Download Tissue Factor: A Conventional or Alternative

Mini-Review
Clinical Chemistry 62:4
563–570 (2016)
Tissue Factor:
A Conventional or Alternative Target in Cancer
Therapy
Andreas Eisenreich,1* Juliane Bolbrinker,1 and Ulrike Leppert2
BACKGROUND: Tissue factor (TF) is an evolutionary conserved glycoprotein that plays an important role in the
pathogenesis of cancer. TF is expressed in 2 naturally occurring protein isoforms, membrane-bound full-length (fl)TF
and soluble alternatively spliced (as)TF. Both isoforms have
been shown to affect a variety of pathophysiologically relevant functions, such as tumor-associated angiogenesis,
thrombogenicity, tumor growth, and metastasis. Therefore,
targeting TF either by direct inhibition or indirectly, i.e., on
a posttranscriptional level, offers a novel therapeutic option
for cancer treatment.
CONTENT: In this review we summarize the latest findings regarding the role of TF and its isoforms in cancer
biology. Moreover, we briefly depict and discuss the therapeutic potential of direct and/or indirect inhibition of
TF activity and expression for the treatment of cancer.
SUMMARY: asTF and flTF play important and often distinct roles in cancer biology, i.e., in thrombogenicity and
angiogenesis, which is mediated by isoform-specific signal transduction pathways. Therefore, both TF isoforms
and downstream signaling are promising novel therapeutic targets in malignant diseases.
© 2016 American Association for Clinical Chemistry
Cancer is a leading cause of death worldwide (1, 2 ). Lung
cancer and colorectal cancer are among the most common malignant diseases (3, 4 ). Tissue factor (TF)3 is an
evolutionary highly conserved glycoprotein expressed in
humans and several other species (5, 6 ). Human TF is
1
Charité-Universitätsmedizin Berlin, CC04, Institut für Klinische Pharmakologie und
Toxikologie, Berlin, Germany; 2 Charité-Universitätsmedizin Berlin, CC02, Institut für
Physiologie, Berlin, Germany.
* Address correspondence to this author at: Charité-Universitätsmedizin Berlin, CC04,
Institut für Klinische Pharmakologie und Toxikologie, Charitéplatz 1, 10117 Berlin,
Germany. Fax +49-30-4507525112; e-mail [email protected].
Received May 24, 2015; accepted January 14, 2016.
Previously published online at DOI: 10.1373/clinchem.2015.241521
© 2016 American Association for Clinical Chemistry
3
Nonstandard abbreviations: TF, tissue factor; pre-mRNA, premature messenger RNA; fl,
full-length; as, alternatively spliced; SR, serine/arginine rich; Clk, cdc2-like kinase;
miRNA, microRNA; PAR, protease-activated receptor; PKC, protein kinase C; MCP-1,
monocyte chemotactic protein-1; F, coagulation factor; TFPI, TF pathway inhibitor;
r, recombinant.
genetically coded by the TF gene coagulation factor III,
tissue factor (F3)4 and transcribed to TF prematuremessenger RNA (pre-mRNA; Fig. 1) (7 ). Because of alternative splicing TF is expressed in 3 mRNA splice variants: full-length (fl)TF, alternatively spliced (as)TF, and
a third variant named TF-A. Translation of flTF and
asTF mRNA splice variants leads to the generation of the
flTF isoform, a membrane-bound and highly procoagulant protein (8 ), and soluble asTF with low prothrombogenic potential but strong proangiogenic, cell proliferation–
facilitating, and prosurvival activities (2, 9, 10 ). The
TF-A mRNA splice variant is not translated to a protein
(Fig. 1) because of termination sequences within alternative exon 1A, leading to an early translation stop (11 ).
TF-A mRNA expression has been detected in several cancer cell lines as well as in human endothelial cells (5, 11 ).
