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.

The Effects of the Breast Cancer Mammography Screening Programme in Women aged 40 to 84 years in Turku, Finland (1987-2009)

The objective of this thesis was to evaluate whether a more extensive mammography screening programme (TurkuMSP) conducted by the city of Turku, had an effect on breast cancer (BC) incidence, survival, or mortality in years 1987 to 2009. Despite the fact that some studies have suggested a 20 percent reduction in BC mortality due to mammography screening, there are findings of harm to subjects, which are claimed to negate the benefits of screening. Thus, the aims of this study are most pertinent. A total of 176 908 screening examinations were performed in 36 000 women aged 40−74 during the years 1987−1997. In all, 685 primary BCs were found in the screened women, either screen-detected (n=531) or during screening intervals (n=154). Survival and BC recurrence rate of women with screen-detected BC was compared to 184 women with clinical BCs detected among individuals who did not take part in the screening. The invitation interval, which may influence the outcome, was studied in the age group 40 to 49 by inviting those born in even calendar years annually for mammography screening and those born in odd years, triennially. In addition, BC incidence and mortality in the total female population of Turku aged 40 to 84 years was compared with the respective figures of Helsinki and the rest of Finland, both during the pre-screening era (1976-1986) and the screening era (1987-2009). The study was designed to compare women by age groups, because women aged 50 to 59 were generally screened in all of Finland, whereas only in Turku women aged 40 to 49 and 60 to 74 were screened in addition. Data regarding cancer recurrence were derived from the Finnish Cancer Registry and data on deaths were collected from Statistics Finland. In survival analyses, screened women with invasive BC had a significantly higher survival rate than the women with clinical BC. The survival benefit started to appear already during the first follow-up years and was evident in all age groups. A marginal survival extension was also seen in screened women when BC had spread to ipsilateral axillary nodes already at diagnosis. Recurrence-free survival rate after BC treatment was significantly more favorable among the screened women compared with women with BC found clinically. The screening invitation interval did not significantly influence BC mortality in the subset of women aged 40 to 49 years. There were no consistent differences in the changes of BC incidence between Turku and the comparison areas during the screening era. In Turku, the BC mortality incidence in women aged 55−69 years was significantly lower during the screening era (from 1987 to 1997) compared with the pre-screening era, whereas no such change was found in the city of Helsinki or Tampere. When comparing the changes in incidence-based BC mortality during years 1987 to 2009 in Turku to those of Helsinki and the rest of Finland, there was a suggestion of more than 20 percent lower mortality in Turku among oldest age group (75-84 years) compared with the reference residential areas, but the differences were not consistently significant. Interpretation of the study results should be made with caution because there were no random control groups, and on the other hand, the number of cases in subgroups was fairly low to yield definite conclusions. Also due to the many statistical analyses, some of the findings may be due to chance. The results are, however, suggestive for a decrease of BC mortality in the elderly age groups due to wide mammography screening. This finding needs confirmation in further studies before recommending an expansion of mammography screening to women up to the age of 74 years

Novel Roles of HCG/LH Signalling in the Mouse Mammary Gland and its Tumorigenesis.

Breast cancer is the most common cancer in women, and its development is intimately related to hormonal factors, but how hormones affect breast physiology and tumorigenesis is not sufficiently known. Pregnancy elicits long-term protection from breast cancer, but during the first ten years after pregnancy, breast cancer risk is increased. In previous studies, there has been conflicting data on the role of human chorionic gonadotropin (HCG) and the functionality of its receptor in extragonadal tissues. The aim of this study was to elucidate the role of chronically elevated HCG in mouse physiology. We have created a transgenic (TG) mouse model that overexpresses HCG. HCG is similar to lutenizing hormone (LH), but is secreted almost solely by the placenta during pregnancy. HCG and LH both bind to the LH receptor (LHR). In the current study, mammary gland tumors were observed in HCG TG mice. We elucidated the role of HCG in mammary gland signalling and the effects of LHR mediated signalling in mouse mammary gland gene expression. We also studied the effects of HCG in human breast epithelial cell cultures. Several endocrine disturbances were observed in HCGβ TG female mice, resulting in precocious puberty, infertility, obesity and pituitary and mammary gland tumors. The histology of the mammary gland tumors of HCGβ TG females resembled those observed in mouse models with activated Wnt/β-catenin signalling pathway. Wnts are involved in stem cell regulation and tumorigenesis, and are hormonally regulated in the mammary gland. We observed activated β-catenin signalling and elevated expression of Wnt5b and Wnt7b in TG tumors and mammary glands. Furthermore, we discovered that HCG directly regulates the expression of Wnt5b and Wnt7b in the mouse mammary gland. Pharmacological treatment with HCG also caused upregulation of several Wnt-pathway target genes in ovariectomized wild type (WT) mice in the presence of physiological concentrations of estradiol and progesterone. In addition, differential expression of several metabolic genes was observed, suggesting that HCG affects adipocyte function or glucose metabolism. When WT mice were transplanted with LHR deficient or wild type WT mammary epithelium, differential expression of several genes affecting the Wnt-signalling pathway was observed in microarray analysis. Diminished expression of several genes associated with LHR function in other tissues, such as the ovary, was observed in mammary glands deficient of epithelial LHR. In cultured human mammary epithelial cells HCG upregulated the expression of WNT5B, WNT7B similar to mouse, suggesting that the observations found are relevant in human physiology. These studies suggest that HCG/LHR signalling affects gene expression in non-gonadal tissues, and that Wnt-signalling is regulated by HCG/LH in human and mouse mammary glands.

