Lääketieteellinen tiedekunta


Recent Submissions

  • Haikala, Heidi (2017)
    MYC is a transcription factor and a proto-oncogene, which is overexpressed in almost 50% of all breast cancers. Cell cycle promoting oncogenic MYC supports the altered energetic needs of continuously proliferating cancer cells, since there is an immense requirement for nucleic acids, proteins, and lipids to support the cell division. MYC supports the metabolic switch by enhancing the main nutrient (glucose and glutamine) uptake, increasing glycolysis and glutamine utilization, as well as by instructing the normally ATP generating citric acid cycle to serve biosynthetic growth promoting reactions. In this study we found that the metabolic changes induced by MYC lead to decreased production of ATP and the consequent activation of a key cellular energy sensor protein AMP-activated kinase (AMPK). AMPK is a metabolic guardian, which instructs cells to perform catabolic reactions to restore the ATP levels. However, persistent AMPK activity directs MYC-transformed cells towards apoptotic cell death. We found that MYC-activated AMPK phosphorylated the tumor suppressor protein p53, which accumulated on the mitochondrial surface. In the mitochondria, p53 primed the cell death promoting protein BAK for apoptotic cell death induction, thus connecting MYC-driven metabolic transformation to apoptotic sensitivity. In addition, we discovered a novel synthetic lethal and potentially druggable interaction between MYC activation and inhibition of a small GTPase RhoA. We found that RhoA assists MYC to upregulate glutamine metabolism, which highlights the relevance of metabolism as a sweet spot for targeting oncogenic MYC. Furthermore, we found that supraphysiological, highly expressed MYC is globally repressing the transcriptional targets of serum response factor (SRF), a RhoA downstream target, thus exposing new pathways needed for cancer cell survival. In addition, the same study contributed to a finding that supraphysiological MYC represses a set of genes related to luminal identity of breast epithelial cells, which possibly affects the cell differentiation and apoptotic sensitivity in vivo. The described mechanisms suggest a possible answer to the old paradox why cancerous MYC expression levels, but not physiological MYC, sensitize cells to apoptosis. Finally we studied how the altered metabolism-derived sensitivity to apoptosis could be exploited as a potential therapeutic strategy to kill MYC expressing cancer cells. In a rationalized drug combination screen, we found that the pharmacological superactivation of AMPK is able to potentiate the anticancer activity of apoptosis targeting therapies, which usually do not show great efficacy in solid tumors as single agents. The described combination proved substantial efficacy against highly MYC expressing breast cancer models in vitro, in vivo and in ex vivo. Thus, we describe the first pharmacological MYC apoptosis activation strategy that could be easily translated to the benefit of breast cancer patients, and currently clinical trials to test the combination are under consideration. In addition to the described scientific findings, the ex vivo and in vivo preclinical research models of breast cancers developed during this study are likely to serve breast cancer research community by providing more predictive and more translationally relevant platforms for studies of breast cancer biology and drug discovery.
  • Boyd, Sonja (Unigrafia, 2017)
    Primary sclerosing cholangitis (PSC) is particularly common in the Nordic countries. Most (70-80%) PSC patients have concomitant inflammatory bowel disease (IBD). The majority of patients are male. Increased levels of serum cholestasis markers in IBD patients lead to PSC suspicion, and diagnosis is verified with magnetic resonance cholangiopancreatography or endoscopic retrograde cholangiography (ERC). PSC patients usually become symptomatic over time, and PSC may lead to liver fibrosis and finally to cirrhosis. Cholangiocarcinoma (CCA) develops in 7-13% of PSC patients. The prognosis of CCA is poor. Dysplasia of the bile ducts is thought to precede the development of CCA. Liver transplantation (LT) may be performed in end-stage PSC cirrhosis, but premalignant biliary changes may also be a partial indication for LT even if the patient is asymptomatic. At Helsinki University Hospital, PSC patients regularly undergo systematic dysplasia surveillance with ERC and brush cytology (BC) to detect dysplastic changes of the bile ducts and to determine disease progression. ERC changes are graded according to the modified Amsterdam ERC score. If suspicion of dysplasia is repeatedly seen in BC, patients are referred to liver surgeons for consideration of LT. Our aims were to evaluate the relevance of the screening protocol at PSC diagnosis and later during the disease course to identify risk factors for biliary neoplasia, and to evaluate the utility of ERC, BC and ploidy analysis of biliary epithelium in detecting biliary neoplasia. In their first, diagnostic ERC (n=261), 81% of PSC patients were asymptomatic, but 43% already had advanced ERC changes. Seven percent had malignancy or malignancy suspicion in BC. Those who had elevated liver enzymes and advanced ERC changes were most likely diagnosed with biliary neoplasia during follow-up. Female patients had milder ERC changes and a lower cumulative risk of biliary neoplasia. During the follow-up period (median 4.7 years), 2.7% were diagnosed with CCA and 3.1% with biliary dysplasia. Independent risk factors for biliary neoplasia were elevated extrahepatic ERC score, suspicious/malignant BC and elevated serum alanine aminotransferase. During surveillance (median 6 years) with ERC and BC, 3.8% out of 447 PSC patients were diagnosed with CCA, and 5.1% had dysplasia in the explanted liver. ERC score, male gender, elevated liver enzymes, serum tumour markers, suspicious BC and inflammation or aneuploidy in BC were risk factors for CCA. All patients with biliary neoplasia had intra- and extrahepatic ERC changes. 126 PSC patients underwent liver transplantation in Helsinki University Hospital during 1984-2012, and 28% of these LTs were due to neoplasia suspicion for asymptomatic patients. The combined sensitivity of BC and ploidy analysis to detect biliary neoplasia was 68% with 86% specificity. The ten-year survival of asymptomatic patients after LT was 91%. Screening and surveillance of PSC patients with ERC and BC may help find patients that are most prone to develop CCA. Patients with advanced extrahepatic ERC changes and elevated liver enzymes are most likely to be later diagnosed with CCA. On the other hand, patients with intrahepatic ERC changes only have a negligible CCA risk. If BC is repeatedly suspicious, even asymptomatic patients should be referred to evaluation of LT.
