Coagulation factor V in breast cancer: a p53-regulated tumor suppressor and predictive marker for treatment response to chemotherapy.

BACKGROUND: Patients with cancer are at an increased risk of developing coagulation complications, and chemotherapy treatment increases the risk. Tumor progression is closely linked to the hemostatic system. Breast cancer tumors express coagulation factor V (FV), an essential factor in blood coagulation. The functional role of FV during treatment with chemotherapy is poorly understood and was explored in this study. OBJECTIVES: We aimed to investigate the role of FV in breast cancer progression by exploring associations with treatment response, gene regulation, and the functional effects of FV. METHODS: The receiver operating characteristic plotter was used to explore the predictive value of FV mRNA (F5) expression for treatment with FEC (5-fluorouracil, anthracycline, and cyclophosphamide). Breast cancer cohorts were analyzed to study treatment response to FEC. The effect of chemotherapy on F5 expression, the regulation of F5, and the functional effects of FV dependent and independent of chemotherapy were studied in breast cancer cell lines. RESULTS: F5 tumor expression was significantly higher in responders to FEC than in nonresponders. In vitro experiments revealed that anthracycline treatment increased the expression of F5. Inhibition and knockdown of p53 reduced the anthracycline-induced F5 expression. Mutation of a p53 half-site (c.158+1541/158+1564) in a luciferase plasmid reduced luciferase activity, suggesting that p53 plays a role in regulating F5. FV overexpression increased apoptosis and reduced proliferation slightly during anthracycline treatment. CONCLUSION: Our study identified F5 as a p53-regulated tumor suppressor candidate and a promising marker for response to chemotherapy. FV may have functional effects that are therapeutically relevant in breast cancer.

Coagulation Factor V (F5) is an Estrogen-Responsive Gene in Breast Cancer Cells.

Most breast cancers express estrogen receptor (ER) where estrogen signaling plays an important role. Cancer contributes to activation of the coagulation system leading to an imbalance in the hemostatic system, and coagulation factor (F) V, which is a key regulator of blood coagulation, has been shown to be increased in breast tumors. Thus, the molecular association between estrogens and FV was explored. Stimulation with 17-ß-estradiol (E2) or 17-ß-ethinylestradiol (EE2) resulted in a time- and dose-dependent increase in F5 messenger RNA and FV protein in ERα-positive MCF-7 cells. Pretreatment with the ER antagonist fulvestrant or knockdown of ERα prior to stimulation with E2 counteracted this effect. Three ERα-binding half-sites were identified in the promoter region of the F5 gene in silico. Reporter gene analysis showed that all three half-sites were involved in the estrogen-induced gene regulation in vitro, as the effect was abolished only when all half-sites were mutated. High F5 levels in ER-positive breast tumors were associated with increased relapse-free survival of breast cancer patients.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding.

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

Indirect regulation of TFPI-2 expression by miR-494 in breast cancer cells.

TFPI-2 has been shown to be involved in breast cancer pathogenesis by inhibiting extracellular matrix degradation, and low levels are associated with disease progression. As microRNA-494 (miR-494) protects against breast cancer progression, we investigated whether miR-494 is involved in the regulation of TFPI-2 in MCF-7 breast cancer cells. TFPI-2 mRNA and protein levels increased after transfection with miR-494 mimic, and TFPI-2 mRNA and miR-494 levels correlated positively in tumors from breast cancer patients. No specific binding sites for miR-494 in the 3'-untranslated region (UTR) of TFPI2 were identified; however, miR-494 was predicted in silico to bind 3'-UTR of the transcription factors AHR and ELF-1, which have potential binding sites in the TFPI2 promoter. ELF-1 mRNA was downregulated whereas AHR mRNA levels were upregulated after transfection with miR-494 mimic. Knockdown of ELF-1 and AHR increased and reduced TFPI-2 mRNA levels, respectively. Increased luciferase activity was seen when TFPI-2 promoter constructs containing the potential AHR or ELF-1 binding sites were co-transfected with miR-494 mimic. In conclusion, TFPI-2 mRNA levels were upregulated by miR-494 in MCF-7 breast cancer cells most likely by an indirect association where miR-494 targeted the transcription factors AHR and ELF-1. This association was supported in a breast cancer cohort.

Syndecan-3 and TFPI colocalize on the surface of endothelial-, smooth muscle-, and cancer cells.