The application of cDNA and tissue microarray methods in the study of human carcinomas

Currently, numerous high-throughput technologies are available for the study of human carcinomas. In literature, many variations of these techniques have been described. The common denominator for these methodologies is the high amount of data obtained in a single experiment, in a short time period, and at a fairly low cost. However, these methods have also been described with several problems and limitations. The purpose of this study was to test the applicability of two selected high-throughput methods, cDNA and tissue microarrays (TMA), in cancer research. Two common human malignancies, breast and colorectal cancer, were used as examples. This thesis aims to present some practical considerations that need to be addressed when applying these techniques. cDNA microarrays were applied to screen aberrant gene expression in breast and colon cancers. Immunohistochemistry was used to validate the results and to evaluate the association of selected novel tumour markers with the outcome of the patients. The type of histological material used in immunohistochemistry was evaluated especially considering the applicability of whole tissue sections and different types of TMAs. Special attention was put on the methodological details in the cDNA microarray and TMA experiments. In conclusion, many potential tumour markers were identified in the cDNA microarray analyses. Immunohistochemistry could be applied to validate the observed gene expression changes of selected markers and to associate their expression change with patient outcome. In the current experiments, both TMAs and whole tissue sections could be used for this purpose. This study showed for the first time that securin and p120 catenin protein expression predict breast cancer outcome and the immunopositivity of carbonic anhydrase IX associates with the outcome of rectal cancer. The predictive value of these proteins was statistically evident also in multivariate analyses with up to a 13.1- fold risk for cancer specific death in a specific subgroup of patients.

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.

First-Line Chemotherapy with Anthracycline and Taxane Combination in Metastatic Breast Cancer. Detection of bone metastases with TRACP 5b

Background: In Finland, breast cancer (BC) is the most common cancer among women, and prostate cancer (PC) that among men. At the metastatic stage both cancers remain essentially incurable. The goals of therapy include palliation of symptoms, improvement or maintenance of quality of life (QoL), delay of disease progression, and prolongation of survival. Balancing between efficacy and toxicity is the major challenge. With increasing costs of new treatments, appropriate use of resources is paramount. When new treatment regimes are introduced into clinical practice a comprehensive assessment of clinical benefit, adverse effects and cost is necessary. Both BC and PC show a predilection to metastasize to bone. Bone metastases cause significant morbidity impairing the patients´ QoL. Diagnosis of bone metastases relies mainly on radiological methods, which however lack optimal sensitivity and specificity. New tools are needed for detection and follow-up of bone metastases. Aims: Anthracyclines and taxanes are effective chemotherapeutic agents in the treatment of metastatic breast cancer (MBC) with different mechanisms of action. Therefore, evaluation of the combination of anthracyclines with taxanes was a justifiable approach in the treatment of MBC patients. We assessed the efficacy, toxicity, cost of treatment and QoL of BC patients treated with first-line chemotherapy for metastatic disease with the combination epirubicin and docetaxel. We also evaluated the diagnostic potential of tartrate-resistant acid phosphatase 5b (TRACP 5b) and carboxyterminal telopeptides of type I collagen (ICTP) in the diagnosis of bone metastases in BC and TRACP 5b in PC patients. Results: The combination of epirubicin and docetaxel was effective in this phase II study, but required individual dose adjustment to avoid neutropenic infections, and the use of growth factors to maintain a feasible dose level. The response rate was 54 % (95 % CI 37-71) and the median overall survival (OS) was 26 months. Of the patients, 87 % were treated for infections. The treatment of adverse events required additional use of health resources mainly due to neutropenic infections, thereby raising direct treatment costs by 20 %. Despite adverse events, the global QoL was not significantly compromised during the treatment. Clinically evident acute cardiac toxicity was not observed. The combination of serum TRACP 5b and ICTP was at least equally sensitive and specific in detection of of bone metastases as commonly used total alkaline phosphatise (tALP) in BC patients. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC patients. Conclusions: Treatment with epirubicin and docetaxel showed high efficacy in first-line chemotherapy of MBC. The relatively high incidence of neutropenic infections requiring hospitalization increased the treatment costs. Despite adverse events, the global QoL of the patients was not significantly compromised. The combination of TRACP 5b and ICTP showed similar activity as tALP in detecting bone metastases in MBC. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC.