  • Räsänen, Minna (Helsingin yliopisto, 2017)
    Aims: Rectal cancer, one of the most common cancers worldwide, causes significant mortality. After curative rectal cancer surgery, local recurrences (LR) are detected in 4-10% and distant metastases in 20-31% of the patients. For diagnosing recurrence, the surveillance is recommended, but the optimal frequency, length and methods are not well established. This study analysed the pattern of rectal cancer recurrence, the usefulness of surveillance and the different surveillance instruments available. Nowadays, low colorectal or coloanal anastomosis is performed whenever possible because most patients prefer anus-preserving surgery over a permanent stoma. The covering stoma is used to facilitate anastomosis healing and to protect against fatal complications. In this study, we determined the cumulative risk of and the risk factors for failure of low colorectal or coloanal anastomosis. In locally advanced rectal cancer, when the tumour has invaded the adjacent organs, a multivisceral en bloc -resection is needed. In these resections, morbidity and mortality are higher than in standard resections. An attempt is made to decrease tumour size by neoadjuvant long-course (LC) chemoradiotherapy (CRT); the goal is to achieve clear resection margins. The oncological results of multivisceral resections for rectal cancer were evaluated, as were the risk factors for LR and survival. The response to CRT seen in response magnetic resonance imaging (MRI) and the histopathology report was analysed. In addition, the usefulness of the response seen in MRI and the pathological specimen in predicting curative resection, risk of LR and survival was investigated. Neoadjuvant short-course radiotherapy (SCRT) has been reported to diminish the rate of LR. Anyhow, meticulous total mesorectal excision (TME) surgery has good local control without RT in so-called good T2-T3N0-1 tumours , and the use of SCRT has been questioned in many countries because of side-effects and uncertainty of utility. The aim of this study was to determine, whether SCRT prevents LR in pT3N1-2 patients compared with those operated on without RT. Patients and methods: A total of 952 rectal cancer patients were operated on at Helsinki University Hospital between January 2005 and June 2014. Study I comprised 481 consecutive rectal cancer patients treated with radical intention during 2005-2011. Study II comprised all patients (n= 273) operated on with anterior resection (AR) combined with low colorectal or coloanal anastomosis and covering stoma during 2005-2011. The Study III population consisted of 94 patients, operated on because of locally advanced rectal cancer by multivisceral resection between 2005 and 2013. In Study IV, 151 patients operated on because of pT3N1-2 rectal cancer during January 2005-June 2014 were included. Of these 151 patients, 57 did not have radiotherapy (RT) before the operation and 94 had received short-course RT before surgery. Data were retrospectively collected from patient records. Results: Local recurrence occurred in 8.3% and distant metastases in 23.3% of patients treated with curative intention. The median time to recurrence was 1.3 years after operation, and 75% of recurrences were diagnosed during the first two postoperative years. Recurrences were detected significantly earlier by the follow-up visits than by symptoms. All of the surveillance instruments used, were equally useful for finding recurrences. Curative re-operation was possible in 41.1% of patients with disease recurrence. The colorectal or coloanal anastomosis was switched to a permanent stoma in 23 (8.5%) of 271 patients. Protective stoma was not closed in five patients (1.8%). Total permanent stoma rate was 10.3%. The risk factors for failure of low colorectal or coloanal anastomosis were tumour location under 6 cm from anal verge, coloanal anastomosis, early anastomotic complication, anastomotic fistula, anal incontinence, and LR. After multivisceral resection, LR occurred in 10.6%. The median time for detecting a recurrence was 1.4 years (0.7-5.2). The 5-year overall survival (OS) for the whole population was 51.8% and for curatively treated patients (n=83) 59.2%; the 5-year disease-free survival (DFS) for the latter group was 64.2%. A significant risk factor for LR and survival was R1 resection. Poor or no response in post-treatment MRI predicted LR and also seemed to predict DFS in curatively treated patients. Of patients having pT3N1-2 rectal cancer, LR occurred in altogether 11.3%. The rate of LR in the surgery only group was 14% and in the RT + surgery group 9.6%. Risk factors in univariate analysis for LR were tumour location under 6 cm from anal verge, positive circumferential resection margin (CRM), tumour perforation and mucinous histology. In multivariate analysis, the risk factors were tumour location under 6 cm from anal verge and positive CRM. The short-course RT did not prevent LR. Conclusions: The most intensive follow-up should fall within two to three years for all patients treated with curative intent, and thereafter, only patients with known risk factors for recurrence should be followed. The risk of permanent stoma after low anterior resection in our study population was low, most probably due to routine use of covering stoma. Multivisceral resection is safe and has an over 50% 5-year OS and almost 90% local control. Poor response for CRT in post-treatment MRI predicts LR and seems to be associated with decreased DFS in curatively treated patients. Achievement of R0 multivisceral resection is most important concerning LR and also survival. Short-course radiotherapy did not affect LR risk in pT3N1-2 rectal cancer patients. Larger studies allowing analysis of subgroups with different T3 classes and N1 and N2 groups separately are needed to evaluate the role of short-course neoadjuvant radiotherapy in T3N+ tumours.
