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

1.9 MB·English

by  Stavik, Benedicte

Staviketal.JournalofHematology&Oncology2013,6:5
http://www.jhoonline.org/content/6/1/5 JOURNAL OF HEMATOLOGY
& ONCOLOGY
RESEARCH Open Access
α β
TFPI and TFPI are expressed at the surface of
breast cancer cells and inhibit TF-FVIIa activity
Benedicte Stavik1,2,3†, Mari Tinholt1,2,3,4†, Marit Sletten1, Grethe Skretting2,3, Per Morten Sandset2,3,4
and Nina Iversen1*
Abstract
Background: Tissuefactor(TF) pathway inhibitor-1 (TFPI) is expressed inseveral malignanttissues- and cell lines
and werecently reported that it possesses anti-tumor effects in breast cancer cells, indicating a biological roleof
TFPIin cancer. The two main splice variantsof TFPI; TFPIα and TFPIβ,are both able to inhibit TF-factor VIIa (FVIIa)
activity in normal cells, but only TFPIαcirculatesin plasma. The functional importanceof TFPIβis therefore largely
unknown, especially incancer cells. We aimed to characterize theexpression and function of TFPIα, TFPIβ, and TF in
a panelof tumor derived breast cancer cell lines in comparison to normal endothelial cells.
Methods: TFPIα,TFPIβ,and TF mRNAand protein measurements were conducted using qRT-PCR and ELISA,
respectively. Cell-associated TFPI was detected after phosphatidylinositol-phospholipase C(PI-PLC)and heparin
treatment byflow cytometry, immunofluorescence, and Western blotting. The potential anticoagulant activity of
cell surface TFPIwas determined ina factor Xaactivityassay.
Results: The expression ofboth isoforms of TFPIvaried considerably among the breast cancer cell lines tested,
from no expression inSum149 cells to levels above or in thesame range as normal endothelial cells inSum102
and MDA-MB-231cells. PI-PLC treatment released both TFPIα and TFPIβ from the breast cancer cell membrane and
increased TF activity onthe cell surface, showingTF-FVIIainhibitory activity of the glycosylphosphatidylinositol-
(GPI-) anchored TFPI. Heparin treatment released TFPIαwithout decreasingthecell surfacelevels, thus indicating
thepresence ofintracellular storage pools of TFPIα in thebreast cancer cells.
Conclusion: GPI-attached TFPIlocated atthe surface of breast cancer cells inhibited TFactivityand could possibly
reduce TF signaling and breast cancer cell growth locally, indicating a therapeutic potentialof the TFPIβisoform.
Keywords: TFPIα, TFPIβ, TF-FVIIa, Breast cancer cells, Endothelial cells, Heparin, PI-PLC, Anticoagulant activity
Introduction translationally modified in the ER and in Golgi, and are
Tissue factor (TF) pathway inhibitor-1 (TFPI) is a then transported by vesicles to the exterior of the cell
plasma serine protease inhibitor, which is mainly known [5]. The mature TFPIα protein comprises 276 amino
for its role in the coagulation cascade, being responsible acids and contains an N-terminal end, three Kunitz-type
for the modulation of TF induced blood coagulation. inhibitor domains, and a positively charged C-terminal
The human TFPI gene is positioned on chromosome 2 tail [1]. Intracellular TFPIα is thought to either remain
and spans about 70kb [1,2]. Two main splice variants soluble or bind to an unknown co-factor containing a
are transcribed from TFPI; TFPIα and TFPIβ [1,3,4]. glycosylphosphatidylinositol (GPI) anchor [5]. Depend-
Both mRNAs encode a signal peptide that redirects the ing on the nature of TFPIα inside the cell, the protein is
translatingribosometotheER,insertingtheproteininto either secreted or indirectly attached to the outer cell
the ER lumen as it is translated. The proteins are post- wall after transportation to the outer cell membrane
[5,6]. In comparison, the mature TFPIβ protein com-
*Correspondence:[email protected] prises223aminoacids.Itsharesaminoacids1–181with
†Equalcontributors TFPIα, and thus the N-terminal end and the first two
1DepartmentofMedicalGenetics,OsloUniversityHospitalandUniversityof
Oslo,BOX4956Nydalen,OsloN-0424,Norway Kunitz-type domains, but has a different C-terminal end
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Staviketal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Staviketal.JournalofHematology&Oncology2013,6:5 Page2of14
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containinga GPI attachment signal that exclusively loca- indicating the presence of intracellular storage pools of
lizesittothe cellmembrane[6]. TFPIαinthebreastcancercells.