However, the biological function of TF-A is still unknown (5, 11 ). flTF and asTF are expressed in several
types of cancer cells and tumors and play important roles
in cancer biology, i.e., in thrombogenicity, survival, tumor growth, angiogenesis, signaling, invasion, and metastasis (2– 4, 8, 12, 13 ). Moreover, both TF isoforms
are also involved in other pathologies, such as cardiovascular diseases (14, 15 ). Therefore, targeting specific TF
isoforms offers novel potential therapeutic options for
the treatment of cancer patients.
In this review, we summarize the latest findings regarding the role of TF isoforms in cancer biology. Moreover, we briefly discuss the therapeutic potential of direct
and indirect inhibition of TF activity and/or isoform expression for cancer treatment.
TF Isoform Expression in Cancer
The expression of TF and its isoforms is induced and
highly regulated in several types of cancer, i.e., in lung
and breast cancer (3, 4, 16 ). Several factors are involved
in enhanced TF expression in tumor tissues, such as hypoxia or genetic modifications of oncogenes and tumor
suppressor genes, e.g. tumor protein p53 (TP53) and
Kirsten rat sarcoma viral oncogene homolog (KRAS)
4
Human genes: F3, coagulation factor III, tissue factor; TP53, tumor protein p53; KRAS,
Kirsten rat sarcoma viral oncogene homolog.
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Mini-Review
Fig. 1. Scheme of the TF isoform expression.
The TF premature (pre)mRNA is generated by transcription of the human TF gene F3. The arrow depicts the transcription start. Because of
constitutive or alternative splicing, 3 TF mRNA splice variants are generated on the posttranscriptional level. Removal of all introns (constitutive
splicing) leads to the generation of the flTF variant. Additional removal of exon 5 by alternative splicing leads to the production of asTF.
Retention of a part of intron 1 as alternative exon 1A in the mature transcript (alternative splicing) leads to the generation of the third splice
variant, TF-A. The mRNA variants flTF and asTF are translated in membrane-bound flTF or soluble asTF protein, respectively. Because of
termination sequences within the alternative exon 1A no protein is generated from the TF-A mRNA variant.
(2, 4, 17 ). These stimuli and events were shown to induce the transcription of the TF gene F3. Yu et al. demonstrated that mutation of the oncogene KRAS led to
increased TF expression in colorectal cancer cells (17 ). In
2009, Regina and colleagues showed that mutation of the
oncogene TP53 increased F3 transcription in non–small
cell lung cancer (4 ). Moreover, they found that increased
TF expression was associated with reduced survival of
patients with non–small cell lung cancer (4 ). In 2013,
Sun et al. showed hypoxia to induce the generation of
whole TF mRNA in breast cancer cells (18 ).
TF isoform expression is also modulated on a posttranscriptional level. Differential TF isoform expression
is regulated by several factors, i.e., serine/arginine-rich
(SR) proteins and SR protein kinases (19 ). In 2010,
Chandradas et al. showed that the SR proteins SRp40,
SC35, ASF/SF2, and SRp55 modulate TF isoform expression in the human monocytic leukemia cell line
THP-1. They found that binding of SRp40 and SC35 to
regulatory motifs in the TF pre-mRNA sequence increased the generation of asTF (20 ). Moreover, they depicted binding of ASF/SF2 and SRp55 to regulatory premRNA sites to facilitate flTF expression in THP-1 cells
(20 ). The authors suggested that SR protein-mediated
control of asTF and flTF generation could potentially
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Clinical Chemistry 62:4 (2016)
affect cancer-related thrombogenicity and angiogenesis
(20 ). Recently, we demonstrated hypoxia to induce asTF
and flTF expression in A549 lung cancer cells (2 ). In this
context, we found the SR protein kinases cdc2-like kinase
(Clk)1 and 4 to be involved in posttranscriptional regulation of hypoxia-induced TF isoform expression in
A549 cells (2 ). MicroRNAs (miRNAs) are also involved
in posttranscriptional expression control of TF and its
isoforms. In 2011, Zhang et al. demonstrated that inhibition of miR-19a led to increased expression of total TF
in breast cancer cells (16 ). They showed that increased
TF concentrations were associated with increased breast
cancer invasiveness (16 ). Recently, we demonstrated that
inhibition of miR-19a and miR-126 increased flTF and
asTF generation in human endothelial cells. This led to
increased flTF-mediated procoagulant activity (15 ).