  • Hämäläinen, Satu (Helsingin yliopisto, 2017)
    Osteoarthritis (OA) is the most common joint disease and a frequent cause of pain and disability, with the joints of the hand being the most frequently affected site. OA is a complex disorder that results from the interplay of genetic and environmental factors. It becomes more prevalent with age, and after the age of 50 a higher proportion of women are affected than men. We examined some possible genetic risk factors of hand OA with consideration of previously proposed risk mechanisms, namely: loading of cartilage structure, inflammation, and overweight. We also attempted to replicate previously found associations between hand OA and susceptibility genes, and to analyse in our material the findings of earlier publications that reported associations of gene variants with OA in joint sites other than the hand. The participants in this study were 542 dentists and teachers from the Helsinki metropolitan region. The single nucleotide polymorphism (SNP) in the COL2A1 gene, which encodes for the main collagen present in normal cartilage, and was previously associated with generalised OA, was also associated with OA in the hand; among dentists the risk was doubled in carriers of this allele compared with those with the major allele. A history of repetitive hand loading tasks combined with carrying the minor allele further increased the risk. Minor alleles of the pro-inflammatory TNFα SNPs, and their haplotype, were associated with an increased risk of hand OA. Further, interactions between TNFα, IL4R, and IL10 SNPs were found, which suggest that the effect of the TNFα polymorphisms on hand OA was modified by the IL4R and IL10 gene variants. Minor alleles of the adipokine RETN SNPs, and the haplotype containing these minor alleles, decreased the risk of ROA. The association of the haplotype with the decreased risk of hand OA was stronger in overweight women. In contrast, the RARRES2 SNP minor allele and LEPR haplotype increased the risk of hand OA. The LEP haplotype, however, was associated with a lower risk of ROA only among overweight women. These findings suggests that BMI is an effect modifier of these associations. A replication study found that the A2BP1 SNP minor allele decreased the risk of ROA, which confirms the original finding. The minor allele of the spinal OA candidate gene TGFB1 almost doubled the risk of symptomatic DIP OA. Teachers with the minor allele of ESR1 rs9340799 SNP had an almost tripled risk of symptomatic DIP OA. Furthermore, we observed interactions between SNPs and occupation in their relation to hand OA. In conclusion, 43 SNPs from 27 candidate genes were analyzed in this study and SNPs in eight of them (COL2A1, TNFα, LEP, LEPR, RARRES2, RETN, A2BP1, and TGFB1) were found to be associated with susceptibility to hand OA. Interactions between the genotypes, occupational loading and BMI were also observed.
  • Hilander, Taru (Hansaprint, 2017)
    Proteins, consisting of amino acids, work as building blocks in the cells. In addition, they carry out vast amounts of cellular functions. Accurate protein synthesis is thus crucial for the normal function of cells. The first step of protein synthesis is the charging of transfer-RNAs (tRNAs) with their cognate amino acids. Evolutionarily conserved and extremely old proteins, aminoacyl-tRNA synthetases (aaRSs), carry out this step and each amino acid-tRNA pair has its own synthetase for the task. However, in some cases the amino acids are so similar in size and structure that they cannot be separated well enough by the aaRSs. To avoid mischarging and the subsequent protein misfolding, some of the synthetases have an editing domain, which recognizes and hydrolyses incorrect amino acid-tRNA pairs. In addition, cells have other important quality control mechanisms to ensure protein homeostasis, the capacity of cells to maintain internal stability of the proteome. Patient mutations in genes connected to protein synthesis and quality control are found to cause different diseases, the mechanisms of which are not yet, however, well known. Research on these topics is thus important. The aim of this thesis was to study the molecular mechanisms of different tRNA-charging defects and protein quality control mechanisms in both protein-translating compartments of a eukaryotic cell, cytosol and mitochondria. The first part of the thesis describes the molecular mechanism and the clinical phenotype of a special cytosolic tRNA charging defect caused by mutations in SEPSECS gene. The corresponding protein is involved in charging of the 21st amino acid, selenocysteine, to its tRNA. We identified mutations in this gene and showed them to lead to a decreased amount of selenocysteine-containing proteins, selenoproteins, in the brain of a patient with a severe early onset encephalopathy. Our study also indicated increased protein oxidation in the patient brain. This study extends the clinical phenotypes connected to SEPSECS mutations, and indicates that selenoprotein synthesis defect can resemble mitochondrial disease with lactate elevation. In the second part of this thesis, the potential of an amino acid analogue of arginine, canavanine, to induce protein misfolding in mitochondria was studied. The results demonstrated that mitochondrial protein translation machinery does not distinguish canavanine from arginine. The amino acid analog was incorporated into mitochondrially encoded proteins causing protein instability and formation of aberrant polypeptides. Surprisingly, however, canavanine did not induce mitochondrial unfolded protein response (UPRmt), a previously described signalling pathway induced by accumulation of misfolded proteins inside mitochondria. The study showed that none of the protein quality control mechanisms were able to solve protein misfolding caused by canavanine, which led to a severe respiratory chain defect. Canavanine has been used previously in a large number of studies to induce cytosolic protein misfolding, but the impact of canavanine for mitochondrial function has been largely ignored. Canavanine can be used in future as a tool to study further the consequences of protein misfolding in mitochondria, and for studying how mitochondria solve stalled ribosomes, which was also detected to be a consequence of canavanine in our study. The goal of the third part of the thesis was to study UPRmt in an animal model. The purpose was to generate a mouse model that has a mutation in the editing domain of mitochondrial alanyl-tRNA synthetase, leading to amino acid mischarging and formation of unfolded proteins inside mitochondria in vivo. The result of the study indicated for the first time the importance of amino acid editing by a tRNA synthetase as an essential quality control mechanism in mammalian mitochondria. The work presented in this thesis provides new information concerning the mechanisms of different tRNA charging defects and their consequences for the cell and organism. Special emphasis was on mitochondrial function.