In vivo, only TFPIα is detected in circulating blood
and this isoform is therefore considered to be important Results
inarresting the coagulationinitiation.Heparintreatment TFPIαandTFPIβmRNAandproteinexpressionlevels
rapidly increase the release of TFPI from endothelial Quantitative real-time PCR using reverse primers spe-
cells both in vivo [7] and in vitro [8,9], which may be an cific for TFPIα and TFPIβ was performed to determine
important anticoagulant effect of heparins [10,11]. Cell- the relative mRNA expression of the two isoforms in the
associated TFPIα and TFPIβ are both able to inhibit the variousbreastcancercelllines.Theresultswerenormal-
activity of the TF-factor VIIa (FVIIa) catalytic complex ized against the TFPI expression level in the primary
in normal cells in vitro [12] although their contribution human coronary artery endothelial cells (HCAECs)
to the anticoagulant effect is not completely understood. (Table 1), while the TFPI negative non-human Chinese
In cancer, TF expression and function has been exten- hamster ovary (CHO) cell line was used as a negative
sively studied and the non-haemostatic effects of TF in control in Figure 1A. The breast cancer cell lines
promoting cancer cell metastasis and angiogenesis are expressed TFPIα mRNA in the following high to low
well documented [13-19]. In contrast, TFPI has shown order: Sum102 > MDA-MB-231 > MCF-7 > SK-BR-3 >
anti-tumor effects. We recently investigated the bio- BT-474 > ZR-75-1 > Sum149. TFPIβ mRNA was
logical relevance of TFPI in breast cancer cells through expressed in the same high to low order as TFPIα
overexpression and knockdown studies and found that (Table 1), and the expression of the two isoforms corre-
both isoforms exerted tumor suppressing properties, lated significantly (r = 0.94, p < .001). As illustrated in
such as increased apoptosis and reduced proliferation- Figure 1A, the Sum102 breast cancer cells expressed
and migration/invasion in vitro [20-22]. Furthermore, twice as much TFPIα and β mRNA as the MDA-MB-
several cancer cell lines and tumors have been reported 231 cells, and 17- and 4-fold more TFPIα and TFPIβ
to express TFPIα [23,24], and enhanced levels of both mRNA, respectively, than the non-cancerous breast epi-
TFPIα and TFPI-factor Xa (FXa) complexes have been thelial cell line ME16C2.TheSum102 cell line expressed
detected in plasma of patients with solid tumors [25,26], twice as muchTFPIα and similar levels of TFPIβ as the
supporting that TFPI may be involved in malignant dis- HCAECs and the endothelial cell line EA.hy926, while
easemechanisms. the MDA-MB-231 cells expressed similar levels of
To further explain the role of TFPI in malignant dis- TFPIα, but only half the amount of TFPIβ as the
ease, we have in the present study aimed to characterize HCAECs and EA.hy926 cells (Table 1 and Figure 1A).
TFPI by investigating the expression and localization of Compared to the HCAECs, the relative TFPIα mRNA
TFPIαand TFPIβ, and also the relation to TF expression expression was 10-fold lower in the non-cancerous
and activity, in a panel of breast cancer cell lines. Since breast epithelial cells ME16C2 and 100 – 1000-fold
the endothelium is considered to be the predominant lower in the breast cancercell lines SK-BR-3 and MCF-7
site of TFPI synthesis, the TFPI characteristics of the (Table 1). Virtually no TFPIα or TFPIβ mRNA was
breast cancer cells were compared to normal endothelial expressed by the BT-474, ZR-75-1, and the Sum149 cell
cells. The selected breast cancer cell lines have been lines(Figure 1AandTable1).