These data show that both transcriptional and posttranscriptional regulation play an important role for cancerrelated increase and modulation of the expression and
activity of TF and its isoforms.
Impact of TF Isoforms on Signaling in Cancer
Both flTF and asTF differentially modulate cancer cell
signaling and other biological functions, such as cell pro-
Tissue Factor Isoforms and Cancer
liferation and angiogenesis (13, 21 ). There is genetic evidence indicating that protease-activated receptors (PARs)
are crucial for flTF-induced effects in cancer (22 ). The proposed mechanisms and aspects of potential pharmacological
treatment strategies have been reviewed in detail elsewhere
(22 ). flTF has been shown to mediate cell signaling via
PAR-2 and downstream signaling proteins, i.e., protein kinase C (PKC) and extracellular signal-regulated kinase 1
and 2 (13, 23 ). In this context, Hu et al. found that flTF
induced cell proliferation and migration of colon cancer
cells via PAR-2–mediated signaling (23 ).
In contrast to flTF, asTF was demonstrated to mediate
cell signaling independently of PAR-2 (10, 21, 24 ). In
2009, van den Berg and colleagues found that asTF directly
binds to ␤3 and ␤1 integrins. This induced angiogenesis in
vitro and in vivo independent of PAR-2 (24 ). We also
showed that asTF induced the proangiogenic potential and
proliferation rate of immortalized cardiomyocytes in a PAR2–independent manner (10 ). Moreover, we found asTF to
induce chemotaxis of human monocytic THP-1 leukemia
cells as well as cell proliferation of A549 lung cancer cells
(2, 10 ). Finally, Kocatürk and colleagues demonstrated that
asTF, but not flTF, increased breast cancer cell proliferation
via binding to ␤1 integrins in vitro (21 ). Furthermore, they
showed that asTF inhibition reduced tumor growth and
proliferation in vivo (21 ). These data indicate that both TF
isoforms are able to activate distinct signaling pathways,
leading to an isoform-specific modulation of cancer-related
biologic processes, such as tumor growth and angiogenesis
(13, 21 ).
The Role of TF Isoforms in Cancer-Related
Thrombosis
Thrombotic events play a clinically significant role in the
pathophysiology of cancer (3, 8 ). The procoagulant activity of asTF is controversial (7, 8, 25 ). We found no
detectable effect of increased asTF expression on endothelial thrombogenicity (5 ). Moreover, Yu and Rak demonstrated that flTF, rather than asTF, mediated the prothrombogenic activity of colorectal carcinoma cells (8 ).
In line with this, Hobbs et al. showed that asTF overexpression had no detectable impact on the procoagulant
activity of pancreatic MiaPaCa-2 cancer cells (12 ). In
contrast, Bogdanov and colleagues found that asTF exhibited, at a minimum, low procoagulant activity when
exposed to phospholipids in conditioned medium (7 ).
Similarly, Unruh et al. showed that overexpression of
asTF slightly increased the prothrombotic potential of
pancreatic ductal adenocarcinoma cells (25 ). Davila et al.
also found that asTF, which was secreted by pancreatic
tumor cells, exhibited low procoagulant activity (26 ). In
both studies, the low procoagulant potential of asTF was
dependent on its association with phospholipid surfaces
in vitro and in vivo (25, 26 ). These findings indicate
Mini-Review
that, if at all, asTF exhibits only low prothrombogenic
potential (7, 25 ). Thus, the influence of asTF on cancerrelated thrombosis remains unclear.