  • Porkholm, Mikaela (Unigrafia Oy, 2017)
    Although most children with cancer can be cured, every fifth patient still succumb from their disease. The need for new treatments and more refined diagnostic methods are thus understandable. Angiogenesis, the formation of new blood vessels, is an essential phenomenon in many malignancies. The first study in this thesis evaluated the role of two regulators of angiogenesis, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), during allogenic hematopoietic stem cell transplantation (HSCT) in 67 children. High levels of VEGF and Ang-2 in plasma samples collected after allogenic HSCT correlated with severe acute graft-versus-host disease (GVHD). High Ang-2 post-HSCT was associated with increased non-relapse mortality and, together with concomitantly high VEGF, correlated with inferior event-free survival. However, methodological challenges should prompt cautious interpretation of these results. The second study summarized results of the anti-angiogenic Angiocomb protocol in the treatment of pediatric diffuse intrinsic pontine gliomas (DIPGs). The survival of study patients (N = 41) did not differ from that of controls (N = 8), but the ability to attend school or daycare and walk was maintained for a longer time after the diagnosis in the study cohort. The Angiocomb protocol was generally well tolerated, with the most frequent adverse event being neutropenia. The third study explored the effect of the Extended Angiocomb protocol in a cohort of 17 heavily pre-treated children with high-risk or end-stage malignancies. Although a significant increase in Karnofsky-Lansky performance scores occurred in patients during therapy, the protocol was more toxic than anticipated. This underlines the importance of careful patient selection and close monitoring during metronomic therapy in heavily pre-treated patients. The fourth study analyzed genetic alterations by next-generation sequencing and protein expression and microvessel density (MVD) by immunohistochemistry in 26 DIPG tumor samples. 87% of the patients had mutations in H3-K27M, excluding two rarely encountered long-term survivors. One of the long-term survivors had an IDH1 mutation, an alteration formerly considered to be absent in DIPGs. Angiogenesis-related genes were altered in 40% of the patients, whereas MVD showed up to 6-fold variation. The clinical significance of these findings requires further studies in the future.
  • Järvinen, Riikka (Unigrafia, 2016)
    Non-muscle-invasive bladder carcinoma (NMIBC) should be treated with a transurethral resection of the bladder tumour (TURBT). The need for an adjuvant intravesical treatment is evaluated after TURBT and is based on various tumour characteristics. The aim of the adjuvant intravesical treatment is to reduce the number of recurrences and the risk of progression. This thesis consists of four individual FinnBladder (FB) studies that compared different intravesical regimens of chemo- and immunotherapy among patients with intermediate- or high-risk NMIBC. The present thesis analyses the long-term results of those studies. Patients in these studies belonged to the intermediate- or to the high-risk group of NMIBC. Patients in study I were treated with mitomycin C (MMC) or bacillus Calmette-Guérin (BCG) instillations for up to one year, whereas the patients in study II had one perioperative plus four weekly MMC instillations followed by MMC or alternating MMC and BCG instillations. The patients in study III had one perioperative MMC plus four weekly MMC instillations followed by either BCG or alternating BCG and interferon-α2b (IFN) instillations. The patients of study IV had one perioperative epirubicin (EPI) followed by BCG or combination of EPI and IFN. The follow-up times of the patients that were still alive were between 8.0 (study IV) and 19.4 years (study II). The median follow-up times of all patients were between 7.2 (study II) and 8.6 years (study III). In study I, we found that BCG significantly reduced the time to recurrence but had no effect on progression, disease specific survival or overall survival compared to MMC. In study II, we found no difference between the study groups (one perioperative MMC and four weekly MMC followed by BCG or alternating MMC and BCG). However, the risk of dying of BC appeared to be markedly lower than observed in the series of untreated patients. In study III, we found that one perioperative and four weekly MMC instillations followed by a monthly maintenance with BCG significantly reduced the probability of recurrence compared to maintenance with alternating BCG and IFN. In study IV, we found that one perioperative EPI administration followed by five weekly BCG and a 1- to 2-yr BCG maintenance treatment was significantly better in reducing recurrence than was a combination of EPI and IFN using the same schedule. Two subgroup analyses of our studies revealed a lower probability of recurrence in patients that had been treated with a single perioperative chemotherapy instillation compared to those without the single instillation. The risk of progression was very low in the best treatment groups of our studies after 10 years, which might question the benefit of a recommended life-long follow-up for all patients. Three out of four patients who progressed died of BC despite the relatively meticulous follow-up. Better methods are still needed to recognize the progression in NMIBC early enough to be able to lower the mortality of this disease.
