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)