Browsing by Subject "cancer"

Growth differentiation factor 15 (GDF15), a member of TGF-β super family is a soluble cytokine that is associated with different pathological conditions including cancer, cardiac and renal failure and obesity. Its high serum levels are linked with symptoms like cachexia/anorexia in cancer patients and can be used as a marker for these diseases. Its crucial role in weight regulation and energy homeostasis has been demonstrated by treating obese mice with GDF15, which results in weight lose along with improved glucose metabolism and increased insulin tolerance. It is now known that GDF15 exerts its metabolic effect by binding to a GDNF receptor -α-Like (GFRAL) receptor along with co-receptor RET. Interestingly, these two receptors co-localize only in the brain stem area of mice and humans indicating involvement of a neuronal circuit in GDF15 mediated effects. Despite its implications in major health disorders, little is known about the interaction of GDF15 with its receptors and how this interaction in turn modulates different cellular signalling and functions. The aim of the thesis was to study the mechanism and factors involved in endocytosis of GDF15. I employed high content imaging and flow cytometry techniques to visualize and analyse the internalization of ligand-receptor complex and investigate the role of actin, dynamin and phosphoinositide 3 kinase in the process. The results suggest that similar to the internalization of other cellular growth factors, the uptake of GDF15 is affected by disruption of the actin cytoskeleton. The role of dynamin is still unclear. I also discovered that the internalization of GDF15 was inefficient even in cells that expressed the receptor GFRAL, with large cell-to-cell variation. By following the intracellular localization of the receptor GFRAL, my results revealed that the receptor GFRAL is not efficiently exported to the plasma membrane and most of the protein is retained in the Golgi compartment of cells. This phenomenon was stronger in murine fibroblast cells, where the receptor was almost exclusively trapped in the secretory compartment, explaining why the uptake of the ligand GDF15 is so inefficient in these cells. The system developed during this project will now be used to analyse different factors involved in the uptake of GDF15 and eventually uncover the possible endocytic pathway. Moreover, the Golgi retention of the receptor opens up new questions to investigate like whether the physiological function of GDF15 is regulated by receptor export signals. This will help deciphering the complex and mysterious interaction of GDF15 with its receptor GFRAL.

Background Merkel Cell Carcinoma (MCC) is a rare neuroendocrine tumor that is associat-ed with old age and immunosuppressive condition. It has two distinct sub-groups differentiated with their Merkel Cell Polyomavirus (MCPyV) positivi-ty/negativity. While both groups are considered aggressive, the Merkel cell Pol-yomavirus negative group has significantly worse prognosis. Traditionally MCC cases have been diagnosed based on their physiological appearance and im-munohistochemical markers such as cytokeratin 20 and tumor transcription factor-1, which differentiate MCCs from small cell carcinomas. It still requires skilled personnel such as dermatologists and pathologists to identify MCCs. More precise and effective biomarkers are required to improve MCC diagnos-tics, to enhance patient survival and in the development of personalized medi-cine for MCC. Neurocan (NCAN) is a chondroitin sulphate proteoglycan that is found mainly in central nervous tissue in adults. The core protein of Neurocan is formed of 3 domains, G1 containing a single immunoglobulin domain, the glycosamino-glycan binding backbone and G3 domain containing regulatory protein-like sequences and epidermal growth factor/lectin-like domains. It is produced mainly by reactive astrocytes and its main function is to guide the growth of ax-ons and to participate in the formation of neural extracellular matrix. Neurocan is linked to inhibition of axonal regeneration and glial scarring in case of neu-ral injury. There are few mentions of Neurocan changes related to cancer out-side of the central nervous system, however, there is clear evidence of chon-droitin sulphate proteoglycan involvement in tumor invasiveness and potential-ly promotion of malignant tumor phenotype. Aim of the study and experimental design This thesis is a part of a project studying novel biomarkers and therapeutic tar-gets for MCC. The aim of the study was to identify a novel cancer specific gene (Neurocan) that would be either a potential biomarker or therapeutic target, and to set up the pipeline for further expanding the parent project. Neurocan was first identified from outlier gene detection methods applied to MCC sample series containing samples of 141 MCC patients. After this Neu-rocan expression levels were studied at protein level using immunohistochem-istry for MCC sample series. NCAN expression levels in MCC cell lines were investigated at mRNA and protein level with qPCR and Western blotting re-spectively. Functional studies of Neurocan such as an effect on cell prolifera-tion were performed with siRNA knockdown assays, and analyzed with West-ern blotting and qPCR. Results 144 FFPE samples in TMA (tissue microarray) format were stained for Neu-rocan protein expression; 31 samples expressed NCAN at low level, 60 at in-termediate level and 53 cases had high NCAN expression. The low NCAN ex-pression correlated with poor MCC specific survival (5-year survival 44%) when compared to intermediate and high expression groups (5-year survival 73% and 65% respectively). Kaplan-Meier survival analysis also implicated a signifi-cant difference in survival between the groups, p-value 0.044. NCAN expres-sion levels had a strong association with Merkel Cell Polyomavirus (MCPyV) status (Pearson Chi-square, p-value = 0.006) with 83% of high NCAN expres-sion cases being MCPyV positive, where as 55% of low NCAN expression cases were MCPyV negative. Cox proportional hazards model revealed that NCAN is unlikely to be an independent variable in patient survival. NCAN expression correlated with the MCPyV status of 9 tested MCC cell lines (Student’s t-test, p-value = 0.041). Protein level studies were inconclusive due to lack of specific antibodies and testing methods. 4 cell lines were tested for NCAN functionality in cell cultures. siRNA knock-down of NCAN did not affect the survival of MCC cell lines, however, it had a reducing effect on Large T-antigen expression of the MCPyV positive cell lines. Likewise, siRNA knockdown of Large T-antigen reduced the expression of NCAN mRNA in MCPyV positive cell lines. No such interactions were found in the MCPyV negative cell lines. siRNA knockdown of sT-antigen significantly reduced the growth of MCPyV positive WaGa cell line (Student’s t-test, p-value = 0.01). NCAN and large T-antigen targeting siRNAs had only a minor growth reducing effect on WaGa cell lines, and MKL1 cell line saw only minor growth reduction with all of the different siRNA treatments. These were not statistically significant findings. Whether Neurocan expression is directly controlled by MCPyV T-antigens, or whether the regulation is due to a signaling cascade of sorts, is still unknown.