  • Taipale, Kristian (2016)
    In the history of oncology scientists have tried to treat cancer in numerous different ways. Traditionally surgery, chemotherapy and radiotherapy have been regarded as the three main treatment modalities. However, harnessing the power of the human immune system by cancer immunotherapy has recently attracted considerable interest, even though studies on immunotherapy started already more than hundred years ago. The crucial feature of immunotherapy is that it reshapes the immunosuppressive environment created by the tumor. Immunosuppression blocks the activity of body s own immune system, which would normally destroy the transformed cells, and it has been identified as one of the hallmarks of cancer. Oncolytic viruses are a promising form of immunotherapy, and the first viruses have already been approved for clinical use. Oncolytic virotherapy is based on the concept of selective viral replication inside tumor cells. Replication causes tumor cell lysis, release of danger signals and immune cell activation. Viruses can also be modified to stimulate the immune system further, for example by inserting immunostimulatory cytokines into their genomes. One of the most popular viruses used as an oncolytic platform is adenovirus, which is a non-enveloped double-stranded DNA virus. Treatments with oncolytic adenoviruses have been previously shown to be safe and able to activate the anti-tumor immunity in pre-clinical and clinical settings. This thesis investigates the influence of adenoviral immunotherapy on the human immune system. Additionally, predictive and prognostic potential of several immune-related biomarkers and clinical variables is evaluated. The patients included in these studies were treated as a part of the Advanced Therapy Access Program (ATAP), which was a clinical access program in contrast to a clinical trial. The thesis includes analyses from all of the 290 patients treated in ATAP. The first part of the thesis includes findings from the T cell subsets in the peripheral blood and tumors of 50 patients treated with an oncolytic adenovirus. When comparing pre-and post-treatment samples, a significant shift in the ratio of CD4- and CD8-positive T cells in peripheral blood was identified. The proportion of CD4-positive T helper cells decreased in the majority patients, while the proportion CD8-positive cytotoxic T cells increased. Interestingly, the changes in peripheral blood T cell levels were correlated with T cell levels in the tumor biopsies. Moreover, this correlation was found to be inverse for CD4-positive T cells and positive for CD8-positive T cells. The second study of the thesis concerned the pre-treatment tumor biopsies of 27 patients treated in the ATAP program. Several pathways related to immune responses, such as B cell receptor signaling, GM-CSF signaling and leukocyte extravasation signaling, were activated in patients surviving a shorter time than their matched controls. In the level of individual genes, several macrophage markers, complement components and complement receptors were also upregulated in this same patient population. To verify findings on a protein level, immunohistochemical stainings were performed for 19 biopsies. In these analyses, helper T cell marker CD4 and macrophage marker CD163 were significantly higher in patients with poor prognosis. Relating to infiltration of T helper cells, many T cell exhaustion markers, such as TIM-3, PD-L1, PD-L2, CTLA-4, were increased in patients with worse than expected survival, possibly suggesting a presence of exhausted T cell population. Third part focused on identifying predictive and prognostic factors for adenoviral immunotherapy. In this study, absence of pre-treatment neutralizing antibodies and low baseline neutrophil-to-lymphocyte ratio were found to correlate with a significantly longer overall survival, whereas absence of liver metastases was associated with an improved disease control rate. In multivariate Cox regression tumor type, gender and WHO performance status were linked to a lower hazard ratio for tumor related mortality. Interestingly, treatment with viruses that were armed with immunostimulatory cytokines was also associated with a lower hazard ratio. In predictive regression analysis intraperitoneal administration route of the viral treatment was correlated with a significantly higher odds ratio for disease control. In addition to improving the understanding on immunologic phenomena related to adenoviral immunotherapy, these results offer interesting directions for the development of biomarkers and clinical criteria for selecting the right patients for the treatments. Moreover, the findings provide a rationale for combination studies with already approved and emerging immune checkpoint inhibitors. In the future this knowledge could possibly be utilized to improve also the design of the viruses by targeting them more specifically to the immunosuppressive pathways that are activated in cancer.
  • Aakula, Anna (Helsingin yliopisto, 2016)
    MicroRNAs (miRNAs/miRs) impact on cellular signaling pathways and biological processes in physiological and pathological states. The expression of miRNAs is often altered in cancer, but the functions and their targets are less well understood. In this thesis project, the miRNA regulation of genes relevant for PCa proliferation was comprehensively mapped through phenotypic screening and tumor expression data. Given the role of androgen receptor (AR) as a key oncogenic driver in PCa and estrogen receptor α (ERα) playing a similar role in BCa, the regulation of these receptors was investigated here. Systematic gain-of-function screens were conducted with 1129 miRNA molecules and changes in AR, proliferation marker Ki67, and the apoptotic marker cPARP were quantified using reverse-phase protein arrays. The results identify miR-19a, -32, -124a, -130b, -148a, and -583 as regulators of proliferation and, as potential regulators of FLNC, MSRB3, PARVA, PCDH7, PRNP, RAB34 and SORBS1 genes. These aberrantly expressed genes were significantly associated with biochemical recurrence-free survival of 140 PCa patients. Altogether 13 miRNAs that regulate the AR 3'-untranslated region (3'UTR) (miR-135b, -185, -297, -299-3p, -34a, -34c, -371-3p, -421, -449a, -449b, -634, -654-5p and -9) were identified and validated in PCa. Interestingly, the RNA-sequencing data revealed a longer 3 UTR of AR, and most of the AR targeting miRNAs were found to act via this extended AR mRNA. MiR-135b and miR-34c showed an inverse association with AR protein levels in 47 clinical PCa samples. Similarly, miR-135b targeted the ERα 3'UTR in BCa and showed significant inverse correlation with the steady-state ERα protein levels in a cohort of 101 breast tumors. MiR-135b also inhibited the growth of AR-positive PCa and ERα-positive BCa cells, whereas it failed to affect the AR- and ERα-negative cells. Prolonged miR-135b expression in 3D and xenograft experiments, however, seem to confer a growth advantage and putatively induce an epithelial-to-mesenchymal switch in LNCaP cells. Taken together, this study identifies specific miRNAs as important regulators of AR, ERα, and HIF1AN protein levels, as well as novel regulatory molecules, which impact on PCa and BCa proliferation and are aberrantly expressed in tumors. This reveals novel regulatory nodes with potential as diagnostic biomarkers and that could be considered as targets for therapy or as therapy if delivery of miRNA as therapy becomes feasible.
