and SOX1. To further classify the cell types produced as well as identify potential differences between cell lines, gene expression of the obtained cells was profiled with single cell RNA sequencing of ~22,000 cells, which uncovered the heterogeneity of neural progenitors produced. Further, although two differentiation batches produced similar cell-type compositions on a whole, a fraction of the lines showed inter-individual differences in cell type composition, which correlated with expression variability of known marker genes. Additionally, the cell types produced in vitro were compared to those produced in vivo by mapping our dataset to a reference fetal brain dataset (Polioudakis et al., 2019). It was observed that the in vitro dataset represented a subset of the cell types present at mid-gestation. Overall, the single cell characterization of differentiated cells allowed greater resolution in understanding cell-type heterogeneity of cortical neurogenesis, which is of key relevance for future applications such as disease modeling.

Acute leukemia is a life-threatening disease of blood and bone marrow, which is caused by malignant transformation of immature white blood cells. These malignant white blood cells invade space in bone marrow decreasing its ability to produce normal blood cells, eventually leading to death within weeks after the diagnosis without treatment. The acute leukemia can be broadly divided into its lymphoblastic and myeloid form, based on the affected cell lineage. Furthermore, acute leukemias can be classified based on different genomic features, such as gene fusions. Fusion genes are strong drivers in various cancers such as acute leukemias, and they are formed when two or more original genes join together forming a novel hybrid gene. If the novel hybrid gene is transcribed, it can lead to a translation of an abnormal fusion protein with altered function. The detection of the gene fusions is very important, since it affects to diagnosis and treatment of the patient. Various techniques can be used for fusion gene detection, of which the RNA sequencing is the method of choice, due to its ability to provide an unbiased identification of all known and novel gene fusions from the sample in a single experiment. In this thesis, the overarching aim was to develop an optimal sampling protocol for fusion gene detection using RNA sequencing for acute leukemia diagnostics. First, the whole blood samples in EDTA-tubes were collected from acute leukemia patients based on the findings from routine diagnostics. Next, the RNA was extracted at three different timepoints (0h, 8h, and 32h). The samples were stored at 4°C between the extractions. Finally, the RNA sequencing libraries were constructed, and the RNA sequencing was performed. After the sequencing, the data was analyzed using the FusionCatcher algorithm for fusion gene detection and the EdgeR-package for differential expression analysis. The FusionCatcher detected the same gene fusion in all the four fusion gene positive patients compared to routine diagnostics. However, the FusionCatcher failed to recognize the gene fusion in some of the samples with very low number of fusion breakpoint-spanning reads. These reads were visualized with IGV, suggesting that the detection failure resulted from the very low number of break-point-spanning reads. Furthermore, the sample storage did not affect on gene fusion detection. In addition, FusionCatcher detected PIK3AP::BLNK gene fusion from one of the fusion gene negative patients, suggesting a possibility that the patient truly was fusion gene positive. The differential expression analysis revealed changes in gene expression between the different timepoints. The results showed changes in various pathways related for example to cell death and protein biosynthesis, but also to pathways related to cancer. The results showed that prolonged sample storage alters the gene expression profile thus affecting the results of a gene expression study.

Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases and has the worst clinical outcome. Characterizing features of TNBC are high recurrence and mortality rates, and the absence of three commonly targetable breast cancer biomarkers estrogen receptor, progesterone receptor, and HER2, limiting the number of targetable therapy options. Cytotoxic CD8 positive T cells play a crucial role in the anticancer immune response and act as a major component of successful cancer immunotherapies. However, cancer cells can evade T cell-mediated killing by overexpressing programmed death-ligand 1 (PD-L1) resulting in T cell exhaustion and limited immune response via the interaction with programmed death protein 1 (PD-1). Systemic anti-PD-L1/PD-1 therapies aim to prevent this immunosuppressive mechanism, but they are burdened with potentially life-threatening autoimmunity-type adverse effects. Therefore, cancer cell-specific targets to downregulate PD-L1 could offer efficacious and less harmful ways to overcome PD_L1/PD-1 mediated immunosuppression. Serine protease hepsin is commonly overexpressed in many solid tumors where it is responsible for the activation of HGF/MET signaling pathway as well as degradation of desmosomes and hemidesmosomes leading to the loss of epithelial integrity, invasion, and metastasis. Earlier studies have linked hyperactive HGF/MET pathway to the upregulation of immune checkpoint molecule PD-L1. In this thesis, I show how pharmacological inhibition of hepsin leads to decreased MET activity and downregulation of PD-L1 in a panel of TNBC cell lines. My results demonstrate the potential of hepsin-mediated regulation of PD-L1 in tumor immunosuppression, and hint at the potential of hepsin as a therapeutic avenue towards safe and efficacious immunotherapy in the future. These results are part of a larger study addressing the role of hepsin as a regulator of PD-L1 breast cancer.