Next-generation sequencing has evolved during the past 10 years to become the go-to method for genome-wide analysis projects. Based on parallelizable PCR methods adopted from the traditional Sanger sequencing, NGS platforms can produce massive amounts of genetic information in a single run and read an entire DNA molecule within a day. The immense amount of nucleotide sequence data produced by a single sample has brought us to an era of algorithmic optimization for analysis and guring out parallelization schema. For cohort projects generally cloud based systems are used due to vast computing power requirements. Anduril is an integration and parallelization framework well suited for NGS analysis, as is shown in this study. After a brief review of the golden standard methods of NGS analysis, we describe the incorporation of the main tools into the new sequencing bundle for Anduril. Tools for alignment (BWA, Bowtie), recalibration (GATK, Picard-tools) and variant calling (GATK, Samtools, VarScan) are in main focus. The Best Practice of Broad Institute, creators of The Genome Analysis Toolkit (GATK), has been a big inspiration in the creation of our sequencing pipeline. The evolution of sequencing bundle tools into a pipeline is discussed through three separate project examples. First, a small group of 8 chronic myeloid leukemia patient samples were analysed after implementation of the main tools of the pipeline. The results were consistent with previous results, but no novel relevant mutations were found. Second, exome sequencing data from 180 breast cancers with controls available in TCGA (The Cancer Genome Atlas) were processed for use in various projects in our lab. The example showed the power of Anduril in gross cohort analysis projects, enabling automatic parallelization and intelligent work ow management system. Third, we analysed exome data from 330 TCGA ovarian cancers with controls and created a prototypical set of database components for creation of a database of annotated variants for use in analytical queries. Compared to other integration frameworks (e.g. GATK, Crossbow and Hadoop), Anduril is a robust contender for the programming oriented scientist. As cloud computing is becoming at an increasing rate a requirement in large genome-wide analysis projects, Anduril provides an e ective generalizable framework for adding tools, creating pipelines and executing entire work ows on multi-nodal computing servers. As technology advances and available computational resources grow, fast multi-processor analysis can be incorporated into health care more and more for detection of disease causing genes, medication kinetics altering polymorphisms and cancer driving mutations in an everyday setting.

