Fibroblast Growth Factor 8 and its Receptors in The Growth and Angiogenesis of Experimental Breast Cancer . With Special Reference to Thrombospondin-1 as a Novel Target Gene for FGF-8

The growth of breast cancer is regulated by hormones and growth factors. Recently, aberrant fibroblast growth factor (FGF) signalling has been strongly implicated in promoting the progression of breast cancer and is thought to have a role in the development of endocrine resistant disease. FGFs mediate their auto- and paracrine signals through binding to FGF receptors 1-4 (FGFR1-4) and their isoforms. Specific targets of FGFs in breast cancer cells and the differential role of FGFRs, however, are poorly described. FGF-8 is expressed at elevated levels in breast cancer, and it has been shown to act as an angiogenic, growth promoting factor in experimental models of breast cancer. Furthermore, it plays an important role in mediating androgen effects in prostate cancer and in some breast cancer cell lines. We aimed to study testosterone (Te) and FGF-8 regulated genes in Shionogi 115 (S115) breast cancer cells, characterise FGF-8 activated intracellular signalling pathways and clarify the role of FGFR1, -2 and -3 in these cells. Thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis, was recognised as a Te and FGF-8 regulated gene. Te repression of TSP-1 was androgen receptor (AR)-dependent. It required de novo protein synthesis, but it was independent of FGF-8 expression. FGF-8, in turn, downregulated TSP-1 transcription by activating the ERK and PI3K pathways, and the effect could be reversed by specific kinase inhibitors. Differential FGFR1-3 action was studied by silencing each receptor by shRNA expression in S115 cells. FGFR1 expression was a prerequisite for the growth of S115 tumours, whereas FGFR2 expression alone was not able to promote tumour growth. High FGFR1 expression led to a growth advantage that was associated with strong ERK activation, increased angiogenesis and reduced apoptosis, and all of these effects could be reversed by an FGFR inhibitor. Taken together, the results of this thesis show that FGF-8 and FGFRs contribute strongly to the regulation of the growth and angiogenesis of experimental breast cancer and support the evidence for FGF-FGFR signalling as one of the major players in breast cancers.

CLEVER-1 as an immune suppressive molecule

The immune response and immune suppression are equally essential for the immune system to protect the host against an infection and to protect self-molecules in different pathophysiological conditions. Pregnancy is one of the conditions where the maternal immune system remains resistant against microbes and yet attains tolerance to protect the fetus, whose genetic material differs partially from the mother’s. However, if the balance of immune suppression is not precise in the host it can favor conditions which lead to diseases, such as cancer and autoimmune disorders. This study was initiated to investigate the expression and functions of CLEVER-1/Stabilin-1, a multifunctional protein expressed on subsets of endothelial cells and type II macrophages, as an immune suppressive molecule. Firstly, the expression of CLEVER-1/stabilin-1 and its function in human placental macrophages were examined. Secondly, the expression profile and functional significance of stabilin-1 on healthy human monocytes was investigated. The results clarified the expression of CLEVER-1/stabilin-1 on placental macrophages, and verified that CLEVER-1/stabilin-1 functions as an adhesion and scavenging molecule on these cells. The data from normal monocytes revealed that the monocytes with low stabilin-1 expression carried a pro-inflammatory gene signature, and that stabilin-1 can directly or indirectly regulate pro-inflammatory genes in monocytes. Finally, it was shown that monocyte CLEVER-1/stabilin-1 dampens IFN production by T cells. To conclude, CLEVER-1/stabilin-1 is defined as an immune suppressive molecule on monocytes and macrophages. Strikingly, anti-stabilin-1 antibodies may have the potential to promote the Th1 dependent inflammatory response and counteract the tumor induced immune suppression.

Morphometric Study of Cerebral Arterioles in CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL) is the most common hereditary small vessel disease (SVD) leading to vascular dementia. The cause of the disease is mutations in NOTCH3 gene located at chromosome 19p13.1. The gene defect results in accumulation of granular osmiophilic material and extracellular domain of NOTCH3 at vascular smooth muscle cells (VSMCs) with subsequent degeneration of VSMCs. This arteriopathy leads to white matter (WM) rarefaction and multiple lacunar infarctions in both WM and deep grey matter (GM) visible in magnetic resonance imaging. This thesis is focused on the quantitative morphometric analysis of the stenosis and fibrosis in arterioles of the frontal cerebral WM, cortical GM and deep GM (lenticular nucleus (LN), i.e. putamen and globus pallidus). It was performed by assessing four indicators of arteriolar stenosis and fibrosis: (1) diameter of arteriolar lumen, (2) thickness of arteriolar wall, (3) external diameter of arterioles and (4) sclerotic index. These parameters were assessed (a) in 5 elderly CADASIL patients with the mean age of onset 47 years and of death 63 years, (b) in a 32-year-old young CADASIL patient with the first ischemic episode at the age of 29 years and (c) a very old CADASIL patient aged 95 years, who suffered the first stroke at the age of 71 years. These measurements were compared with age-matched controls without stroke, dementia, hypertension, and cerebral amyloid angiopathy. Morphometric analyses disclosed that in all age groups of CADASIL patients compared to corresponding controls there was significant narrowing of arteriolar lumen (stenosis) and fibrotic thickening of the walls (fibrosis) in the WM arterioles, although the significance of stenosis in the very old patient was marginal. In the LN arterioles there was only significant fibrosis without stenosis. These results suggest that the ischemic lesions and lacunar infarcts in the cerebral WM are mainly attributable to the stenosis of arterioles, whereas those in the LN are probably mainly due to hemodynamic changes of the cerebral blood flow. In conclusion: The SVD of CADASIL is characterized by narrowing of lumina and fibrotic thickening of walls predominantly in the cerebral WM arterioles. On the other hand, in the LN the ischemic lesions and lacunar infarcts are most probably hemodynamic due to impaired autoregulation caused by the rigidity of fibrotic arterioles. The pathological cerebral arteriolar alterations begin to develop already at a relatively young age but the onset may be delayed to a remarkably old age. This underlines the well known great variability in the clinical picture of CADASIL. The very late onset of CADASIL may cause its underdiagnosis, because the strokes are common in the elderly and are attributed to common risk factors.