derived from different breast tumors, and are classified TFPI antigen levels were measured using the free and
as basal- or luminal-like, and primary or metastatic total TFPI enzyme-linked immunoabsorbent assay
according to the subtype and origin of the tumor, re- (ELISA) kits. The freeTFPI kit detects an epitope in the
spectively. We report here considerable variations in the third Kunitz domain of TFPI and thereby measure only
expression of both TFPI isoforms among the various TFPIα. The total TFPI kit detects an epitope between
breast cancer cell lines tested. The expression ranged the first two Kunitz domains and it therefore able to
from not detected to levels comparable to and even measure both TFPI isoforms. ELISA of recombinant
higher than observed in normal, primary endothelial TFPI , which lacks the third Kunitz domain, con-
1-161
cells. TFPIα was detected in the cell medium and on the firmed this, as only the total, but not the free kit
surface of breast cancer cells, attached through a GPI detected the truncated TFPI (data not shown). Secreted
anchor, while TFPIβ was exclusively located on the cell TFPIα was measured in the cell medium, and TFPIα
surface. The GPI-attached, cell bound TFPI was found mRNA and protein levels correlated significantly
to be functionally active, and to our knowledge, demon- (r = 0.94, p = .002). The breast cancer cell lines secreted
strating for the first time anticoagulant properties of TFPIα in the following high to low order: Sum102 >
TFPI expressed on the surface of breast cancer cells. MDA-MB-231 > MCF-7 and SK-BR-3. No detectable
Heparin treatment increased the TFPIα levels in the levels of TFPIα protein were secreted by the BT-474,
supernatant without altering the cell surface levels, ZR-75-1, and Sum149 cells (Figure 1B and Table 1). The
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Table1CharacterizationofTFPIandTFinaselectionoftumorderivedbreastcancercelllinesandnormalcells t/6/1/5y&On
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Celltypes Description/Subtype Origin Invasiveness TFPIαmRNA(RQ) TFPIβmRNA(RQ) RelativeFreeTFPIα TFmRNA(RQ) Relativefl†TF olo
(Invitro)* (secreted) (cell-associated) gy
2
SUM102 Breastepithelial,basal-like Tumor(primary) Invasive 1.85 1.02 1.41 129 105 0
1
3
MDA-MB-231 Breastepithelial,basal-like Tumor(metastatic) Invasive 0.87 0.46 0.31 894 705 ,6
:5
MCF-7 Breastepithelial,luminal-like Tumor(metastatic) Non-invasive 0.01 0.02 0.04 0.45 0.96
SK-BR-3 Breastepithelial,luminal-like Tumor(metastatic) Non-invasive 3E-03 0.02 0.05 1.58 1.64
BT-474 Breastepithelial,luminal-like Tumor(primary) Non-invasive 8E-04 6E-03 n.d 2.63 1.32
ZR-75-1 Breastepithelial,luminal-like Tumor(metastatic) Non-invasive 7E-04 5E-03 n.d 3.55 2.33
SUM149 Breastepithelial,basal-like Tumor(primary) Invasive 2E-04 4E-03 n.d 4373 948
HCAEC Coronaryarteryendothelial Primary(normal) Non-invasive 1.00 1.00 1.00 1.00 1.00
EaHy926 Umbilicalveinendothelial Transformed Non-invasive 1.11 0.83 0.38 0.62 0.61
ME16C2 Breastepithelial,basal-like Primary(immortalized) Invasive 0.11 0.25 0.28 336 170
*fromreferences[38-42].
RQ-relativequantificationcalculatedusing18sRNAasendogenouscontrol.AllvalueswerecalculatedagainstHCAECexpressionasanormalendothelialreference.
†fulllength.
n.d.-notdetected,belowloweststandard.
TFPIandTFmRNAandproteinlevelsweremeasuredusingqRT-PCRandELISAinthebreastcancercellsandinthenon-cancerousbreastepithelialcellsME16C2andumbilicalveinendothelialcelllineEaHy926.All
valueswerecomparedtotheprimaryhumancoronaryarteryendothelialcellsHCAECandpresentedasrelativevalues,mRNAresultsbythecomparativeCtmethodandantigenlevelsbydirectcomparison.
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Figure1TFPIexpressionindifferentbreastcancercelllinesandnormalcells.Cellswereseededin6-wellstraystoreach~80%confluence
inthreedaysbeforemediawerecollectedandcellslysedforRNAisolation.(A)RelativeTFPIαandTFPIβmRNAexpressionmeasuredusingqRT-
PCR.ΔΔCtvalueswerecalculatedusing18srRNAasanendogenouscontrolandtheTFPInegative,non-humanCHOcellsasanegativecontrol.
