Prognostic Factors In Breast Cancer. With Special Reference to Cyclins A, B1, D1 and E, MMP-1 and Decorin

Breast cancer is a highly heterogenous malignancy, which despite of the similar histological type shows different clinical behaviour and response to therapy. Prognostic factors are used to estimate the risk for recurrence and the likelihood of treatment effectiveness. Because breast cancer is one of the most common causes of cancer death in women worldwide, identification of new prognostic markers are needed to develop more specific and targeted therapies. Cancer is caused by uncontrolled cell proliferation. The cell cycle is controlled by specific proteins, which are known as cyclins. They function at important checkpoints by activating cyclin-dependent kinase enzymes. Overexpression of different cyclins has been linked to several cancer types and altered expression of cyclins A, B1, D1 and E has been associated with poor survival. Little is known about the combined expression of cyclins in relation to the tumour grade, breast cancer subtype and other known prognostic factors. In this study cyclins A, B1 and E were shown to correlate with histological grade, Ki-67 and HER2 expression. Overexpression of cyclin D1 correlated with receptor status and non-basal breast cancer suggesting that cyclin D1 might be a marker of good prognosis. Proteolysis in the surrounding tumour stroma is increased during cancer development. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading extracellular matrix proteins. Increased expression and activation of several MMPs have been found in many cancers and MMPs appear to be important regulators of invasion and metastasis. In this study MMP-1 expression was analysed in breast cancer epithelial cells and in cancer associated stromal cells. MMP-1 expression by breast cancer epithelial cells was found to carry an independent prognostic value as did Ki-67 and bcl-2. The results suggest that in addition to stromal cells MMP-1 expression in tumour cells control breast cancer progression. Decorin is a small proteoglycan and an important component of the extracellular matrix. Decorin has been shown to inhibit growth of tumour cells and reduced decorin expression is associated with a poor prognosis in several cancer types. There has been some suspicion wheather different cancer cells express decorin. In this study decorin expression was shown to localize only in the cells of the original stroma, while breast cancer epithelial cells were negative for decorin expression. However, transduction of decorin in decorin-negative human breast cancer cells markedly modulated the growth pattern of these cells. This study provides evidence that targeted decorin transduction to breast cancer cells could be used as a novel adjuvant therapy in breast malignancies.

From epithelial to mesenchymal: regulation of invasive cancer cell motility

Metastasis is the main cause of death among cancer patients. In order to initiate the metastatic cascade cancer cells have to undergo epithelial-to-mesenchymal transition (EMT). In EMT epithelial cells lose their cell-cell and cell-extracellular matrix (ECM) contacts and become more motile. The expression of the transcription factor Slug and of the mesenchymal intermediate filament vimentin is induced during EMT. Vimentin is often overexpressed in malignant epithelial cancers but the functional role of vimentin remains incompletely understood. In addition, kinases such as AKT and ERK are known to be involved in the regulation of EMT and cancer cell motility but the mechanisms underlining their functions are often unclear. Integrins are heterodimeric receptors that attach cells to the surrounding tissue and participate in regulating cell migration and invasion. Changes in integrin activity are linked to increased cell motility and further cancer metastasis. The aim for my PhD studies was to investigate the role of cellular signalling pathways and vimentin in the regulation of cancer cell motility and EMT. Our results revealed that in prostate cancer the downregulation of AKT1 and AKT2, but not AKT3, induces activation of cell surface 1-integrins leading to enhanced cell adhesion, migration and invasion. In addition, our findings demonstrated a reciprocal regulatory interaction between vimentin and ERK2 facilitating ERK-mediated phosphorylation of Slug at serine-87 (S87) in breast cancer. Surprisingly, Slug S87 phosphorylation is dispensable for E-cadherin repression but essential for the induction of vimentin and Axl expression in early onset of EMT. Our findings reveal previously unknown mechanistic information of how prostate and breast cancer cell motility and disease progression is regulated

Filopodia and stromal extracellular matrix as regulators of cancer cell invasion and growth

Bidirectional exchange of information between the cancer cells and their environment is essential for cancer to evolve. Cancer cells lose the ability to regulate their growth, gain the ability to detach from neighboring cells and finally some of the cells disseminate from the primary tumor and invade to the adjacent tissue. During cancer progression, cells acquire features that promote cancer motility and proliferation one of them being increased filopodia number. Filopodia are dynamic actin-rich structures extending from the leading edge of migrating cells and the main function of these structures is to serve as environmental sensors. It is nowadays widely appreciated, that not only the cancer cells, but also the surrounding of the tumor – the tumor microenvironment- contribute to cancer cell dissemination and tumor growth. Activated stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs) actively participate on tumor progression. CAFs are the most abundant cell type surrounding the cancer cells and they are the main cell type producing the extracellular matrix (ECM) within tumor stroma. CAFs secrete growth factors to promote tumor growth, direct cancer cell invasion as well as modify the stromal ECM architecture. The aim of this thesis was to investigate the function of filopodia, particularly the role of filopodia-inducing protein Myosin-X (Myo10), in breast cancer cell invasion and metastasis. We found that Myo10 is an important regulator of basal type breast cancer spreading downstream of mutant p53. In addition, I investigated the role of CAFs and their secreted matrix on tumor growth. According to the results, CAF-derived matrix has altered organization and stiffness which induces the carcinoma cell proliferation via epigenetic mechanisms. I identified histone demethylase enzyme JMJD1a to be regulated by the stiffness and to participate in stiffness induced growth control.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.