  • Leppälahti, Suvi (Helsingin yliopisto, 2016)
    Teenage pregnancy is recognized by the World Health Organization (WHO) as one of the main public health concerns worldwide. Teenage childbirth and motherhood are associated with substantial socioeconomic and health inequalities. Research concerning those who undergo abortion at a young age remains limited, partly due to lack of reliable data. The present register-based study was designed to assess teenage pregnancy in Finland from 1987 to 2012. The aim was to investigate the trends, determinants and health and social consequences of teenage abortion and childbirth. The trends in teenage abortion between 1987 and 2009 (n=52 968) were examined. The proportion of abortions among all teenage pregnancies remained steady throughout the study period, being 82% among 13- to 15-year-olds and 41% among 18- to 19-year-olds in 2007–2009. Great fluctuation in the incidence of teenage abortion was seen (8.0/1000 in 1993, 11.5/1000 in 2003 and 9.7/1000 in 2009 among all teenagers). Repeat abortions almost doubled among the 16- to 19-year-olds. Obstetric outcomes were compared between all nulliparous teenagers (13–19 years [n=7305], further analyzed in groups by specific age) and control women (25–29 years, n=51 142) with singleton deliveries in 2006–2011. Teenagers faced increased risks of maternal complications during pregnancy, including a risk of urinary tract infection (UTI) (adjusted OR 2.9 [1.8-4.8]), pyelonephritis (6.3 [3.8-10.4]) and eclampsia (3.2 [1.4-7.3]). However, they were more likely to have uncomplicated vaginal deliveries (1.9 [1.7-2.0)]. The 1987 Finnish Birth Cohort was used to analyze the determinants and consequences of teenage pregnancy up to 25 years of age: girls who had experienced underage (<18 years) abortion (n=1041, 3.6%) or childbirth (n=395, 1.4%) were compared with each other and with girls who had no pregnancies. Five percent of all the girls in the cohort experienced underage pregnancy. Early-onset behavioral and emotional disorders and poor socioeconomic background were associated with higher risks of childbirth, and to a lesser degree, abortion. Specific risk factors of underage induced abortion were psychoactive substance use disorders (2.2 [1.3-3.5]), and having a mother who smoked during pregnancy (1.5 [1.3-1.8]) or had undergone induced abortion (1.8 [1.5-2.2.]) When the early adulthood outcomes of those who had experienced underage abortion were compared with those who gave birth, no significant differences in the risks of psychiatric disorders were found. Those who underwent abortion were more likely to achieve higher educational levels (2.4 [1.2-5.0]). When compared with those with no pregnancies, both pregnancy groups had elevated risks of most adverse conditions, but the risks were similar before and after conception. In conclusion, the low incidence of teenage pregnancy in Finland suggests that reproductive health services function comparatively well. To add, abortion or childbirth per se was not associated with mental ill-health. However, a substantial proportion of girls who experience a teenage pregnancy have a disadvantaged background and mostly for that reason, they seem to be a risk group for adverse outcomes in adulthood. These girls should be offered extra attention by social and healthcare professionals to prevent unplanned teenage pregnancies and further marginalization.
  • Martelius, Laura (Helsingin yliopisto, 2016)
    The occurrence and significance of vertical lung ultrasound artifacts known as B-lines were investigated in children. B-lines are a non-specific sign of lung pathology. They can be used to estimate lung liquid and to diagnose lung disease. Healthy term infants, children with congenital heart disease undergoing cardiac surgery, and children undergoing computed tomography of the chest were included in the study. We examined whether the mode of delivery influences B-lines in healthy term infants and compared lung ultrasound with static lung compliance, computed tomography, and chest radiographs. B-lines were more numerous in infants born by elective cesarean section than in those born vaginally. Lung edema scores from lung ultrasound and chest radiographs correlated significantly, but B-lines and static lung compliance did not. A consistent association was present between the number of B-lines in ultrasound and the extent of parenchymal changes in computed tomography. B-lines appear suitable for documenting resolution of lung liquid during postnatal adaptation. They may indicate lung edema after cardiac surgery and detect lung disease in children.