(B)SecretedTFPIαproteinmeasuredincellmediaandnormalizedtototalproteinamounts.Meanvalues+SD(n=3)arepresented.
Sum102 cell line secreted 40% more TFPIα than the Cleavage of the GPI anchors by phosphatidylinositol-
HCAECs, while the MDA-MB-231 cells secreted TFPIα phospholipase C (PI-PLC) treatment resulted in a sig-
levels within the range of the endothelial cell line EA. nificant 1.5- and 5.3-fold increase in free and total TFPI
hy926 and the non-cancerous breast epithelial cells antigen levels, respectively, in the Sum102 cell super-
ME16C2 (Figure 1B and Table 1). The breast cancer cell natant (Figure 2A), and a corresponding 49% decrease in
lines that expressed abundant TFPIα and TFPIβ origi- total, but not free TFPI antigen levels, in the cell lysate
nated from both primary and metastatic basal-like (Figure 2B). PI-PLC treatment reduced the amount of
tumors and have previously been shown to display inva- TFPI positive Sum102 cells from 31.8 (± 3.9) to 3.9
sive characteristicsinvitro(Table1). (± 0.2) as measured by flow cytometry, corresponding to
an approximately 75% removal of the cell surface TFPI
TFmRNAandantigen (Figure 2C). Immunostaining demonstrated a clear, but
TF protein levels were measured in the cell lysate and not a complete removal of cell surface TFPI after PI-
correlated significantly with mRNA expression in all the PLC treatment of the cells (Figure 2D). This effect was
cell lines (r = 0.99, p < .001). Moreover, the expression also apparent when a TFPIα specific antibody was used
of TF associated withTFPI expression in all breast can- (data not shown). Western blot analysis of deglycosy-
cer cell lines tested except the Sum149 cells. Compared lated PI-PLC supernatants showed that both isoforms of
to the endothelial cells HCAEC, high levels of TF TFPI were released after PI-PLC treatment of Sum102
mRNA were detected in the basal-like, invasive breast cells (Figure 2E). Slightly heavier bands than previously
cancer cell lines Sum102, MDA-MB-231, and Sum149, reported for TFPI was observed, which may be due to
and also in the non-cancerous breast epithelial cells cancer cell specific modifications.No significant increase
ME16C2. In contrast, the luminal-like, non-invasive in free and a minor increase in total TFPI antigen was
breast cancer cell lines MCF-7, SK-BR-3, BT-474, and detected in the MDA-MB-231 cell supernatants after PI-
ZR-75-1 expressed low levels of TF mRNA, in the same PLC treatment (Figure 2F), and a slight reduction in cell
range as the endothelial cells HCAEC and EA.hy926 surface TFPI levels was seen by flow cytometry
(Table 1). The breast cancer cells Sum149 and MDA- (Figure 2G). No significant decrease in free or total TFPI
MB-231 expressed 13- and 3-fold more TF mRNA, re- was detected in the cell lysate of these cells after PI-PLC
spectively, compared to the non-cancerous ME16C2 treatment(data not shown).
cells, while the Sum102 cell line expressed less TF Heparin treatment of Sum102 cells increased the free
mRNA relative toME16C2 cells,but 130-foldmorethan and total TFPI antigen levels 2.9- and 2.6-fold, respect-
theHCAECs(Table 1). ively, in the cell supernatant (Figure 3A). A correspond-
ing 48% and 32% decrease in free and total TFPI,
Cell-associatedTFPIinbreastcancercellsversusnormal respectively, was seen in the cell lysate (Figure 3B). PI-
endothelialcells PLC pre-treatment prior to heparin treatment resulted
The breast cancer cell lines Sum102 and MDA-MB-231, in an additional 60-70% increase in free and total TFPI
possessing high TFPI expression, were further analyzed antigen levels in Sum102 supernatant compared to hep-
to determine the nature of any cell-associated TFPI. arin treatment alone (Figure 3A). A reduction in TFPI
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(Seefigureonpreviouspage.)