BACKGROUND: Tissue factor (TF) pathway inhibitor (TFPI) exists in two isoforms; TFPIα and TFPIß. Both isoforms are cell surface attached mainly through glycosylphosphatidylinositol (GPI) anchors. TFPIα has also been proposed to bind other surface molecules, like glycosaminoglycans (GAGs). Cell surface TFPIß has been shown to exert higher anticoagulant activity than TFPIα, suggesting alternative functions for TFPIα. Further characterization and search for novel TFPI binding partners is crucial to completely understand the biological functions of cell associated TFPI. METHODS AND RESULTS: Potential association of TFPI to heparan sulphate (HS) proteoglycans in the syndecan family were evaluated by knock down studies and flow cytometry analysis. Cell surface colocalization was assessed by confocal microscopy, and native PAGE or immunoprecipitation followed by Western blotting was used to test for protein interaction. Heparanase was used to enzymatically degrade cell surface HS GAGs. Anticoagulant potential was evaluated using a factor Xa (FXa) activity assay. Knock down of syndecan-3 in endothelial,- smooth muscle- and breast cancer cells reduced the TFPI surface levels by 20-50%, and an association of TFPIα to syndecan-3 on the cell surface was demonstrated. Western blotting indicated that TFPIα was found in complex with syndecan-3. The TFPI bound to syndecan-3 did not inhibit the FXa generation. Removal of HS GAGs did not release TFPI antigen from the cells. CONCLUSIONS: We demonstrated an association between TFPIα and syndecan-3 in vascular cells and in cancer cells, which did not appear to depend on HS GAGs. No anticoagulant activity was detected for the TFPI associated with syndecan-3, which may indicate coagulation independent functions for this cell associated TFPI pool. This will, however, require further investigation.

Global gene expression analysis reveals a link between NDRG1 and vesicle transport.

N-myc downstream-regulated gene 1 (NDRG1) is induced by cellular stress such as hypoxia and DNA damage, and in humans, germ line mutations cause Charcot-Marie-Tooth disease. However, the cellular roles of NDRG1 are not fully understood. Previously, NDRG1 was shown to mediate doxorubicin resistance under hypoxia, suggesting a role for NDRG1 in cell survival under these conditions. We found decreased apoptosis in doxorubicin-treated cells expressing NDRG1 shRNAs under normoxia, demonstrating a requirement for NDRG1 in apoptosis in breast epithelial cells under normal oxygen pressure. Also, different cellular stress regimens, such as hypoxia and doxorubicin treatment, induced NDRG1 through different stress signalling pathways. We further compared expression profiles in human breast epithelial cells ectopically over-expressing NDRG1 with cells expressing NDRG1 shRNAs in order to identify biological pathways where NDRG1 is involved. The results suggest that NDRG1 may have roles connected to vesicle transport.

TFPIα and TFPIß are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity.

BACKGROUND: Tissue factor (TF) pathway inhibitor-1 (TFPI) is expressed in several malignant tissues- and cell lines and we recently reported that it possesses anti-tumor effects in breast cancer cells, indicating a biological role of TFPI in cancer. The two main splice variants of TFPI; TFPIα and TFPIß, are both able to inhibit TF-factor VIIa (FVIIa) activity in normal cells, but only TFPIα circulates in plasma. The functional importance of TFPIß is therefore largely unknown, especially in cancer cells. We aimed to characterize the expression and function of TFPIα, TFPIß, and TF in a panel of tumor derived breast cancer cell lines in comparison to normal endothelial cells. METHODS: TFPIα, TFPIß, and TF mRNA and protein measurements were conducted using qRT-PCR and ELISA, respectively. Cell-associated TFPI was detected after phosphatidylinositol-phospholipase C (PI-PLC) and heparin treatment by flow cytometry, immunofluorescence, and Western blotting. The potential anticoagulant activity of cell surface TFPI was determined in a factor Xa activity assay. RESULTS: The expression of both isoforms of TFPI varied considerably among the breast cancer cell lines tested, from no expression in Sum149 cells to levels above or in the same range as normal endothelial cells in Sum102 and MDA-MB-231 cells. PI-PLC treatment released both TFPIα and TFPIß from the breast cancer cell membrane and increased TF activity on the cell surface, showing TF-FVIIa inhibitory activity of the glycosylphosphatidylinositol- (GPI-) anchored TFPI. Heparin treatment released TFPIα without decreasing the cell surface levels, thus indicating the presence of intracellular storage pools of TFPIα in the breast cancer cells. CONCLUSION: GPI-attached TFPI located at the surface of breast cancer cells inhibited TF activity and could possibly reduce TF signaling and breast cancer cell growth locally, indicating a therapeutic potential of the TFPIß isoform.

TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIß cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIß, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIß, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes facilitating cancer cell growth and immunologic response, possibly by signal transduction involving the EGFR pathway.

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility.

BACKGROUND: Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231). METHODS: Stable cell lines with TFPI (both α and ß) and only TFPIß downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion. RESULTS: Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIß was downregulated, revealing a novel function of this isoform in cancer metastasis. CONCLUSIONS: Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.

Overexpression of both TFPIα and TFPIß induces apoptosis and expression of genes involved in the death receptor pathway in breast cancer cells.