Colorectal cancer is one of the most common cancers in the world, and in 2020 it was the cause of nearly 1 million deaths. A major reason for the high death rate is treatment resistance; eventually, almost all colorectal cancer patients with metastatic disease stop responding to chemotherapy. The problem of treatment resistance is not specific to this type of cancer, but it is a widespread issue for all cancer treatments. Chemotherapy resistance is the sum of several cellular and non-cellular factors that together enable sustained cell growth despite the treatment. The non-cellular factors are related to the tumor microenvironment, whereas the cellular factors are related to changes in gene expression, which facilitate e.g. the repair of drug-induced damage or lead to changes in drug metabolism. Lately, researchers have turned their interest to translational control in chemotherapy resistance. This is because translational control plays a major role in stress adaptation. During cellular stress, global translation rates are reduced and those messenger RNAs that are most important for cell survival are translated efficiently. Moreover, translation is fine-tuned by transfer RNA (tRNA) modifications. These modifications are chemical groups that are added to the ribose and the nucleobase of the tRNA molecule, and they affect all aspects of tRNA function, ranging from the structure and stability of the molecule to reading frame maintenance and rate of translation. tRNA modifications are dynamic and change in response to the cellular state, thus facilitating adaptation by translational control. Given the major role of translational control and tRNA modifications in cellular stress responses, their role in the chemotherapy response and adaptation should be thoroughly investigated. The aim of this thesis was to study how chemotherapy affects translation and tRNA modifications in a colon adenocarcinoma model. The cell lines SW480 (from a primary colorectal tumor) and SW620 (from a metastasis) were treated with 5-fluorouracil, oxaliplatin, and leucovorin (FOLFOX), a common combination of chemotherapeutics used in colorectal cancer treatment. The cells were subjected to long-term cyclic treatment as well as 24 h pulse treatment. Chemotherapy resistant cell lines were established by increasing the concentration of FOLFOX for each round of treatment. The effect on translation was studied by polysome profiling, which revealed that FOLFOX treatment causes immediate translational stress, as evidenced by the “shoulders” in the polysomal fractions in the profiles of the pulse treated cells. We hypothesized that these shoulders represent halfmers, polyribosomes without the large subunit. No difference was observed between the long-term treated cells and controls, possibly indicating that the cells had adapted to FOLFOX. The resistant cells exhibited slightly reduced translational activity, which might be due to altered function of ribosomes following the exposure to 5-fluorouracil. Changes in tRNA modification levels were quantified by liquid chromatography mass spectrometry. Several anticodon loop modifications exhibited altered levels after the pulse treatment. In addition, 5-FUrd, a metabolite of 5-fluorouracil, was incorporated into the tRNA. The long-term treated or resistant cells exhibited no differences in the modification levels. In conclusion, this study provided insights on the immediate effects of FOLFOX treatment on translation. This constitutes the first step towards understanding how RNA-based regulatory mechanisms may contribute to the effect and possible resistance to chemotherapy.