The Effects of Anesthetic Induced Loss of Consciousness on Quantitative Electroen Cephalogram, and Bispectral and Spectral Entropy Indices. Studies on Healthy Male

Changes in the electroencephalography (EEG) signal have been used to study the effects of anesthetic agents on the brain function. Several commercial EEG based anesthesia depth monitors have been developed to measure the level of the hypnotic component of anesthesia. Specific anesthetic related changes can be seen in the EEG, but still it remains difficult to determine whether the subject is consciousness or not during anesthesia. EEG reactivity to external stimuli may be seen in unconsciousness subjects, in anesthesia or even in coma. Changes in regional cerebral blood flow, which can be measured with positron emission tomography (PET), can be used as a surrogate for changes in neuronal activity. The aim of this study was to investigate the effects of dexmedetomidine, propofol, sevoflurane and xenon on the EEG and the behavior of two commercial anesthesia depth monitors, Bispectral Index (BIS) and Entropy. Slowly escalating drug concentrations were used with dexmedetomidine, propofol and sevoflurane. EEG reactivity at clinically determined similar level of consciousness was studied and the performance of BIS and Entropy in differentiating consciousness form unconsciousness was evaluated. Changes in brain activity during emergence from dexmedetomidine and propofol induced unconsciousness were studied using PET imaging. Additionally, the effects of normobaric hyperoxia, induced during denitrogenation prior to xenon anesthesia induction, on the EEG were studied. Dexmedetomidine and propofol caused increases in the low frequency, high amplitude (delta 0.5-4 Hz and theta 4.1-8 Hz) EEG activity during stepwise increased drug concentrations from the awake state to unconsciousness. With sevoflurane, an increase in delta activity was also seen, and an increase in alpha- slow beta (8.1-15 Hz) band power was seen in both propofol and sevoflurane. EEG reactivity to a verbal command in the unconsciousness state was best retained with propofol, and almost disappeared with sevoflurane. The ability of BIS and Entropy to differentiate consciousness from unconsciousness was poor. At the emergence from dexmedetomidine and propofol induced unconsciousness, activation was detected in deep brain structures, but not within the cortex. In xenon anesthesia, EEG band powers increased in delta, theta and alpha (8-12Hz) frequencies. In steady state xenon anesthesia, BIS and Entropy indices were low and these monitors seemed to work well in xenon anesthesia. Normobaric hyperoxia alone did not cause changes in the EEG. All of these results are based on studies in healthy volunteers and their application to clinical practice should be considered carefully.

The Effects of Fibroblast Growth Factor 8b on Reproductive Organs and Prostate Tumorigenesis

Fibroblast growth factors (FGFs) are involved in the development and homeostasis of the prostate and other reproductive organs. FGF signaling is altered in prostate cancer. Fibroblast growth factor 8 (FGF8) is a mitogenic growth factor and its expression is elevated in prostate cancer and in premalignant prostatic intraepithelial neoplasia (PIN) lesions. FGF8b is the most transforming isoform of FGF8. Experimental models show that FGF8b promotes several phases of prostate tumorigenesis - including cancer initiation, tumor growth, angiogenesis, invasion and development of bone metastasis. The mechanisms activated by FGF8b in the prostate are unclear. In the present study, to examine the tumorigenic effects of FGF8b on the prostate and other FGF8b expressing organs, an FGF8b transgenic (TG) mouse model was generated. The effect of estrogen receptor beta (ERβ) deficiency on FGF8binduced prostate tumorigenesis was studied by breeding FGF8b-TG mice with ERβ knockout mice (BERKOFVB). Overexpression of FGF8b caused progressive histological and morphological changes in the prostate, epididymis and testis of FGF8b-TG-mice. In the prostate, hyperplastic, preneoplastic and neoplastic changes, including mouse PIN (mPIN) lesions, adenocarcinomas, sarcomas and carcinosarcomas were present in the epithelium and stroma. In the epididymis, a highly cancer-resistant tissue, the epithelium contained dysplasias and the stroma had neoplasias and hyperplasias with atypical cells. Besides similar histological changes in the prostate and epididymis, overexpression of FGF8b induced similar changes in the expression of genes such as osteopontin (Spp1), connective tissue growth factor (Ctgf) and FGF receptors (Fgfrs) in these two tissues. In the testes of the FGF8b-TG mice, the seminiferous epithelium was frequently degenerative and the number of spermatids was decreased. A portion of the FGF8b-TG male mice was infertile. Deficiency of ERβ did not accelerate prostate tumorigenesis in the FGF8b-TG mice, but increased significantly the frequency of mucinous metaplasia and slightly the frequency of inflammation in the prostate. This suggests putative differentiation promoting and anti-inflammatory roles for ERβ. In summary, these results underscore the importance of FGF signaling in male reproductive organs and provide novel evidence for a role of FGF8b in stromal activation and prostate tumorigenesis.