During the past few decades, the explosion of discovery in cancer and immunological research has led to the increased understanding of the interactions between the immune system and tumors. These developments have provided vital information about the immune system’s role in cancer development. It is evidenced that the immunity system is capable to distinguish tumor cells from normal tissue by recognizing tumor antigens that are exclusively expressed on tumor cells or are presented in greater amounts on tumor cells than normal cells. Consequently, the immune cells start to attack tumors for protecting the host. The possibility to use the immune system as a weapon against cancer cells leaded to the promising innovation – cancer immunotherapy – which aims to activate the body’s own immune system and its components to mount antitumor immune responses for eliminating cancer cells. The antitumor efficacy and high safety profile of several immunotherapeutic strategies have already been demonstrated thereby resulting in their integration into clinical practice. However, most patients have not benefited from cancer immunotherapy as a single treatment. In this regard, new innovative methods are clearly needed to overcome the obstacles hindering the clinical success of this field. Therapeutic cancer vaccines are emerging as attractive immunotherapies currently being evaluated in both pre-clinical and clinical studies. The purpose of cancer vaccines is to eradicate tumor cells by eliciting antitumor CD8+ T cell responses against the injected tumor antigens. Due to the ability to specifically kill tumor cells and simultaneously trigger immune responses against tumor antigens via direct oncolysis and by encoding transferred tumor antigens, oncolytic viruses are of significant interest for being used as in situ cancer vaccines. Despite these unique properties, several factors such as tumor immunosuppression and immune tolerance to targeted tumor antigens resembling antigens of normal tissues hamper the use of oncolytic vaccines in clinic. Instead of focusing only on CD8+ T cells, it has been suggested that giving more attention to CD4+ T helper cells, which are required for priming and expansion of CD8+ T cell responses, could be the key to improve the efficacy of cancer vaccines. Researchers have also demonstrated that an ongoing antigen-specific CD4+ T cell response can lead to the bystander activation of surrounding T cells with unrelated antigen specificities. Based on this theory, the hypothesis of this study was to employ the pre-existing immunological CD4+ memory against infectious pathogens in generating bystander CD8+ immunity against solid tumors. In this study, mice transplanted with poorly immunogenic B16-OVA tumors were pre-immunized with the chosen vaccine to induce immunological CD4+ memory against an infectious pathogen. Tumors were then treated with already developed cancer vaccine, which was peptide-coated conditionally replicating adenovirus (PeptiCRAd) complex. PeptiCRAd was constructed by electrostatically coating adenovirus with both pathogen-derived and tumor-derived peptide. The intratumorally injected double-coated PeptiCRAd complex was assumed to activate peptide-specific T cells and thus, result in anti-pathogen CD4+ T cell recall responses and the following bystander activation of antitumor CD8+ T cells, which can then mount an effective immune response to destroy cancer cells. The efficacy of this treatment was observed in pre-immunized mice by measuring the growth of injected tumors. The experiment was repeated identically with non-immunized naïve mice to see the difference in the results. The immunological background of this treatment approach was investigated by analyzing mouse tissue samples with standard immunological techniques including ELISA, IFN-γ ELISPOT and flow cytometry. This study showed that long-term immunological memory against the pathogen was successfully accomplished and the strongest inhibition of tumor growth in pre-immunized mice was achieved with double-coated PeptiCRAd, whereas the antitumor efficacy was not seen in naïve mice. Additionally, a new ex vivo method to detect pathogen-specific CD4+ T cells from spleen was developed and the stimulation of cell-mediated immunity by this treatment was supported by finding the highest levels of pathogen-specific CD4+ Th1 cells from mice treated with double-coated PeptiCRAd. Some encouraging results concerning the beneficial immune cell composition of tumors and tumor draining lymph nodes were also obtained from other performed experiments. Though further immunological analyses are required for understanding the precise mechanisms of action behind the treatment, the increased immunogenicity and antitumor efficacy of double-coated PeptiCRAd can still be considered as a consequence of the bystander effect, which can possibly be utilized for developing improved strategies to win the fight against cancer.