Figure2PI-PLCtreatmentofSum102andMDA-MB-231cells.Serum-starvedcellsweretreatedwithSFM(untreated)orPI-PLC(1U/mL)for2
hoursbeforethesupernatantwasremovedandcellslysedorcollectedforflowcytometryanalysis.TotalandfreeTFPIantigenmeasuredinthe
supernatant(AandF)andlysate(B)ofuntreatedandPI-PLCtreatedSum102(AandB)andMDA-MB-231(F)cells.Meanvalues+SD(n≥8)of
threetosixindependentexperimentsarepresented.AllstatisticalcomparisonswereconductedbetweenPI-PLCtreatedsamplesanduntreated
controls.Flowcytometryanalysisofuntreated(black,solid)andPI-PLCtreated(black,broken)Sum102(C)andMDA-MB-231(G)cells.One
representativeexperimentofthreeisshown.(D)FluorescenceimagesofSFM+glycerol(untreated,leftpanel)andPI-PLC(middlepanel)treated
Sum102cellsstainedwithantibodyagainstbothisoformsofTFPI.Therightpanelshowsasecondaryantibodybackgroundcontrolstaining.One
representativeexperimentofthreeisshown.(E)WesternblotofsupernatantsfromSFM+glycerol(untreated)andPI-PLCtreatedSum102cells.
Samplesweredeglycosylatedandincubatedwithapolyclonalanti-humanTFPIantibody.
levels was also observed in cell lysates, especially appar- TF-FVIIaactivityandco-localization
ent in the total TFPI levels (Figure 3B). A 2.1- and 2.3- To explore the potential anticoagulant activity of the
fold increase in free and total TFPI antigen levels, breast cancer and endothelial cell-associated TFPI, we
respectively, was observed in the supernatant of MDA- measured theTF activity on the Sum102, MDA-MB-231
MB-231 cells after heparin treatment, though no add- and HCAEC cell surface indirectly by a chromogenic
itional increase was seen when the cells were pre-treated FXa activity assay. The generation of FXa on the surface
with PI-PLC (Figure 3C). Heparin reduced free and total of Sum102 and HCAEC cells increased significantly by
TFPI levels in the cell lysate by 40% and 34%, respect- 1.3- and 2-fold, respectively, following PI-PLC treatment
ively, and pre-treatment with PI-PLC reduced the levels (Figure 5A and C). A small 10% increase in FXa levels
even further (Figure 3D). Flow cytometry showed no re- was observed on MDA-MB-231cells after PI-PLC treat-
duction of cell surface TFPI levels after heparin treat- ment,althoughnot significant probably duetotheabun-
ment in either cell line (Figure 3E), and no additional dant TF levels in these cells (Figure 5B). Treating cells
reductionincellsurfaceTFPIwasobservedafterheparin with anti-TFPI blocking antibody resulted in higher FXa
treatmentofPI-PLC pre-treatedcells (datanot shown). activity than PI-PLC treatment in all cells tested
TheTFPI characteristics in the breast cancer cell lines (Figure 5A-C), indicating that additional non-GPI bound
were verysimilar tothose observedintheprimaryendo- TFPI with anticoagulant activity is present on the cell
thelial cells HCAEC. In the supernatant, free and total surface. ELISA results confirmed that PI-PLC treatment
TFPI antigen levels were increased 1.3- and 2.4-fold, re- did not alter theTF antigen levels in the cells compared
spectively (Figure 4A) after PI-PLC treatment. Comple- to untreated controls (data not shown). Control experi-
mentary, the free and total TFPI antigen levels in the ments performed in the absence of FVIIa showed no
cell lysate were reduced by 27% and 40%, respectively generation of FXa (data not shown), and the addition of
(Figure 4B). Measured by flow cytometry, TFPI positive anti-TF blocking antibody prior to analysis suppressed
cells were reduced from 200.4 (±16.9) to 43.1 (±1.9) fol- the FXa generation significantly, except in the HCAEC
lowing PI-PLC treatment, corresponding to a 90% re- cells (Figure 5A-C). Immunostaining with both anti-
moval of TFPI from the endothelial cell surface TFPI and anti-TF antibodies simultaneously demon-
(Figure 4E, left panel). As demonstrated for Sum102 and strated a co-localization of these proteins at the surface
MDA-MB-231 cells, no reduction in cell surface TFPI of Sum102 cells (Figure 5D, upper panels), which was
was observed by flow cytometry after heparin treatment diminished after PI-PLC treatment (Figure 5D, bottom
of HCAECs (Figure 4E, right panel), though total TFPI panels).
