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. 2015 Sep;13(9):1683-93.
doi: 10.1111/jth.13049. Epub 2015 Jul 31.

Alternatively spliced tissue factor synergizes with the estrogen receptor pathway in promoting breast cancer progression

B Kocatürk  1 , C Tieken  1 , D Vreeken  1 , B Ünlü  1 , C C Engels  2 , E M de Kruijf  2 , P J Kuppen  2 , P H Reitsma  1 , V Y Bogdanov  3 , H H Versteeg  1
Affiliations

Alternatively spliced tissue factor synergizes with the estrogen receptor pathway in promoting breast cancer progression

B Kocatürk et al. J Thromb Haemost. 2015 Sep.

Abstract

Background: Procoagulant full-length tissue factor (flTF) and its minimally coagulant alternatively spliced isoform (asTF), promote breast cancer (BrCa) progression via different mechanisms. We previously showed that flTF and asTF are expressed by BrCa cells, resulting in autoregulation in a cancer milieu. BrCa cells often express hormone receptors such as the estrogen receptor (ER), leading to the formation of hormone-regulated cell populations.

Objective: To investigate whether TF isoform-specific and ER-dependent pathways interact in BrCa.

Methods: Tissue factor isoform-regulated gene sets were assessed using ingenuity pathway analysis. Tissues from a cohort of BrCa patients were divided into ER-positive and ER-negative groups. Associations between TF isoform levels and tumor characteristics were analyzed in these groups. BrCa cells expressing TF isoforms were assessed for proliferation, migration and in vivo growth in the presence or absence of estradiol.

Results: Ingenuity pathway analysis pointed to similarities between ER- and TF-induced gene expression profiles. In BrCa tissue specimens, asTF expression was associated with grade and stage in ER-positive but not in ER-negative tumors. flTF was only associated with grade in ER-positive tumors. In MCF-7 cells, asTF accelerated proliferation in the presence of estradiol in a β1 integrin-dependent manner. No synergy between asTF and the ER pathway was observed in a migration assay. Estradiol accelerated the growth of asTF-expressing tumors but not control tumors in vivo in an orthotopic setting.

Conclusion: Tissue factor isoform and estrogen signaling share downstream targets in BrCa; the concomitant presence of asTF and estrogen signaling is required to promote BrCa cell proliferation.

Keywords: blood coagulation; cell movement; cell proliferation; integrin beta1; tumors.

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Figures

Fig. 1
Fig. 1
asTF expression positively associates with BrCa grade and stage in patients with ER+, but not ER− tumors. A) Associations of asTF or flTF expression with BrCa grade and stage in patients with ER+ tumors. B) Associations of asTF or flTF expression with BrCa grade and stage in patients with ER+ tumors. The cohort comprised 574 non-metastatic BrCa patients; Χ2 statistical tests were used to evaluate associations. Please see supplementary tables 3 and 4 for the associations between asTF or flTF expression and other clinical parameters.
Fig. 2
Fig. 2
Estrogens and asTF cooperate to induce BrCa cell proliferation. A) Control, flTF- and asTF-expressing cells were cultured in phenol red-containing media in the presence or absence of ER inhibitor ICI-182,780 (final [C] = 100 nM). After 3 days, proliferation rates were determined using MTT assay. B) Control, flTF- and asTF-expressing cells were cultured in phenol red free medium. Cells were treated with either 1 nM E2 or solvent control (EtOH). After 6 days, proliferation rates were determined using MTT assay. C) As in B, but using 10 nM of E2. D) ERα transcript levels in control, flTF-and asTF-expressing cells were determined using real-time PCR. E) ERα protein levels in control, flTF-and asTF-expressing cells were determined by western blotting. F) β1 integrin was downregulated in asTF-expressing cells using lentiviral shRNA. Scrambled shRNA was used as a control. Reduction of β1 integrin protein levels was verified by western blotting. G) Cells were treated with 1 nM E2 or solvent control. Proliferation rates were assessed using MTT assay at days 3 and 6. *P < 0.05, **P <0.01, and ***P < 0.001. H) Proliferation of asTF-expressing cells in the presence or absence of ER and the asTF-blocking antibody Rb1.
Fig. 3
Fig. 3
E2 and asTF independently promote BrCa cell migration. A) Cells were seeded to confluency in silicone inserts. The following day, inserts were removed to leave a gap (demarcated using a dashed line). Gap closure was monitored at regular time intervals. B) The remaining gap area was calculated as percent closure of the area at t=0 by using Image J software. Scale bars: 300μm.
Fig. 4
Fig. 4
E2 increases the growth of asTF+ BrCa cells in vivo. A) pcDNA or asTF cells were injected into mammary fat pads of NOD/SCID mice. Estrogen pellets were placed subcutaneously. Tumor growth was monitored for 12 weeks by measuring tumor volume. B) Final tumor volumes are shown. C) Proliferating cells were detected using Ki67 staining. Scale bars: 50μm. D) Ki67+ cells were counted and represented as percent of the total cell number. *P < 0.05, **P <0.01, and ***P < 0.001. E) Vessel structures were detected using anti-vWF antibody F) vWF signal strength is shown as fold increase over control, scale bars: 50μm, *P < 0.05.
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