Diffuse large B-cell lymphoma (DLBCL) represents the most common diagnostic entity of lymphoid malignancies. As only 60% of the patients are cured with the current standard of care R-CHOP immunochemotherapy, the quest for better biomarkers and targeted therapies continues. Non-synonymous mutations in the WWE1 domain of an uncharacterized E3 ubiquitin ligase Deltex-1 have been associated with poor outcomes in DLBCL patients. Thus, to elucidate molecular features underlying this observation, this Master’s thesis set out to characterize the expression and subcellular localization of Deltex-1 in a panel of DLBCL cell lines, and to investigate the interaction partners of Deltex-1 in the activated B-cell like (ABC) DLBCL cell line context. The study aimed to gain further knowledge to understand the role that Deltex-1 plays in the pathogenesis of DLBCL, which could be used for inspecting its future possibilities as a prognostic marker or a drug target. Western blot analysis of the cell lysates revealed variable levels of Deltex-1 expression, especially between the ABC-DLBCL cell lines in comparison to germinal centre B-cell like (GCB) DLBCL cell lines. Western blots of separate cytoplasmic and nuclear fractions of the cells showed that Deltex-1 was expressed both in the cytoplasmic and the nuclear fractions of the cells, and the expression levels were reflecting the levels of the whole cell lysates of the same cell lines. The more exact localization of Deltex-1 was observed with immunofluorescence staining and microscopy of fixed cells from a few chosen cell lines. A distinct plasma membrane localization was detected in an ABC-DLBCL cell line U2932. The protein-protein interaction partners of Deltex-1 in the U2932 cell line were screened using proximity-dependent biotin labelling and affinity purification mass spectrometry. The experiments revealed novel associations between Deltex-1 and B-cell receptor signalling regulators, such as B- lymphocyte antigen CD20 and tyrosine protein kinase Lck. Though additional research is needed to define the functional mechanisms of these interactions, these findings might lead to the discovery of the connection between Deltex-1 and lymphomagenesis. In conclusion, this study provides novel information on Deltex-1 expression in the DLBCL context and describes previously unidentified associations of Deltex-1 with B-cell receptor signalling. Yet, more functional experiments are required to clarify the nature of these interactions.

Tumor cells exhibit uncontrolled proliferation, which is supported and accelerated by a constant supply of nutrients carried by blood vessels. Tumor angiogenesis, the formation of new blood vessels, besides its contribution to tumor growth, also allows the dissemination of tumor cells into distant organs. In addition to the hematogenous routes, the tumor cells metastasize through lymphatic vasculature as well. Tumor-associated lymphangiogenesis, the formation of new lymphatic vessels, is a key process in this regard. Multiple growth factor pathways regulate angiogenesis and lymphangiogenesis. Among the most important vascular growth factors implicated in this regulation are vascular endothelial growth factors (VEGFs) and angiopoietin growth factors (Angs). It has been shown that targeting VEGF/VEGFR-2 pathway could inhibit tumor growth. Many studies during the last decade have demonstrated that attenuating VEGFR-3 function inhibits primary tumor growth and also metastasis. Selective antibodies against Ang2 were shown to inhibit tumor growth and metastasis in different tumor models in mice. However, the question regarding whether combining different therapeutic methods, namely anti-VEGFR-2, anti-VEGFR-3 and anti-Ang2, will have additive benefits in comparison to single-agent therapies still remains. We aimed to test the inhibitory effects of simultaneous targeting of all VEGF pathways and Ang2 on primary tumor growth in human lung carcinoma xenografts in immunodeficient mice. To achieve this, we used soluble VEGF – trap (Aflibercept), VEGF-C/D – trap and antibodies against Ang2. Our results show that triple therapy significantly improves the inhibition of primary tumor growth in comparison to monotherapies and dual therapies. Combination of all 3 treatments also improved the reduction of intra-tumor blood vessels and lymphatic vessels. The effects of triple targeting on controlling metastasis, however, was not significant in orthotopic breast cancer model, mainly due to great variation in tumor growth in this model. However, a clear trend of reduced metastasis in several organs (liver, kidneys) was observed. Overall, this study suggests that attenuating all VEGF pathways and Ang2 could improve the inhibitory effects of anti-angiogenic treatments.