levels were increased 2-fold in the cell supernatant and
reducedby38%inthecelllysate.Furthermore,freeTFPI Discussion
was increased 2.9-fold in the supernatant and reduced In the present study, we have characterized the mRNA
by 46% in the cell lysate (Figure 4C and D). Sequential and protein expression of two isoforms of TFPI (α and
treatment with PI-PLC followed by heparin did not re- β) and also TF, in a panel of breast cancer cell lines in
duce TFPI cell surface levels any further than PI-PLC comparison to normal endothelial cells. We have pro-
alone (datanot shown),whichisequivalenttothe obser- vided novel evidence that non-cancerous and malignant
vations in the breast cancer cell lines. Moreover, PI-PLC breast epithelial cells express TFPIβ protein. The breast
pre-treatment of HCAEC cells followed by heparin cancer cell lines are classified as either basal- or
released 50% more free TFPI into the supernatant and luminal-like according to the subtype of the tumor they
reduced cell lysate levels additionally compared to hep- were derived from. In general, basal-like breast cancer
arin alone (Figure 4C and D) as seen in the Sum102 cells tend to lack hormone receptors and are more inva-
breastcancercells. sive than luminal-like cells [27,28]. TheTFPI expression
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Figure3HeparintreatmentofSum102andMDA-MB-231cells.Serum-starvedcellsweretreatedwithSFM(untreated)orheparin(5U/mL)
for2hoursorwithPI-PLC+heparin(2+2hours)beforethesupernatantwasremovedandcellslysedorcollectedforflowcytometryanalysis.
ThesupernatantwasdiscardedafterPI-PLCpre-treatment.FreeandtotalTFPIantigenmeasuredinthesupernatant(AandC)andlysate
(BandD)ofuntreated,heparinorPI-PLC+heparintreatedSum102(AandB)andMDA-MB-231(CandD)cells.Meanvalues+SD(n≥9)of
threetosixindependentexperimentsarepresented.Statisticalcomparisonswereconductedbetweenheparin/PI-PLC+heparintreatedsamples
anduntreatedcontrols.(E)Flowcytometryanalysisofuntreated(black,solid)andheparintreated(black,dotted)Sum102(leftpanel)andMDA-
MB-231(rightpanel)cells.Onerepresentativeexperimentofthreeisshown.
seemed to correlate well with invasiveness and basal-like cell type dependent differences in the expression of a
origin of the cells, except for the TFPI negative Sum149 GPI-attached cofactor for TFPIα. Intriguingly, no reduc-
cell line. Among the cell lines tested,TFPI expression was tion in free TFPI was observed in Sum102 cell lysates
detected in the following high to low order; Sum102 > after PI-PLC treatment as seen in the HCAEC cell
MDA-MB-231>MCF-7>SK-BR-3>BT-474>ZR-75-1> lysates. This may be due to higher background levels of
Sum149, which is comparable to data from Kao and co- free TFPI in the Sum102 cell lysate than in the super-
workers[28]whenweextractedtheTFPIexpressionresults natant. Thus it seems likely that the amount of GPI-
from their publicly available data file of gene expression attached TFPIα was insignificant compared to the total
profiles of different breast cancer cell lines. The TFPI ex- amount of cell-associated TFPIα in these cells. Low
pression also seemed to correlate with the growth rate of levels of TFPI seemed to be released from the MDA-
the cells as MDA-MB-231, MCF-7, BT-474, and ZR-75-1 MB-231 cells after PI-PLC treatment, which may be due
have a doubling time of approximately 23, 29, 72, and 80 to the relative low TFPIβ expression in these cells com-
hours, respectively. Moreover, high expression of TF was pared to the Sum102 and endothelial cells, or to other
also observed in cells expressing TFPI which may contrib- structural differences on the surface of these cells that
ute to the growth of the cells. Previously, the MDA-MB- madetheGPIanchors inaccessible toPI-PLC.