  • Hyvönen, Maija (2016)
    Gliomas, malignant brain tumors, are among the most aggressive cancers in adults. Due to their invasive growth, resistance mechanisms and tendency to relapse, the prognosis of high-grade glioma patients is very poor. Therefore, to improve therapeutic strategies it is essential to study molecular mechanisms underlying the progression of gliomas and identify novel, glioma-specific proteins that could be therapeutically targeted. In this study we first mapped vascular markers of malignant gliomas to find potential candidates for tumor targeting. By using in vivo phage display method we identified a peptide, CooP, which, after systemic delivery, specifically homed to brain tumor satellites and their vasculature in mice. Coop was successfully used in tumor imaging and targeted drug delivery. We also identified mammary-derived growth inhibitor (MDGI) as an interacting partner for CooP in the brain tumor tissue and tumor-associated vasculature. Homing peptide-conjugated nanocarriers have shown great potential in targeting various tumors, including gliomas. Therefore, we estimated the potential of CooP peptide in the surface functionalization and targeted delivery of porous silica nanoparticles in vitro and in vivo. CooP-functionalized particles were shown to be stable, non-toxic and suitable for targeting MDGI expressing subcutanous xenografts. In the last part of this study we characterized the expression and function of MDGI in gliomas. Our immunohistochemical analyses revealed abundant MDGI expression in clinical brain tumor specimens and patient-derived gliospheres. In lower-grade glioma patients MDGI expression correlated with poorer overall survival. Importantly, also endothelium-associated expression of MDGI in the clinical samples was observed. Functional in vitro and ex vivo assays demonstrated that MDGI overexpression enhanced the aggressive growth of glioma cells, whereas even more striking changes occurred when MDGI was genetically silenced. MDGI silencing compromised the growth of human gliospheres, altered the expression of stress-related proteins, disrupted mitochondrial function and eventually caused apoptotic cell death. In addition, mass spectrometric analyses revealed significant changes in the intracellular metabolites after MDGI silencing. Together our results show that the glioma-specific CooP peptide can be utilized both in glioma imaging and targeted drug delivery as well as in biofunctionalization and targeting of nanoparticles in vivo. In addition, MDGI, the interacting partner of CooP, is abundantly expressed in malignant gliomas and essential for glioma cell survival. Since MDGI is reachable via intravenously injected agents, it could be a potential candidate for the targeted treatment of gliomas.
  • Holopainen, Tanja (Helsingin yliopisto, 2016)
    Angiopoietins (Angs) and vascular endothelial growth factors (VEGFs) regulate angiogenesis, the formation of new blood vessels, and lymphangiogenesis, the formation of new lymphatic vessels. Angiogenesis is important in cancer, because for continuous growth a primary tumor needs a supply of oxygen and nutrients delivered via blood vessels. In physiological conditions, the lymphatic vasculature serves to collect interstitial fluid as well as to absorb lipid particles. In the context of cancer, lymphatic vessels serve as a route for the metastatic dissemination of tumor cells. This dissertation aimed to explore the role of vascular endothelial growth factor receptor 3 (VEGFR3) and angiopoietin-2 (Ang2) in cancer progression. In particular, the roles of these proteins in the context of tumor angiogenesis and lymphangiogenesis as well as metastasis were investigated in two separate studies. In addition, the effects of the photodynamic ablation of intralymphatic cancer cells and lymphatic vessels on the development of metastases were explored. Furthermore, the effect of the endothelial bone marrow tyrosine kinase in chromosome X (Bmx) on tumor angiogenesis was investigated. We found that the inhibition of VEGFR3 reduces tumor blood vasculature and primary tumor growth. In the second study we found that in-transit tumor cells can be targeted with verteporfin-induced photodynamic therapy. The photodynamic ablation of lymphatic vessels was improved by combining this strategy with anti-lymphangiogenic therapies via the adenovirally mediated soluble expression of the VEGF-C/D trap. Bmx, a member of the Tec non-receptor tyrosine kinase family, was previously shown to promote tumor cell survival, but relatively little is known about the effect of this kinase on tumor biology. Here, we studied the effects of Bmx tumor angiogenesis by using Bmx gene-deleted mice. Some reduction of primary tumor growth in Bmx-/- mice was detected in several isogenic and oncogenic tumor models, along with some attenuation of tumor angiogenesis. Conversely, overexpression of Bmx resulted in increased tumor progression and angiogenesis. Ang2 is known to be a context-dependent agonist of the tyrosine kinase of the Tie2 receptor. Here, we analyzed the effect of Ang2 on metastatic dissemination into the lungs, finding that the overexpression of Ang2 enhanced lung metastasis. In contrast, Ang2 inhibition decreased the occurrence of lung metastases. In the ultrastructural analysis of the metastatic lungs using transmission electron microscopy, anti-Ang2 treatment attenuated tumor-associated changes in metastasis-associated lung capillaries. This dissertation demonstrates that the blockade of VEGFR3 inhibits tumor angiogenesis, and that the inhibition of Ang2 inhibits lymphatic and lung metastasis and improves endothelial integrity. Furthermore, we demonstrated the ability of photodynamic therapy to eradicate lymphatic vessels and intralymphatic cancer cells. These data provide a rationale for developing new cancer therapies targeting the lymphatics in order to reduce metastasis and tumor progression. Taken together, these results provide new insights into endothelial tyrosine kinase-mediated angiogenesis, tumor lymphangiogenesis, and vascular-based therapeutic strategies in cancer.
