Coagulation Factors: Role in the Development of Thrombosis, Inflammation and Cancer

 

Grethe Skretting
Group Leader

 

Our group has lately focused on the molecular mechanisms related to the role of coagulation inhibitors for the development of thrombosis and cancer. In particular we have concentrated on the involvement of TFPI and PC in these processes. TFPI is the physiological inhibitor of TF induced coagulation. Low levels of TFPI in plasma are associated with increased risk of thrombosis and SNPs in the TFPI gene have been shown to influence the TFPI plasma levels. Significantly increased levels of TFPI have been found in plasma from cancer patients and expression of TFPI in many cancer cells has been demonstrated. The mechanism behind this is as yet unknown. We are also studying the role of TFPI in endothelial cell activation and atherosclerosis.

Estrogens can influence the pathological processes of many hormone-dependent cancers such as breast and ovarian cancers. Women using oral contraception or postmenopausal hormone therapy are at increased risk of venous thrombosis. These women have decreased plasma TFPI levels indicating a link between estrogens and TFPI, both in cancer and venous thrombosis. At present we are focusing on the effect of estrogens on TFPI and the underlying mechanisms. The majority of human breast cancers are estrogen dependent and the cancer cells express the estrogen receptor (ER). Using breast cancer cell lines, both ER expressing and non-expressing, we have demonstrated that estrogens can downregulate the expression of TFPI in a process dependent on the presence of the estrogen receptor ERα. By studying the 5’ flanking region of the TFPI gene, we found that this effect partly was due to binding of the receptors to specific elements in the DNA. In collaboration with a research group in Murcia, Spain, we have revealed that in addition to the direct transcriptional regulation of the TFPI gene by estrogens, microRNAs also participate in the regulation of the TFPI expression. A homologue to TFPI named TFPI-2 is a matrix-associated protein inhibiting the activation of matrix metalloproteinases involved in tumor progression, invasion and metastasis as a tumor suppressor. Methylation of the TFPI-2 gene promoter is associated with reduced transcription of the gene. Estrogens have been shown to regulate the expression of DNA methyltransferases (DNMT) in ER positive cells. Our data strongly indicates that like TFPI, the TFPI-2 expression is also affected by estrogens, evidenced by increased TFPI-2 mRNA levels in breast cancer cells after treatment with estrogens. This is also an ERα dependent process and we are now studying whether DNA or protein methylation is involved.

Hypoxia is a hallmark of several pathophysiological conditions including cancer, atherosclerosis and ischemic cardiovascular disease, conditions characterized by activation of coagulation and increased risk of thromboembolism. Hypoxia is defined as an inadequate oxygen supply to the cells and tissues of the body and hypoxia due to low atmospheric pressure triggers activation of coagulation, most probably due to tissue factor (TF) production by intravascular cells. Another phenomenon related to hypoxia is multidrug resistance (MDR), an acquired phenotype of certain cancers that results in inadequate response to chemotherapy (chemoresistance) and reduced survival. To date, the relationship between hypoxia, coagulation and metastasis, particularly chemoresistance, has remained largely unexplored. To develop new targeted therapies towards avoiding thrombotic complications in cancer and to increase the rate of successful treatment of cancer by reducing the rate of MDR, basic knowledge into the underlying mechanisms is essential. In a study we have investigated the role of hypoxia in the regulation of the TFPI expression in breast cancer cells on the transcriptional level. We found that both hypoxia and overexpression of the hypoxia inducible factor (HIF)-1α caused a strong repression of the TFPI promoter activity. This was caused by binding of HIF-1α to a hypoxia response element (HRE) within the TFPI promoter located at -1065 to -1060 relative to the transcriptional start point. In tissue samples from breast cancer patients, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and HIF-1α. This study has thus demonstrated that HIF-1α is involved in the transcriptional regulation of the TFPI gene and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients. A similar study on another factor that mediates the cellular response to hypoxia, namely HIF-2α, is on-going and the results so far, indicate that HIF-2α can affect the TFPI expression.

Hypoxia is also evident in advanced atherosclerotic plaques most likely due to thickness and level of oxidative burden within the plaques when they are growing. The extrinsic coagulation activator TF and its inhibitor TFPI are expressed by endothelial cells overlaying the plaques, and plaque rupture leads to activation of coagulation and the formation of a thrombus, which is the main contributor to acute manifestations, morbidity, and mortality in atherosclerotic disease. In a project (collaboration with Bente Halvorsen, RIIM), we have used endothelial cells to investigate the impact of hypoxia on the expression of TF and TFPI, which is important for the initiation of coagulation. Hypoxic conditions resulted in an increased TF expression and a reduced TFPI expression accompanied by an increased pro-coagulant activity of the endothelial cells, a situation that might promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

In the same project, we have available carotid plaque material removed from patients during surgery and control carotids. Since both TF and TFPI previously have been shown to be present in atherosclerotic lesions where they are expressed by macrophages, smooth muscle cells, and endothelial cells overlaying the plaques, we want to examine the expression of TFPI, which is expressed as two different isoforms, TFPIα and TFPIβ, in atherosclerotic plaques and associated cells. For this we are using the monocytic cell line THP-1 differentiated into macrophages and further polarized into M1 and M2 macrophages. Recently, we have extended this project to studies on endoplasmic reticulum (ER) stress since this is associated with atherosclerosis. The objective for this part is to investigate the expression of TFPI in response to ER stress and which role TFPI plays in foam cell formation and plaque stability.

A large number of human diseases are caused by defects in protein folding as a result of genetic mutations or adverse physiological conditions. The maintenance of the protein homeostasis in blood requires regulation of coagulation and fibrinolysis and protein deficiencies in these processes lead to hemorrhagic or thrombotic tendency. Many of the coagulation factor deficiencies are caused by reduced circulating protein levels resulting from a broad spectrum of gene mutations. This can cause impaired secretion due to increased intracellular degradation or accumulation of misfolded proteins, processes that have been reported for some factor VII (FVII) and factor VIII (FVIII) deficiencies, and also in deficiencies of protein C and plasmin inhibitor. For a number of cases of diseases caused by protein misfolding, drugs acting directly on the affected protein have been found to prevent misfolding and restore biosynthesis and function. In collaboration with a research group in Ferrara, Italy and several groups at the University of Oslo, we are investigating the intracellular fate of a group of FVII mutations previously reported in both Norwegian patients and also in patients from elsewhere, in order to elucidate the cellular mechanisms implicated in these mutations. Patients with these mutations have bleedings and the present treatment is replacement therapy, which has several limitations. The project includes both studies by overexpressing the FVII variants in a non-FVII expressing cell line, but also genomic editing using a hepatic cell line Huh7, and human embryonic stem cells, which will be differentiated into hepatocytes being the main site of FVII expression. Accumulation of misfolded proteins within the ER might cause ER stress and can trigger the unfolded protein response (UPR) and apoptosis. We have results strongly indicating that the FVII mutant proteins indeed evoke ER stress when overexpressed in cells. We are now investigating the functional consequences of ER stress by mapping affected pathways, apoptosis etc. By these studies we hope to envisage possible therapeutic approaches that can substitute the present replacement therapy. In addition, we are extending a previous study from our group on a protein C mutation, now from a therapeutic point of view.

 
Photo Øystein H. Horgmo, University of Oslo

In front-from left: Christiane Filion Myklebust, Marie-Christine Mowinckel, Grethe Skretting, Maria Eugenia Chollet and
Benedicte Stavik.
Back- from left: Huda Omar Ali, Sandra Espada Serrano, Elisabeth Andersen, Marianne S. Andresen, Xue-Yan Cui and Ann Døli.