In contrast to asTF, flTF was demonstrated to be the
major source of procoagulant activity in cancer settings
(8 ). The expression of flTF is increased in many types of
cancer, i.e., in lung cancer patients or pancreas carcinomas (3, 8 ). These increased flTF concentrations are associated with an increased rate of thrombotic events, which
significantly contributes to morbidity and mortality of
cancer patients (3, 8 ). In 2009, Zwicker et al. found that
the amount of flTF-bearing microparticles was increased
in patients with advanced malignancy and pancreatic
cancer (27 ). This was associated with an increased risk of
venous thromboembolic events (27 ). Substantiating this,
Yu and Rak demonstrated that shedding of flTF-containing
microparticles into the medium increased the prothrombogenic activity of A431 squamous cell carcinoma and
HCT116 colorectal carcinoma cells (8 ). In agreement with
this, Davila et al. showed flTF to induce thrombogenicity of
breast cancer cells (28 ). The role of microparticles in cancerassociated thrombosis has been well summarized in a review
by Geddings and Mackman (29 ).
TF Isoforms and Angiogenesis in Cancer
Angiogenesis is crucial for tumor growth (2, 12 ). Both
TF isoforms were demonstrated to induce angiogenesis
in cancer (2, 10, 12, 30 ). Hobbs et al. showed that asTF
overexpression increased the microvascular density in a
pancreatic cancer tumor model and thus promoted
cancer-related angiogenesis in vivo (12 ). In line with this,
we and other groups also found asTF to mediate proangiogenic processes (2, 10, 24 ). We demonstrated that
asTF overexpression in murine HL-1 cells as well as in
A549 lung cancer cells increased the proangiogenic potential of these cells (2, 10 ). In 2009, van den Berg et al.
demonstrated in endothelial cells that asTF-induced
proangiogenic processes were mediated via integrin ligation, which was independent of PAR-2 signaling (24 ).
flTF was also found to induce angiogenesis in cancer
(13, 30 ). In 2008, Versteeg and colleagues found that
flTF induced angiogenesis via PAR-2 signaling in human
MDA-MB-231 breast cancer cells (30 ). Moreover, they
showed that flTF-activated PAR-2 signaling leads to increased angiogenesis in a mammary tumor mouse model
in vivo (13 ). These data demonstrate that flTF and asTF
play an important role in cancer-related angiogenesis.
The Influence of asTF and flTF on Cancer Cell
Proliferation and Tumor Growth
Both TF isoforms have been shown to enhance cancer
cell proliferation and tumor growth (2, 12, 30 ). In 2008,
Versteeg and colleagues demonstrated that inhibition of
Clinical Chemistry 62:4 (2016) 565
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flTF-mediated PAR-2 signaling reduced tumor growth
in an in vivo breast cancer model (30 ). In vitro, blocking
of flTF had no detectable impact on MDA-MD-231 cell
proliferation (30 ). In line with this, Yu et al. found that
flTF expression in colorectal cancer had no influence on
cell proliferation in vitro but specifically modulates tumor growth in vivo (17 ).
In contrast to flTF, asTF was shown to promote cell
proliferation in vitro as well as tumor growth in vivo
(2, 10, 12, 21 ). Hobbs et al. found asTF overexpression
to enhance tumor growth in vivo in a pancreatic cancer
model (12 ). Substantiating this, we demonstrated asTF
overexpression to increase cell proliferation of murine
HL-1 cells and human A549 lung cancer cells in vitro
(2, 10 ). This was mediated via asTF-induced expression
of proliferation-facilitating factors, such as monocyte
chemotactic protein-1 (MCP-1) (2, 10, 31 ). In accordance with this, Kocatürk and colleagues demonstrated
that increased asTF expression enhanced breast cancer
cells proliferation in vitro and tumor growth in vivo via
␤1 integrin signaling (21 ).
