PLoS One. 2012;7(10):e47184.

TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

Stavik B, Skretting G, Olstad OK, Sletten M, Dehli Vigeland M, Sandset PM, Iversen N.

Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway.

 

Abstract

Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes facilitating cancer cell growth and immunologic response, possibly by signal transduction involving the EGFR pathway.

PMID: 23071754

 

SUPPLEMENT:

An association between cancer and thrombosis has been recognized since the mid 19th century when Bouillaud and later Trousseau, two French physicians, described the occurrence of thrombosis in cancer patients and the development of cancer in patients with thrombotic disease. Today, it is widely recognized that especially solid tumors favor a pro-thrombotic environment, and this association between the hemostatic system and cancer seems to be related to the many processes involved in the development of tumor cells and malignant disease. The physiological role of tissue factor pathway inhibitor (TFPI) as an inhibitor of coagulation is well established, however, recently the significance and impact of the different isoforms of TFPI has been increasingly investigated, also in relation to cancer development. The two main isoforms of TFPI-1, TFPIa and TFPIb, share the first 181 amino acids, which contain the two Kunitz-type inhibitor domains (K1-2) responsible for the anticoagulant activity. In addition, TFPIa consists of a third Kunitz-type domain and a positively charged C-terminal end while TFPIβ has a different shorter C-terminal end encoding a glycosylphosphateidylinositol (GPI) anchor. TFPIa either remains associated with the endothelial cells (80-85 %) or is secreted (-10 %), and in vivo, secreted TFPIa circulates in plasma either as a free, full-length form or as truncated forms, which exist primarily in complex with lipoproteins. A portion of the cell associated TFPIα is stored intracellularly and can be released immediately upon heparin treatment, while the remainder is present on the cell surface. No direct association between TFPIα and the plasma membrane has been shown, instead the binding seems to be indirectly through other presently unknown cell surface molecules. We have recently investigated the association of TFPI with heparan sulfates and found Syndecan-3 to be a binding molecule of TFPIa on the surface of endothelial, smooth muscle cells, and breast cancer cells (Tinholt et al. under review). Due to its GPI anchor, TFPIbeta is exclusively located on the cell surface (Figure 1, hypothetical illustration).

Nina Iversen-fig1

Figure 1. Translated TFPIalpha and TFPIbeta are located in the endoplasmatic reticulum (ER) lumen. Here, TFPIalpha either remains soluble or binds to an unknown cofactor attached to the ER membrane through a GPI anchor. TFPIbeta remains attached to the ER membrane through a GPI anchor encoded in its C-terminal end. Transport vesicles carry the proteins to the cell surface where they are stored intracellularly or secreted (TFPIalpha), or remain attached to the cell membrane (TFPIalpha and beta).

 

We recently reported the expression of TFPI isoforms in different breast cancer cell lines and found considerable differences. It ranged from levels higher than or similar to normal endothelial cells, to levels barely detectable. In all cells tested TFPIa was the predominant isoform (Figure 2). The reason for and mechanism behind the different regulation of TFPI expression in these cancer cells are not known.  We are currently exploring whether SNPs or mutations in the TFPI gene may have any regulatory effects on the expression of TFPI in breast cancer patients.

Nina Iversen-fig2

Figure 2. TFPI expression in different breast cancer cell lines and normal cells. (A) Relative TFPIα and TFPIβ mRNA expression measured using qRT-PCR. ΔΔCt values were calculated using 18s rRNA as an endogenous control and the TFPI negative, non-human CHO cells as a negative control. (B) Secreted TFPIα protein measured in cell media and normalized to total protein amounts. Mean values + SD (n = 3) are presented. Stavik and Tinholt et al. Journal of Hematology & Oncology 2013 6:5  doi:10.1186/1756-8722-6-5

 

In the original article, we overexpressed each isoform of TFPI individually in SK-BR-3 breast cancer cells and investigated the global changes in mRNA and microRNA expression. Although we identified genes exclusively associated with one or the other of the isoforms, and despite their differences in expression and localization, genes involved in the same processes seemed to be affected, and they related closely to cancer cell development and the immune response. Signaling molecules indicated a possible involvement of the EGFR signal transduction pathway and we hypothesize that cell bound TFPI could be able to interfere with or enhance EGFR signaling, thereby altering gene expression and subsequently the processes affected (Figure 3).

Nina Iversen-fig3

Figure 3. Hypothetical illustration of TFPIs interaction with the EGFR signaling pathway (made using SmartDraw).

In 2013, a paper was published showing considerable correlation between RET expression and ASCL1 status in lung cancer samples. The RET expression was high in ASCL1 positive cancers and low in ASCL1 negative cancers. In our paper, RET and ASCL1 were among the 10 most downregulated genes when either of the isoforms of TFPI were overexpressed. RET (Rearranged during transfection) is a proto oncogene and gain-of-function mutations are associated with the development of various types of human cancers. ASCL1 (Achaete-scute homolog 1) is a member of the basic helix-loop-helix transcription factors and activates transcription by binding to the E box (5′-CANNTG-3′) in DNA. The possibility that ASCL1 regulates RET expression has to be investigated but it could provide a partial explanation for the differences in cancer-related gene expression observed in this study.

During the past years we have accumulated considerable evidence for the involvement of TFPI in cancer cell progression in vitro. To test our hypothesis in vivo, we used another breast cancer cell line model with TFPI stably knocked down. The TFPI isoforms were knocked down in MDA-MB-231 cells using siRNA techniques. These cells are able to spontaneously develop tumors when transplanted into the mammary fatpads of mice. When cells with TFPI knocked down was injected, a considerable increase in tumor growth was observed, in comparison to control cells. Future experiments including blocking of the EGFR pathway could elucidate the involvement of this pathway in the TFPI facilitated cancer cell growth.

 

Dr. Benedicte Stavik (Left) and Dr. Nina Iversen (Right)

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