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

BMC Cancer, Aug 2011

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 β) and only TFPIβ 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 TFPIβ 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.

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

BMC Cancer Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility Benedicte Stavik 0 2 Grethe Skretting 1 Hans-Christian Aasheim 0 Mari Tinholt 0 Lillian Zernichow 0 Marit Sletten 0 Per Morten Sandset 1 2 Nina Iversen 0 0 Department of Medical Genetics, Oslo University Hospital , Oslo , Norway 1 Department of Haematology, Oslo University Hospital , Oslo , Norway 2 Institute of Clinical Medicine, University of Oslo , Oslo , Norway 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 a and b) and only TFPIb 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 a2. 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 TFPIb 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. TFPI-1; adhesion; migration; invasion; tumor suppressor - Background An association between cancer and the hemostatic system has been recognized for almost two centuries [1-3]. Thrombosis is nevertheless still among the leading causes of death in cancer patients [3]. The coagulation cascade is triggered by tissue factor (TF) and results in the formation of a fibrin network. TF pathway inhibitor1 (TFPI) is mainly known for its role in the hemostatic system where it is responsible for the inactivation of TF-induced coagulation [4,5]. There is, however, a growing body of evidence of a new role of TFPI in cancer. Elevated levels of plasma TFPI in cancer patients have previously been reported [6,7], and TFPI expression was demonstrated in several tumors, including breast cancer tissue and cells [8,9], indicating a possible involvement of TFPI in cancer biology [9]. Two main isoforms of TFPI are recognized, TFPIa and TFPIb. TFPIa consists of three Kunitz-type domains and a basic C-terminus, and is either secreted or attached to the cell membrane through an unknown glycosylphosphatidylinositol (GPI) linked molecule. TFPIb contains only the first two Kunitz-domains and has a different C-terminus with a GPI anchor that attaches it to the cell membrane [10-13]. The functional role of TFPIb is not yet completely understood [14]. In a recent study, we demonstrated a pro-apoptotic effect of both TFPIa and TFPIb in breast cancer cells in vitro, while corresponding downregulation of endogenous TFPI resulted in reduced apoptotic activity [15]. It has previously been reported that treatment of cancer cells with TFPIa, either recombinant or through ectopic overexpression, resulted in reduced primary and metastatic tumor growth and tumor cell adhesion in murine models [16,17]. The non-hemostatic activity of TFPIa has in several cases been reported to be dependent on the C-terminal part of the protein [18-23], and a peptide corresponding to the C-terminus of TFPIa has been shown to inhibit angiogenesis and tumor growth in vivo [24]. Requirements for malignant cancer cells to form metastases are to escape from the primary tumor, to passage through the circulatory system, and to establish secondary tumors at distant sites. Such movement requires abnormal growth and adhesion characteristics and penetration of the extracellular matrix (ECM) surrounding the tumor and vessel organs [25]. Integrins are cell surface receptors involved in adhering cells to the ECM and mediate cell movement [26]. Binding of the ligand mediates attachment to the matrix, while phosphorylation of the receptor relays spreading of the cells to the matrix [27] through the formation of filopodia, lamellipodia, and stress fibers [28]. Matrix metalloproteinases (MMPs) are proteases which are able to cleave the ECM and basement membrane and are thus important for metastasis of cancer cells [29]. In the present study, we investigated how downregulation of TFPI would affect the growth and metastatic abiliti (...truncated)


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Benedicte Stavik, Grethe Skretting, Hans-Christian Aasheim, Mari Tinholt, Lillian Zernichow, Marit Sletten, Per Sandset, Nina Iversen. Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility, BMC Cancer, 2011, pp. 357, 11, DOI: 10.1186/1471-2407-11-357