Suun mukosiitti on suun limakalvon inflammaatiotila, joka aiheutuu syöpähoidoista. Se on yksi yleisimmistä haittavaikutuksista kemoterapiaa, sädehoitoa tai kemosädehoitoa saavilla syöpää sairastavilla potilailla. Sen patofysiologia perustuu syöpähoitojen kykyyn inhiboida nopeasti jakautuvia soluja, joita syöpäkudoksen lisäksi on myös esimerkiksi suun limakalvolla. Kliinisesti suun mukosiitti aiheuttaa suun limakalvolla turvotusta, punoitusta ja kivuliaita haavaumia, joiden päälle muodostuu herkästi bakteerien muodostama plakkikerros. Pahentuessaan suun mukosiitti vaikuttaa potilaan kykyyn syödä, voi aiheuttaa keskeytyksiä syöpähoitoihin ja voi jopa johtaa sepsikseen sekä kuolemaan. Suun mukosiittia hoidetaan ennaltaehkäisemällä sitä sekä hoitamalla jo olemassa olevia oireita. Ennaltaehkäisyssä tärkeää on potilaan hyvä suuhygienia ja sen lisäksi käytetään esimerkiksi kemoterapian aikana annettua jääpalahoitoa. Erilaisia lääkkeitä sekä suuhuuhteita on tutkittu suun mukosiitin ennaltaehkäisevänä sekä oireenmukaisena hoitona, mutta toistaiseksi niistä ei ole saatu merkittävää hyötyä. Yksittäisistä hoitomuodoista hyvä teho erityisesti ennaltaehkäisevänä hoitona on saatu matala- asteisesta laserterapiasta (LLLT: low-level lasertherapy), mutta sen laaja-alaista käyttöä rajoittaa sen saatavuus ja käytännön toteutus. Koska on huomattu, että suun mukosiitin vakavuusasteeseen vaikuttavat haavaumien päälle muodostuvat bakteeripesäkkeet, on alettu tutkia erilaisia antibakteerisia hoitoja. Antibakteerinen fotodynaaminen terapia (aPDT: antibacterial photodynamic therapy) sekä antibakteerinen sinivalo (aBL: antibacterial blue light) ovat hoitomuotoja, joiden teho perustuu bakteerien epäselektiiviseen tappamiskykyyn. Kaksoisvaloterapia perustuu aPDT:n sekä aBL:n yhteisvaikutukseen. Tämä tutkielma koostuu kirjallisuuskatsauksesta suun mukosiittiin, sen patofysiologiaan ja sen eri hoitokeinoihin sekä tutkimuskatsauksesta, jossa on tutkittu kaksoisvalon antibakteerista tehokkuutta Streptococcus Oralista vastaan. Tutkimuksessa altistettiin S. Oralis pesäkkeitä kaksoisvalolle, samalla kun aPDT:n sekä aBL:n suhteellisia valon energioita muutettiin ja näin selvitettiin onko tällä vaikutusta antibakteriaaliseen tehoon. Kaksoisvalon avulla saatiin hävitettyä kaikki S. Oralis pesäkkeet huolimatta aPDT:n ja aBL:n suhteellisista valon energioista. Tutkielman lopussa liitteenä on käsitelista tutkielmassa käytetyistä lääketieteellisistä termeistä.

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.

Cancer patients have a manifold risk of suffering from both thrombotic events and anticoagulation-related bleeding complications. For this reason, knowledge of their adequate medication is crucial. The aims of this study were to find out are guidelines being followed regarding the treatment of venous thromboembolisms. The emphasis was on the anticoagulation therapy of cancer patients, but also non-cancer patients were analyzed as controls. Data was collected from the clinical information system Uranus CGI. All patient records (with the diagnostic codes I26.0, I67.6, I74.3, I80*, I81*, I83*, K55, N28.0, 022.3) in the hospital district of Helsinki and Uusimaa (Jorvi, Meilahti, Peijas, Lohja, Porvoo, Tammisaari and Hyvinkää hospitals) during the time period 1.1.2014- 29.4.2016 were reviewed. Statistical analysis was performed using the IBM SPSS Statistics and Microsoft Excel computer softwares. The study included 1667 patients, out of whom 163 (9.8%) had active cancer. The recommendation of using low molecular weight heparins as the primary anticoagulants for patients with malignancies has been practiced. More research is necessary in order to find the optimal duration for treatment of, especially, isolated calf muscle venous thromboses and cancer patients’ superficial thrombophlebitides.

The majority of cancers, such as breast cancer, originate from epithelial structures. Highly organized epithelial tissues are comprised of cells which manifest apico-basal polarization. Factors regulating apico-basal polarity and epithelial integrity are often observed deregulated in cancer cells and loss of polarity is often observed in tumors. However, the importance and the specific role of epithelial integrity regulators in tumorigenesis are still not fully defined. This study shows that the key regulators of epithelial cell polarity Par6B and Par6G proteins have a role in the restriction of cell proliferation in mammary epithelial cell lines. Using the shRNA silencing approach, downregulation of PARD6B and PARD6G in the cells lead to the impediment of the cell-cycle exit, verified in proliferation suppressive conditions in which cells normally would enter quiescence. Par6 proteins were shown to regulate cell proliferation via the canonical PI3K/Akt pathway, which is one of the most commonly deregulated cell proliferation promoting pathways in cancer cells. The results demonstrate the unknown function of Par6B and Par6G proteins as cell proliferation regulators and a previously unrecognized relation between Par6 proteins and PI3K/Akt pathway. However, the detailed interaction between Par6 proteins and the PI3K/Akt pathway ought to be investigated further. In addition, the results revealed that Par6 proteins have different effects on cell proliferation suggesting biological differences between Par6 proteins and that certainly bears further investigation. In conclusion, the study presents a previously unknown connection between epithelial integrity regulators and cancer-relevant cell proliferation promoting pathways, which may provide new targets for therapeutic intervention in the future.