The Role of TF Isoforms in Cancer Cell
Migration, Tumor Metastasis, and Invasiveness
Both TF isoforms are known to modulate cancer cell
migration, metastasis, and invasiveness (2, 20, 32 ). In
2011, we found that increased amounts of asTF in cell
culture media induced migration of THP-1 leukemia
cells in vitro (10 ). In line with this, Unruh et al. showed
that increased asTF expression in pancreatic ductal adenocarcinomas led to the generation of metastases in distal
lymph nodes of mice (25 ). In human endothelial cells,
van den Berg and colleagues demonstrated that asTFinduced cell migration is mediated via integrin ␣v␤3
interaction (24 ).
flTF was also found to affect cancer cell migration.
The first evidence for the involvement of TF in tumor
metastasis, via both the TF-triggered coagulation pathway as well as cellular events mediated via the cytoplasmic
domain of TF, came from the work of Palumbo and
Degen (33 ) as well as Ruf and Müller (34 ). Recently,
additional mechanistic insights have come from other
groups. Dorfleutner et al. showed flTF to suppress integrin ␣3␤1-dependent migration of A7 melanoma cells
(32 ). Hu and colleagues reported that flTF promoted
colon cancer cell migration through PAR-2 signaling
(23 ). These finding suggest that asTF as well as flTF
affect cancer cell migration, metastasis, and tumor
invasion.
Available data consistently show that both TF isoforms play an important and often distinct role in cancerrelated hemostatic and nonhemostatic pathophysiological processes, i.e., tumor angiogenesis, thrombosis, and
metastasis.
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Therapeutic Implications of Targeting TF
Both TF isoforms play important roles in the pathophysiology of cancer. flTF, which is highly expressed in several
types of cancer, is involved in cancer-related thrombosis,
tumor growth, and metastasis (2– 4, 8, 12, 13 ). In contrast to flTF, asTF exhibits low prothrombogenic activity
(2, 8, 12 ). Moreover, asTF does not affect physiological
functions of flTF in vessel wall hemostasis and blood
coagulation control (19 ). asTF has been shown to modulate cell survival, proliferation, and angiogenesis in experimental cancer settings (2, 9, 12, 21 ). Targeting flTF
and asTF or their isoform-specific functions may thus
offer novel potential options for the treatment of cancer
(Table 1). Over the last few years, several molecules and
drugs targeting TF, its isoforms, and TF-mediated signaling have been developed and tested for their therapeutic potential for the treatment of cancer and other human
diseases. In this section we will describe and discuss the
therapeutic potential of the most promising tools.
One potential therapeutic approach is to target
flTF-mediated PAR-2 signaling via inhibition of coagulation factors (F) VII and X, because both factors are
needed for efficient PAR-2 signaling. As thoroughly reviewed by Schaffner and Ruf, flTF-mediated signaling
affects different aspects of cancer biology depending on
the tumor type, stage, and receptor types (35 ). Substantiating this, inhibition of direct flTF:FVIIa signaling by
monoclonal anti-flTF antibody 10H10 reduced tumor
growth in aggressive breast cancer, whereas inhibition of
TF-induced coagulation by monoclonal anti-flTF antibody 5G9 had only a minimal effect (30 ). In contrast,
5G9-mediated blocking of coagulation inhibited hematogenous metastasis, whereas inhibition of flTF:FVIIa
signaling by 10H10 had no influence on metastatic tumor homing (30, 36 ). The divergent effects of TF
isoform–mediated direct and/or indirect signaling on
cancer biology were reviewed in detail elsewhere (35 ).
First studies were performed to test the therapeutic
potential of blocking FVII- and FX-mediated effects on
TF in cancer. FVII and FX generation can be reduced via
vitamin K antagonists, which are already approved drugs
and have been used for a long time as anticoagulants
(37 ). Indeed, in a large population-based study, longterm treatment with vitamin K antagonists was associated with reduced risk for cancer development, especially
prostate cancer (37 ). Substantiating this, Nakchbandi et
al. reported that low doses of the vitamin K antagonist
warfarin increased survival of patients with pancreatic
carcinoma (38 ). However, it is thus far unclear whether
these effects depend solely on flTF-mediated processes.