Thrombosis is a major complication and an important cause of death in cancer patients. Tumor cells may trigger coagulation and induce a prothrombotic phenotype, which in return may enhance angiogenesis, tumor growth, and metastasis. Tissue factor pathway inhibitor (TFPI) has been reported to reduce tumor growth and metastasis in vivo and to induce apoptosis and inhibit proliferation in normal cells in vitro. However, no effect has so far been observed in cancer cells. We therefore aimed to characterize the functional effects of ectopic overexpression and endogenous downregulation of TFPI in cancer cells, and to elucidate possible mechanisms involved. The tumor derived breast cancer cells SK-BR-3 and Sum102 were used to construct stable cell lines overexpressing TFPIα and TFPIß, and with TFPI knocked down, respectively. Effects of altered TFPI expression were evaluated by measuring apoptosis and proliferation of the cells, and gene expressions were analyzed using PCR arrays. Increased DNA fragmentation and Caspase 3 activity was observed in SK-BR-3 cells overexpressing TFPIα and TFPIß, while a decrease in apoptosis was seen in Sum102 cells with TFPI expression knocked down. An increase and reduction in expression of pro- and anti-apoptotic genes, respectively, were seen in TFPI overexpressing cells, and the majority of the upregulated genes encoded proteins involved in the death receptor pathway, among them the death receptor ligand TNF-α. In conclusion, TFPIα and TFPIß induced apoptosis in breast cancer cells and increased expression of apoptotic genes indicating a possible involvement of the death receptor pathway.

Reduced LPA expression after peroxisome proliferator-activated receptor alpha (PPARalpha) activation in LPA-YAC transgenic mice.

BACKGROUND:: Apolipoprotein(a) (apo(a)), which is part of the atherogenic lipoprotein Lp(a), shares structural homology with plasminogen (plg). Genes coding for plasminogen (PLG) and apo(a) (LPA) are linked and situated 40kb apart in the telomeric region of the long arm of chromosome 6. LPA is naturally expressed only in primates and hedgehogs. Thus, access to knowledge regarding the mechanism by which LPA expression is regulated is limited due to shortage of appropriate animal models. However, mice transgenic for the human LPA gene have been produced. Lp(a) levels in man are genetically determined and not altered significantly by dietary changes. In contrast, mice transgenic for LPA-yeast artificial chromosome (LPA-YAC) have markedly reduced apo(a) levels after maintenance on a high-fat diet. LPA-YAC carries the 40kb LPA-PLG intergenic region, which includes a putative binding site for peroxisome proliferator-activated receptor alpha (PPARalpha). Therefore, we examined if fibrates, which exert their effect via PPARalpha, could alter LPA expression in transgenic mice. METHODS:: Two LPA transgenic mouse lines with or without the LPA-PLG intergenic region we fed either PPARalpha agonist fenofibrate (FF) or 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY 14643) containing diets for 3 weeks. For the study of serum apo(a) levels, blood were sampled prior the experiment and when the animals were sacrificed. For the study of gene expression pattern pieces of livers were collected and submerged in RNAlater buffer and stored at -70 degrees C until analysis by quantitative PCR. RESULTS AND CONCLUSIONS:: The results showed that fibrates reduce LPA expression in LPA-YAC transgenic mice, but have no impact on hepatic apo(a) mRNA or serum apo(a) protein levels in LPA-cDNA transgenic mice, which lack the LPA-PLG intergenic region. This suggests that the effect of fibrates on LPA expression is mediated upstream of the LPA gene. However, on the basis of current data it is not possible to conclude that PPARalpha is the primary factor that represses LPA expression in LPA-YAC transgenic mice. Negative correlation between FXR and apo(a) mRNA levels, in addition to putative FXR DNA binding sequence in LPA-PLG intergenic region, suggest that it is equally likely that reduced expression of LPA could be a secondary consequence of PPARalpha activation on other genes, such as FXR.

Variations in transfection efficiency of VEGF165 and VEGF121-cDNA: its effects on proliferation and migration of human endothelial cells.

Little is known about the expression pattern of vascular endothelial growth factor (VEGF) among smooth muscle cells of different arterial regions. Therefore, we have conducted studies aimed at increasing expression of VEGF in cultured human smooth muscle cells (SMCs) from different sites: aorta, umbilical artery, and coronary artery. Two plasmids harboring human VEGF121 and VEGF165 isoforms, respectively, were constructed and lipotransfected into vascular SMCs, using the Fu-GENE 6. Extensive optimization of transfection conditions were performed prior to this. Different basal levels of VEGF were observed between cell types: from 0.51-0.95 pg/mL/micrograms protein in umbilical artery, through 2.32-2.39 pg/mL/micrograms protein in coronary artery, to 5.45-7.52 pg/mL/micrograms protein in aortic SMCs. Significant differences in responses to transfection were also observed: The increase in VEGF production was most pronounced in umbilical artery SMCs (e.g., with 4 micrograms VEGF121-cDNA /in the wells)- an approximate 600-fold as opposed to an 18-fold increase in aortic SMCs and a 29-fold increase in coronary artery SMCs. In addition, we observed significant increases in proliferation rate of aortic and coronary endothelial cells (ECs), after incubation with conditioned medium from VEGF-transfected SMCs. Observed changes differed in relation to cell origin and isoform.