231 cells were also shown to express TFPI mRNA and Heparintreatment resultedinthereleaseofTFPIfrom
protein [23]. The relative mRNA ratios between the two the Sum102 and MDA-MB-231 breast cancer cells in a
TFPI isoforms correlated well in all the breast cancer cell similar manner to that observed in normal endothelial
lines in terms of TFPIα being the major isoform, which is cells in this and a previously study [32]. Moreover, our
consistent with our findings and previous results in endo- findings support the growing evidence that the full
thelial cells [6]. A positive correlation between TFPIα length TFPIα is the exclusive TFPI isoform released
mRNAandsecretedantigenlevelswasobservedinthecell upon heparin treatment as similar levels were detected
lines. However, HCAEC cells secreted 3-fold more TFPIα using either ELISA kit. Heparin treatment released TFPI
protein than MDA-MB-231 and EA.hy926 cells, although from breast cancer cells without reducing cell surface
TFPIαmRNAlevelsweresimilar.Thiscouldimplythatless levels, which is consistent with what was observed in
TFPIα is cell-associated in these cells compared to the endothelial cells in this and other studies [6,8,9,30].
MDA-MB-231 breast cancer and EA.hy926 endothelial Thus, our findings indicate that the breast cancer cells
cells. possessed intracellular storage pools of TFPIα in a simi-
Recently, Girard and co-workers reported that TFPIβ lar manner to that suggested for normal endothelial
was the soleTFPI isoform associated with the SK-Hep-1 cells. The basic mechanism and exact cellular
endothelial cell surface through a GPI anchor [29]. In localization of this heparin-releasable TFPI is, however,
contrast to this, our findings indicated that both TFPIα currently unknown. In contrast to what was reported
and TFPIβ were attached to the surface of the Sum102 previously for HUVECs [32], serum was not necessary
breast cancer cells through GPI anchors as confirmed by for the release of TFPIα by heparin in the breast cancer
ELISA and Western blotting of deglycosylated PI-PLC cells or HCAECs, although serum did increase the
treated supernatants. Moreover, a significant increase in amount of TFPI released by heparin. Treatment with PI-
both free and total TFPI was observed after PI-PLC PLC uncovered a heparin-releasable TFPI pool that was
treatment of the normal endothelial cells HCAEC in this not accessible before treatment, since PI-PLC prior to
study, although ELISA results indicated that TFPIβ was heparin treatment released additional TFPIα from the
the most abundant GPI-attached isoform in both cell Sum102 and HCAEC cells. Thus, some GPI-attached
types. Endothelial cells have previously been shown to cell surface protein(s) seemed to interfere with the abil-
expressTFPIα on the cell surface [30], and in a study by ity of heparin to release intracellular TFPIα. This is in
Piro and Broze the endothelial cells ECV304 (later iden- contrast to the findings of Ellery and co-workers in
tified as a bladder carcinoma cell line [31]) were shown HUVECs [32], but consistent with those observed by
to express both TFPIα and TFPIβ on the surface [12]. Zhang et al. [6] and Mast and colleagues [33] in
These results, together with our findings, may indicate HUVEC, ECV406, and EA.hy926 cells and in human
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(Seefigureonpreviouspage.)
Figure4PI-PLCandheparintreatmentofHCAECcells.Serum-starvedcellsweretreatedwithSFM(untreated),PI-PLC(1U/mL),orheparin
(5U/mL)for2hoursorwithPI-PLC+heparin(2+2hours)beforethesupernatantwasremovedandcellslysedorcollectedforflowcytometry
analysis.ThesupernatantwasdiscardedafterPI-PLCpre-treatment.TotalandfreeTFPIantigenmeasuredinthesupernatant(AandC)andcell
lysate(BandD)ofuntreated/PI-PLC(AandB)anduntreated/heparin/PI-PLC+heparin(CandD)treatedHCAECcells.Meanvalues(n≥5;
supernatant,n=4;celllysate)+SDoftwoindependentexperiments.StatisticalcomparisonswereconductedbetweenPI-PLCorheparin/PI-PLC
+heparintreatedsamplesanduntreatedcontrols.(E)Flowcytometryanalysisofuntreated(black,solid),PI-PLC(black,broken,leftpanel),and
heparin(black,dotted,rightpanel)treatedHCAECcells.Onerepresentativeexperimentofthreeisshown.