Nuclear morphometry, apoptotic and mitotic indices, and tubular differentiation in Libyan breast cancer

Aims of this study were to evaluate the relations of nuclear morphometry, mitotic and apoptotic indices, and tubular differentiation with clinicopathological features and survival rate in Libyan women. The data were compared with corresponding results on Finnish, and Nigerian female breast cancer patients. Histological samples of breast cancer (BC) from 131 patients were retrospectively studied. Mitotic activity indices (MAI and SMI), apoptotic index (AI), and fraction of fields with tubular differentiation (FTD) were estimated. Samples were also studied by computerized nuclear morphometry, such as mean nuclear area (MNA). Demographic and clinicopathological features were analyzed from 234 patients. The Libyan BC was dominantly premenopausal, and aggressive in behavior. There were statistically significant correlations between the mean nuclear area, fraction of fields with tubular differentiation, apoptotic index and proliferative indices, and most clinicopathological features. The highest significances were shown between lymph node status and the proliferative and apoptotic indices (p=0.003 with SMI, and p=0.005 with AI). There were significant associations between clinical stage and SMI and AI (p=0.002 and 0.009, respectively). The most significant associations with grade were observed with MNA and FTD (p<0.0001 and 0.001, respectively). The proliferative differences between Libyan, Nigerian and Finnish populations were prominent. These indices in Libyan were lower than in Nigerian, but higher than in Finnish patients. The Libyan patients’ AI is slightly higher than in Nigeria, but much higher than in Finland. The differences between countries may be associated with the known variation in the distribution of genetic markers in these populations. The results also indicated that morphometric factors can be reliable prognostic indicators in Libyan BC patients.

Lymph node transfer in the treatment of postmastectomy lymphedema

Background: Lymphedema is a debilitating disorder with few treatment options. Clinical studies have shown that microvascular lymph node transfer may improve the lymphatic function of the affected limb. This study provides information about the clinical efficacy and safety of this procedure. Further, the biological background of this technique is clarified with an analysis of postoperative production of lymphatic growth factors and cytokines related to lymphangiogenesis. Patients and Methods: The effect of lymph node transfer to recipient and donor sites was analyzed with lymphoscintigraphy, limb circumference measurements, and appearance of clinical symptoms. Axillary seroma samples were analyzed from four patient groups: Axillary lymph node removal (ALND), Microvascular breast reconstruction (BR), lymph node transfer (LN) and combined lymph node transfer and breast reconstruction (LN-BR). Results: The postoperative lymphatic transport index was improved in 7/19 patients. Ten patients were able to reduce or discontinue compression therapy 6 - 24 months postoperatively. The donor lower limb lymphatic flow was slightly impaired (Ti >10) in 2 patients. No donor site lymphedema symptoms appeared during the 8 – 56-month follow-up. A high concentration of the VEGF-C protein was detected in the seroma fluid of all flap transfer groups. The concentration of the anti-inflammatory and anti-fibrotic cytokine IL-10 was increased in the LN-BR group samples when compared to the ALND or BR group. Conclusions: According to this preliminary study, the lymph node transfer seems to be beneficial for the lymphedema patients. However, a randomized study comparing the effect of BR and LN-BR is needed to evaluate the clinical efficacy of lymph node transfer. In addition, the effect of this surgery on the donor site needs to be studied further. The clinical effects of the lymph node transfer might be partly mediated by increased production of the lymphangiogenic growth factor (VEGF-C) as well as the anti-fibrotic cytokine (IL-10).