Furthermore, treatment with vitamin K antagonists bears
the risk of severe bleeding events. This points toward the
need for specifically modifying TF:FVIIa-dependent sig-
Mini-Review
Tissue Factor Isoforms and Cancer
Table 1. Therapeutic implications: strategies directed against TF and its isoforms.
Directed
against
Substance/compound
Effects
Reference
Antibody drug conjugate of flTF
TF and monomethyl
auristatin E
Cytotoxic effect on cancer cells (in vitro, human); Breij et al. (41 )
Antitumor activity in patient-derived
xenograft models (in vivo, mouse, human)
Anti-flTF antibody 10H10
flTF
Reduced breast cancer tumor growth and
metastasis (in vitro + in vivo, human, mouse)
Versteeg et al. (30 )
Anti-TF antibody SunolcH36
TF
Reduced thrombus formation in coronary artery
disease patients (in vivo, human)
Morrow (43 )
FVII/Fc immunoconjugates
TF
Reduced tumor growth of human melanoma in a Hu et al. (40 )
mouse xenograft model (in vivo)
rTFPI
TF
Reduced thrombogenicity and vascular
remodeling (in vivo, rabbit);
Reduced mortality in sepsis (in vivo, human)
Vitamin K antagonists
FVII FX/PAR-2 Long-term use reduces risk of cancer (in vivo,
signaling
human); Low-dose warfarin improved survival
of patients with pancreatic carcinoma
Pengo et al. (37 ),
Nakchbandi et al.
(38 )
Cyclic RGD peptides
asTF/integrin
signaling
Reduced cell proliferation and angiogenesis in
lung cancer cells (in vitro, mouse, human)
Eisenreich et al.
(2,10 )
Anti-␤3/anti-␤1 antibodies
asTF/integrin
signaling
Reduced angiogenesis, endothelium (in vitro +
in vivo, mouse, human)
van den Berg et al.
(24 )
rNAPc2
flTF/FVII
complex
Reduces tumor angiogenesis, growth, and
metastasis in a Lewis lung cancer model
(in vivo, mouse)
Hembrough et al.
(39 )
miR-19, miR19a, miR-126
(mimics/antagomirs)
flTF/asTF
miR-19 mimic: reduced total TF expression in
breast cancer cells (in vitro, human);
miR-19a and miR-126 inhibition: increased
expression (flTF and asTF) and endothelial
thrombogenicity (in vitro, human)
Eisenreich and Rauch
(15 ), Zhang et al.
(16 )
KH-CB19 (Clk inhibitor)
flTF/asTF
Inhibition of Clk1 and 4 (KH-CB19, siRNAs):
Eisenreich et al. (2 ),
reduced expression of flTF and asTF as well as
Zhang et al. (16 )
decreased proangiogenic potential of lung
cancer cells (in vitro, human)
naling without affecting coagulation pathways when targeting TF as a therapeutic option in cancer treatment.
Another promising opportunity is inhibition of
flTF/FVIIa via PCI-27483 or the nematode anticoagulant protein rNAPc2. In vivo, rNAPc2 reduced tumor
growth, angiogenesis, and metastasis in a Lewis lung carcinoma model in mice (39 ). However, the therapeutic
value of these compounds is unclear because of the lack of
adequate human studies. In 2006, Love et al. started a
first safety study of rNAPc2 to prevent tumor progression
and metastases in colon cancer (registered in the National
Institutes of Health database clinicalTrials.gov; ID:
NCT00443573). In 2009, Hedrick et al. initiated a study
of safety and tolerability of the selective FVIIa inhibitor
PCI-27483 in patients with pancreatic cancer receiving
gemcitabine (ClinicalTrials.gov, ID: NCT01020006).
However, for both studies no results were published at the
time this review was written.
In 1999, Hu and colleagues generated immunoconjugates of inactivated FVII and the Fc effector domain of
Zoldhelyi et al. (44 ),
Abraham et al. (45 )
human IgG1 (40 ). The TF-targeting FVII domain mediated highly specific delivery of this molecule to TF.