placenta. Contradictory to aprevious report on endothe- of TFPI in breast cancer cells conducted in previous
lial cells [6] we did not observe any additional release of experiments resulted in an increase in TF, PAR-1, and
cell surface attached TFPI by heparin after PI-PLC pre- PAR-2 mRNA levels, which may indicate that TFPI is in
treatment in our cells. Our methodology is virtually the fact able to affect TF signaling [20]. Pre-treatment with
same, and it is therefore likely that this discrepancy may anti-TFPI blocking antibody increased TF activity even
be due to yet unrevealed cell type specific characteristics more than PI-PLC treatment, indicating the presence of
like differences in the expression of other TFPI binding non-GPI-attached cell surface TFPI that might possess
cellsurfacemoleculessuch assyndecans [34]. anticoagulantactivity.
When looking further into the characteristics of the
different breast cancer cell lines, we discovered a similar Conclusions
substantial variation in the TF mRNA expression as We provide evidence forthe expressionof both isoforms
observed for TFPI. The MDA-MB-231 and MCF-7 cells ofTFPI (TFPIα andTFPIβ)in tumor derived breastcan-
have previously been shown to express high and low cer cell lines. The TFPI expression associated to some
levels of TF [35], respectively, which support our find- extent with the TF expression and the invasiveness and
ings. Interestingly, theTF mRNA and antigen levels cor- basal- or luminal-like subtype of the cells. TFPIα was
related well with the subtype and degree of invasiveness secreted by the cells or remained cell-associated either
of the cells, supporting the tumor growth promoting na- bound to the surface indirectly through a GPI anchor or
ture of TF [13-18,36,37]. In accordance with the expres- located in intracellular, heparin-releasable pools. TFPIβ
sion levels, the TF activity was greater in MDA-MB-231 was exclusively found on the cell surface attached
cells than in Sum102 cells reflecting that TF was in fact through a GPI anchor. PI-PLC-releasable, GPI-attached
more abundant on the surface of these cells. Moreover, cell surface TFPI co-localized withTF and inhibited TF-
the low TF expression in the HCAEC cells was reflected FVIIaactivityandtheremovalofsuchanchoredproteins
by the low levels of FXa generated in the assay and the allowed for the additional release of intracellular TFPIα
inability to further reduce FXa activity after TF blocking. by heparin. Furthermore, flow cytometry, immunofluor-
The increase in TF activity following PI-PLC treatment escence, and FXa activity measurements suggested the
of Sum102 and HCAEC cells also indicated that the presence of a non-GPI bound cell surface associated
GPI-attached, cell surface TFPI was able to inhibit TF- TFPI pool with anticoagulant properties. Similar com-
FVIIa and/or FXa activity on the same cell. The immu- plex features of the two isoforms of TFPI were observed
nostaining results also confirmed association of TFPI in breast cancer cells and in endothelial cells, although
and TF at the cell surface. Thus, GPI-attached TFPI the variation in TFPI levels in the differentbreast cancer
expressed by breast cancer cells was able to directly cell lines demonstrated a tumor-associated regulative
interfere with theTFactivity on the cell surface and may controloftheendogenousTFPIexpression.Ourfindings
therefore have implications for the pro-cancer nature of indicate a possible functional implication of TFPIβ in lo-
TF. Although we were unable to differentiate between cally reducing TF signaling and breast cancer cell pro-
TFPIα and TFPIβ in the procoagulant assay, TFPIβ gression, further endorsing involvement of TFPI in
appeared to be the most abundant isoform on the cell malignantdisease.
surface and we have previously observed that overex-
pression of TFPIα and TFPIβ separately suppressed Materials and methods
breast cancer cell growth by inhibiting cell proliferation Reagents
and increasing apoptotic activity [20], indicating func- RPMI1640,Dulbecco’smodifiedEagle’smedium(DMEM),
tional importance of both isoforms. However, it remains Endothelial Basal Medium-2 (EBM-2), EGM-2-MV Sin-
to clarify whether the GPI-attached TFPI is indeed able gleQuots, fetal bovine serum (FBS) and phosphate buf-
to inhibit TF signaling, which is important in the pro- fered saline (PBS) were purchased from Lonza (Viviere,
tumor effect of TF [18], or if the effects are due to sig- Belgium), while the Human mammary epithelial cell
W
naling mechanisms independent of TF. Overexpression (HuMEC) ready medium, Alexa Fluor 488 goat anti-