  • Lahtela, Jenni (Helsingin Yliopisto, 2016)
    Lung cancer is the leading cause of cancer related deaths worldwide. It is characterised with a high level of intra- and intertumour heterogeneity. Large lung cancer sequencing efforts have identified clear histopathology-specific genetic alteration patterns, which in the cases of lung adenocarcinomas are applied in clinics to direct treatment. Furthermore, lung cancer immunotherapy approaches have recently shown promising results in clinical trials. However, a deeper understanding of the functional importance of novel lung cancer genes as well as the lung cancer-related niche and cell type specific propensities leading to molecular and microenvironmental tumour heterogeneity is needed to better translate the growing amount of information to patient stratified treatments. The first part of this thesis work concentrated on the functional in vitro and in vivo investigation of putative tumour suppressive characteristics of the EPH receptor A3 (EPHA3), a gene commonly mutated in human lung cancers. Our in vitro findings supported the tumour suppressive characteristics of EPHA3 and indicated that EPHA3-mediated tumour suppression was specifically dependent on its kinase activity. However, our in vivo investigation demonstrated that loss of EphA3 does not co-operate with two known genetic alterations of human lung cancer in murine lung tumourigenesis nor it effects lung morphogenesis. Hence, we conclude that our study demonstrates how functional validation of putative cancer genes can be challenged by biological complexity, which may result in acquired compensation or different functional roles in human and mice. The results from the second part of this thesis work showed that cells in the airways of mouse lungs had a higher propensity to develop faster growing and progressing lung tumours than the cells in the distal alveolar space when exposed to known lung cancer genetic alterations, namely expression of oncogenic Kras and loss of Lkb1 (KL). The lung tumours originated from the airways were predominantly classified as adenosquamous carcinomas (ASCs). ASCs showed elevated levels of genes associated with immunosuppression and a notable immune cell infiltration with an increase in the amount of possible myeloid-derived suppressor cells (MDSCs). The KL ASC model may thus represent a relevant preclinical model for the study of anti-MDSC immune therapy as a treatment for ASCs, which in humans represent a rare but aggressive type of lung cancer. Thus, the findings in this thesis work highlight the importance of the functional niche in the progression of lung cancer and, therefore, possibly affecting a response to treatment. Niche-specific investigation of lung cancer genetic alterations thus leads to a more accurate stratification of the preclinical in vivo models, simultaneously revealing relevant molecular mechanisms underlying lung cancer heterogeneity.
  • Saukkonen, Suvi (2015)
    Disruptive behavior is one of the most common psychiatric problems in children and adolescents. Psychopathic-like features designate a group of children and adolescents manifesting a severe, aggressive, and persistent pattern of disruptive behavior. These features have many similarities with adult psychopathy. More research is needed to gain an understanding of the different developmental trajectories for psychopathic-like behavior style in children and adolescents. The current study aimed to investigate cognitive, psychosocial, and personality-related characteristics associated with disruptive behavior in children and adolescents. In addition, psychometric properties of one of the psychopathy measures, Antisocial Process Screening Device Self-Report (APSD-SR), were evaluated. In the first study, the working memory (WM) function of school-aged children with Oppositional Defiant Disorder (ODD)/Conduct Disorder (CD) was compared with age- and gender-matched normative controls. The comorbid diagnosis of Attention-Deficit/Hyperactivity Disorder (ADHD) was controlled for in the patient group. WM function was examined using n-back tasks. The main finding of this study was that patients performed worse on WM tasks than controls, even after controlling for the diagnosis of ADHD. The results suggest that children with disruptive behavior disorders (i.e. ODD and CD) have executive function (EF) deficits, like poor WM, that are not accounted for the comorbid diagnosis of ADHD. The second study, using the data from the Finnish Self-Report Delinquency Study 2012 (FSRD-12), assessed the factor structure and internal consistencies of the APSD-SR in a community sample of 15- to 16-year-old youth (n = 4,855, 51% girls). A three-factor structure of APSD-SR was found to fit the data best, with three meaningful subscales representing narcissism, impulsivity, and callous-unemotional features. The internal consistencies for these subscales were adequate. The results suggest that the self-report version of APSD is a promising screening tool for measuring psychopathic-like features in community youth. The third study used the same FSRD-12 data and examined different types of weapon carrying among youth, and psychosocial and personality-related factors associated with carrying a weapon, especially focusing on the relationship between psychopathic-like features and weapon carrying. The results showed that adolescents carrying weapons had a large cluster of problems in their lives, which were differentiated by the type of weapon carried. In addition, psychopathic-like features were associated with an increased likelihood of carrying any type of weapon (knife, gun, other weapon such as knuckleduster, chain, or pepperspray), but the association was strongest with carrying a gun. This association was significant even after controlling for a variety of psychosocial factors. The findings suggest that psychopathic-like features are strongly related to a higher risk of weapon carrying. Furthermore, the findings underscore the need for a comprehensive evaluation of an adolescent s psychosocial situation, when weapon carrying is identified. Finally, the fourth study investigated the relationship between victimization experiences and psychopathic-like features in youth by using the same FSRD-12 data. The results revealed that victimization was related to psychopathic-like features in youth, even more strongly in girls than in boys. Interestingly, the association between victimization and psychopathic-like features was stronger for recent experiences of victimization than past experiences. The results highlight the need for evaluating victimization experiences when psychopathic-like features are present in youth. Furthermore, some of the juveniles may show psychopathic-like behavior style as a means to cope with trauma.