Specific binding of this immunoconjugate to TF effectively inhibited tumor growth and caused regression of
human melanoma in a mouse xenograft model (40 ).
In 2014, Breij and colleagues analyzed the cytotoxic
potential of a TF antibody– drug conjugate on different
human tumors (41 ). They demonstrated that a conjugate of a TF-specific antibody and the cytotoxic agent
monomethyl auristatin E exhibited potent and TF
expression– dependent cytotoxicity in vitro (41 ). Moreover, this antibody– drug conjugate showed excellent antitumor activity in patient-derived xenograft models of 7
different flTF-expressing solid cancers in vivo with only
marginal effects on coagulation (41 ). In another study,
the therapeutic potential of the flTF-specific antibody
10H10 was determined in cancer (30 ). Versteeg et al.
showed that inhibition of flTF by 10H10 effectively suppressed breast cancer tumor growth and metastasis in
vitro and in vivo (30 ). Recently, Wong et al. initiated a
Clinical Chemistry 62:4 (2016) 567
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study dealing with the effect of ALT-836, a chimeric
anti– human TF monoclonal antibody, in combination
with gemcitabine on locally advanced or metastatic solid
tumors (clinicalTrials.gov, ID: NCT01325558). At the
time of this writing, no direct results for this study have
been published. However, these novel tools need further
investigation to define potential benefits as well as undesirable adverse effects.
The therapeutic value of TF inhibition was also
tested in other human diseases, such as in atherosclerosis
and coronary artery disease (42, 43 ). In vivo studies
showed that overexpression of TF pathway inhibitor
(TFPI), an endogenous inhibitor of TF activity, in
balloon-injured atherosclerotic arteries reduced TFmediated thrombogenicity and vascular remodeling in a
hyperlipidemic Watanabe rabbit model (44 ). Badimon
et al. showed that treatment of human atherosclerotic
plaques with a polyclonal anti-TF antibody significantly
reduced plaque thrombogenicity ex vivo (42 ). In 2005,
the results of the PROXIMATE-TIMI 27 [PROXimal
Inhibition of coagulation using a Monclonal Antibody to
Tissue factor (SunolcH36)–Thrombolysis in Myocardial
Infarction 27] trial revealed that treatment of coronary
artery disease patients with a chimeric mouse/human
monoclonal TF antibody (Sunol-cH36) significantly reduced thrombin generation in a dose-dependent manner
(43 ). Concomitantly, a dose-related incidence of mucosal bleeding was observed which was supposed to result
from effects on platelets (43 ). This might impair the
safety of the investigated antibody.
In 2001, Abraham et al. published results from a
prospective, randomized, placebo-controlled, and multinational phase II clinical trial assessing the safety of recombinant (r)TFPI administration, which indicated that
treatment of sepsis patients with rTFPI reduced 28-day
all-cause mortality compared to controls (45 ).
Taken together, recent antitumor strategies targeting flTF or flTF-mediated PAR-2 signaling show promising results but need further evaluation, especially in
clinical studies, to assess potential benefits as well as to
detect undesirable side effects.
Because asTF has an isoform-specific unique C terminus (7 ), specific antibodies directed against this
unique C terminus could be used to inhibit asTF and its
pathophysiologically relevant functions without affecting
other factors and/or coagulation. Bogdanov and colleagues developed an asTF-specific antibody (7 ). This
antibody was shown to specifically bind asTF in experimental cancer settings (2, 10 ) and to affect breast cancer
cell proliferation in vitro and tumor growth and angiogenesis in vivo (21 ), therefore offering the opportunity to
directly target asTF. However, a potential inhibitory effect of asTF antibodies on asTF-mediated functions
needs to be investigated and mechanistically analyzed in
further studies.