Colorectal cancer (CRC), which refers to the cancer of the colon and the rectum currently ranks as the second leading cause of cancer related death worldwide and as the third most common form of cancer in both males and females. The latest reports show that approximately 10% of all new cancer cases globally are diagnosed as CRC annually. Initiation of sporadic CRC is commonly caused by somatic mutations causing the loss of function of the tumor suppressor gene APC. This leads to aberrant activation of the canonical Wnt signalling pathway. The ApcMin/+ mice model the progression of CRC as they carry a constitutive heterozygous nonsense mutation in Apc allele and develop intestinal adenomas. TCF/LEF transcription factor family members are best known as the main downstream effectors of canonical Wnt signalling. In the presence of nuclear β-catenin, TCF/LEF proteins bind to it through their β-catenin-binding domain and activate the transcription of Wnt target genes. The TCF7 gene encodes several isoforms of TCF1 protein, which are traditionally divided into long and short isoforms, transcribed from different promoters. Previously, it has been shown that Tcf7 deletion (Tcf7-/-) in ApcMin/+ mice increases the formation of adenomas. The aim of my study is to better understand the role of Tcf7 and its isoforms in CRC tumorigenesis. To study the Tcf7 deletion in intestinal adenoma development, ApcMin/+; Tcf7mut/mut; Villin CreERT2 mouse strain was established. The expression of the full-length isoforms (p45) is constitutively prevented in the Tcf7mut/mut mice. Moreover, tamoxifen administration to these mice led to the deletion of all isoforms in the intestinal epithelium. The number of intestinal tumors, their sizes and the survival of the Tcf7 deleted ApcMin/+ mice were analyzed and compared to ApcMin/+ mice. Intestinal tissues of the mice were collected after euthanasia. The tissue samples were preserved in paraffin, and later cut into sections for IHC, stained and imaged. The deletion of Tcf7 was confirmed at the RNA level by qPCR, and at the protein level by immunohistochemistry (IHC). IHC and single-cell RNA sequencing was used to further analyze the effect of Tcf7 deletion in mouse intestinal adenomas. The deletion of all Tcf7 isoforms or only the p45 isoforms in ApcMin/+ mice increased robustly the numbers of intestinal tumors. IHC analysis of the intestinal adenomas showed that the deletion of p45 isoforms was sufficient to cause a dramatic decrease in total Tcf1 expression in the adenoma cells. These results were supported by the qPCR results. In summary, our results lead us to believe that the deletion of p45 isoforms causes an acceleration of tumorigenesis in the adenoma model. Without the Apc mutation, the mice did not develop intestinal adenomas. Interestingly, the expression of the Wnt-target gene Prox1 in intestinal adenomas was decreased when Tcf7 was deleted. We next aim to optimize our protocol for single cell dissociation of adenomas and re-run the single-cell RNA sequencing analysis for further analysis of the mechanisms behind the increased tumorigenesis.

Endometriosis is a common complex disease that affects the quality of life of millions of women worldwide. It is characterized as an inflammatory condition where endometrium-like tissue is found at ectopic sites. The main symptoms are pain and infertility. There is no cure for the disease yet. Diagnosis requires surgery in most cases, the invasiveness is a problem. The costs for societies due to endometriosis are immense. Endometriosis, despite being a benign disease, shares characteristics with malignancies: invasion, proliferation, and angiogenesis. These enigmatic aspects make this disease an interesting subject for research. Endometriosis is shown to have a heritability of 50%. Research on the molecular genetic background is needed for the development of low-invasive diagnostic methods and better treatments for the disease. Genetic research has recently focused on genome-wide association studies of large patient and control cohorts. By design, these studies can only explain a portion of the low-risk genetic variants of common diseases. No causal high-risk gene defects behind endometriosis are found yet. In this study whole exome analysis is utilized for searching a heritable gene defect from a family of four closely related Finnish endometriosis patients in two generations. Two of the patients have a combined phenotype of endometriosis and ovarian or tubal carcinoma. Endometriosis is known to increase the risk for certain types of malignancies, endometriosis-associated ovarian cancers. Four candidate susceptibility genes for endometriosis were identified in this study FGFR4, NALCN, ZNFX1, and NAV2. The findings still need to be validated in patients not related to the study family. The variants found in this study lay a basis for screening additional endometriosis patients and functional analysis of the variants. Subsequent research on these found candidate susceptibility genes may elucidate the pathogenic pathways behind endometriosis or endometriosis-associated ovarian cancer in the future.

Breast cancer is the most common cancer in the world and among women the most cancer deaths causing cancer. MYC is a proto-oncogene, which becomes oncogenic when its expression is deregulated in cancer. MYC is commonly overexpressed in human tumours and this alteration is associated with aggressive cancer phenotype. Furthermore, alterations in the MYC network have been found in the great majority of breast cancers. MYC promotes mitochondrial apoptosis causing a cancer vulnerability, however, in cancer cells the apoptosis is often prevented by antiapoptotic BCL-2 family members. In this study, cell viability and cell death analysis of treated triple-negative breast cancer cell lines together with dendritic cell activation experiments were conducted. This study aimed to find the most potent BCL-2 family antagonist (BH3 mimetic) to combine with metformin to overcome the antiapoptotic BCL-2 family proteins inhibition of MYC-induced apoptosis. In addition, this study determined whether the combinations could induce immunogenic cell death to further intensify cancer cell killing through anti-tumour immunity. In this study, BH3 mimetics combined with metformin were found to induce cell death and reduce cell viability in TNBC cell lines. In addition, metformin and BH3 mimetics were found to activate dendritic cells directly and through immunogenic cell death of cancer cells. However, no MYC-dependent cell death or immunogenic cell death were observed, and this study was unable to indicate the most potent BH3 mimetic to combine with metformin.