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Clinical Chemistry 62:4 (2016)
In contrast to flTF, asTF was found to mediate its
proangiogenic activity independently of PAR-2 via integrins, such as integrin ␣v␤3 (10, 24 ). Thus, asTF-specific
signaling via integrins seems to be another potential target for antiangiogenetic therapy in cancer. This option
was tested in vitro and in vivo (2, 10, 24 ). In 2009, van
den Berg et al. found that blocking of ␤1 and ␤3 integrins as well as treatment with a TF antibody that disrupts
asTF–integrin interaction reduced aortic sprouting ex
vivo in a mouse aortic ring model (24 ). Substantiating this,
we demonstrated that pharmacologic inhibition of integrin
␣v␤3 via cyclic RGD peptides reduced proliferation and the
proangiogenic potential of asTF-overexpressing human
lung cancer cells in vitro (2 ). Further studies are needed to
definitively assess the safety and efficacy of targeting asTFinduced integrin signaling as a therapeutic option for cancer
treatment.
Another therapeutic strategy might be the application of silencing oligos directed against the asTF mRNAspecific exon 4 – 6 transition sequence. However, this option has not been studied so far. Therefore, it is necessary
to focus on the development of such asTF-specific tools.
Moreover, asTF-directed tools and anticancer strategies
deserve to be further studied, especially in adequate in
vivo cancer models. It is essential to determine their potential therapeutic value as well as possible negative and
undesirable side effects.
Manipulation of TF isoform expression on the posttranscriptional level via affecting miRNA-regulated
mechanisms or alternative splicing may offer another
possibility for cancer treatment in the future (Table 1).
Posttranscriptional effects of miRNAs can be modulated
by specific synthetic miRNAs (mimics) or miRNA inhibitors (antagomirs) (15, 16 ). Moreover, several pharmacological tools have already been developed to modulate
alternative splicing, i.e., inhibitors of SR protein kinases,
such as the Clk inhibitor KH-CB19 and the DNA topoisomerase I inhibitor camptothecin (2, 5, 14 ). However,
the biological effects of these substances have only been
tested in vitro so far. Therefore, little is known about
their pathophysiological relevance, therapeutic potential,
and toxicity. Thus, further studies are needed to gain
further insights into posttranscriptional regulation mechanisms in cancer as well as the pathophysiological impact
of such posttranscriptional manipulations in vivo.
Conclusions
Both TF isoforms play essential and often isoformspecific roles in cancer biology and pathophysiology, i.e.,
in cancer-related thrombogenicity, metastasis, and angiogenesis (2, 8, 21, 23, 30 ). Differential TF isoform
functions are suggested to be mediated by isoformspecific signal transduction via PAR-2 or integrins ␣v␤3,
respectively (13, 23, 24 ).
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Tissue Factor Isoforms and Cancer
Several studies indicate that flTF, asTF, and TF
isoform–specific signaling are promising new targets for
the treatment of cancer and other human diseases
(13, 21, 23, 42, 45 ). Moreover, preliminary human
studies and clinical trials were done to characterize the
therapeutic value of drugs and molecules interacting with
TF, its isoforms, and associated signaling pathways, such
as a study indicating that indirect inhibition of TF activity by vitamin K antagonists was associated with a reduced risk for cancer development (37 ). Other clinical
trials have revealed beneficial effects of direct antibody–
or rTFPI-mediated blocking of TF activity in different
human pathologies (43, 45 ). However, the number of
adequate TF isoform–specific tools, such as drugs, antibodies, or silencing oligos, is limited so far. Thus, future
studies should focus on the development of novel therapeutic tools for asTF- and/or flTF-targeting treatment
approaches as well as on the characterization of their therapeutic potential and possible risks. Therefore, such TF
isoform–specific anticancer strategies need to be further
studied, especially in adequate in vivo models as well as in
clinical trials to assess their potential benefits as well as
undesirable side effects in cancer settings.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising
the article for intellectual content; and (c) final approval of the published
article.
Authors’ Disclosures or Potential Conflicts of Interest: No authors
declared any potential conflicts of interest.
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