Tumor expression of alternatively spliced tissue factor is a prognostic marker in non‐small cell lung cancer
2009; Elsevier BV; Volume: 8; Issue: 3 Linguagem: Inglês
10.1111/j.1538-7836.2009.03713.x
ISSN1538-7933
AutoresJérôme Rollin, S. Régina, Y. Gruel,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoTissue factor (TF), a 47‐KDa transmembrane protein, is the essential receptor for factor (F)VII/VIIa and the physiological trigger of blood coagulation [1Monroe D.M. Key N.S. The tissue factor‐factor VIIa complex: procoagulant activity, regulation, and multitasking.J Thromb Haemost. 2007; 5: 1097-105Crossref PubMed Scopus (118) Google Scholar]. In addition, TF is directly involved in cancer progression by promoting tumor growth, angiogenesis, cell migration and development of metastases [2Rak J. Milsom C. Yu J. Tissue factor in cancer.Curr Opin Hematol. 2008; 15: 522-8Crossref PubMed Scopus (45) Google Scholar]. In 2003, Bogdanov et al. [3Bogdanov V.Y. Balasubramanian V. Hathcock J. Vele O. Lieb M. Nemerson Y. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein.Nat Med. 2003; 9: 458-62Crossref PubMed Scopus (392) Google Scholar] described a human alternatively spliced TF (asTF), a TF isoform that lacks exon 5 of the mature TF gene, which encodes the transmembrane domain. asTF is therefore soluble and has been detected both in the blood circulation and in various normal and cancerous tissues [3Bogdanov V.Y. Balasubramanian V. Hathcock J. Vele O. Lieb M. Nemerson Y. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein.Nat Med. 2003; 9: 458-62Crossref PubMed Scopus (392) Google Scholar, 4Goldin‐Lang P. Tran Q.V. Fichtner I. Eisenreich A. Antoniak S. Schulze K. Coupland S.E. Poller W. Schultheiss H.P. Rauch U. Tissue factor expression pattern in human non‐small cell lung cancer tissues indicate increased blood thrombogenicity and tumor metastasis.Oncol Rep. 2008; 20: 123-8PubMed Google Scholar, 5Szotowski B. Goldin‐Lang P. Antoniak S. Bogdanov V.Y. Pathirana D. Pauschinger M. Dörner A. Kuehl U. Coupland S. Nemerson Y. Hummel M. Poller W. Hetzer R. Schultheiss H.P. Rauch U. Alterations in myocardial tissue factor expression and cellular localization in dilated cardiomyopathy.J Am Coll Cardiol. 2005; 45: 1081-9Crossref PubMed Scopus (72) Google Scholar, 6Haas S.L. Jesnowski R. Steiner M. Hummel F. Ringel J. Burstein C. Nizze H. Liebe S. Löhr J.M. Expression of tissue factor in pancreatic adenocarcinoma is associated with activation of coagulation.World J Gastroenterol. 2006; 12: 4843-9PubMed Google Scholar, 7Rauch U. Antoniak S. Boots M. Schulze K. Goldin‐Lang P. Stein H. Schultheiss H.P. Coupland S. Association of tissue‐factor upregulation in squamous‐cell carcinoma of the lung with increased tissue factor in circulating blood.Lancet Oncol. 2005; 6: 254Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar]. Recent studies have proved that asTF does not play a significant role during coagulation [8Censarek P. Bobbe A. Grandoch M. Schror K. Weber A.A. Alternatively spliced human tissue factor (asHTF) is not pro‐coagulant.Thromb Haemost. 2007; 97: 11-4Crossref PubMed Scopus (59) Google Scholar, 9Boing A.N. Hau C.M. Sturk A. Nieuwland R. Human alternatively spliced tissue factor is not secreted and does not trigger coagulation.J Thromb Haemost. 2009; 7: 1423-6Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar] but is likely essential during angiogenesis associated with the development of cancer [4Goldin‐Lang P. Tran Q.V. Fichtner I. Eisenreich A. Antoniak S. Schulze K. Coupland S.E. Poller W. Schultheiss H.P. Rauch U. Tissue factor expression pattern in human non‐small cell lung cancer tissues indicate increased blood thrombogenicity and tumor metastasis.Oncol Rep. 2008; 20: 123-8PubMed Google Scholar, 10Signaevsky M. Hobbs J. Doll J. Liu N. Soff G.A. Role of alternatively spliced tissue factor in pancreatic cancer growth and angiogenesis.Semin Thromb Hemost. 2008; 34: 161-9Crossref PubMed Scopus (25) Google Scholar]. We recently demonstrated that high tumor TF expression in non‐small cell lung cancer (NSCLC) was associated with reduced survival of affected patients and that overexpression of TF was linked to oncogenic events, such as KRAS, p53 and PTEN mutations [11Regina S. Rollin J. Blechet C. Iochmann S. Reverdiau P. Gruel Y. Tissue factor expression in non‐small cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K‐ras mutation.J Thorac Oncol. 2008; 3: 689-97Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Regina S. Valentin J.B. Lachot S. Lemarie E. Rollin J. Gruel Y. Increased tissue factor expression is associated with reduced survival in non‐small cell lung cancer and with mutations of TP53 and PTEN.Clin Chem. 2009; 55: 1834-42Crossref PubMed Scopus (67) Google Scholar]. In the present study, we specifically evaluated the level of asTF transcripts in NSCLC tumors and looked for a relationship between asTF expression and biological and clinical features, that is, tumor TF and VEGF gene expression, oncogenic events and survival of affected patients. We therefore analyzed tumor samples from 57 Caucasian patients with NSCLC who had undergone complete surgical resection as initial treatment (without prior radiotherapy or chemotherapy) between January 2002 and February 2005 in our hospital. This cohort of patients is limited but provided a typical representative of NSCLC (Table S1), as the overall survival (39%) was comparable to those previously described in larger populations of patients [13Mountain C.F. Revisions in the International System for Staging Lung Cancer.Chest. 1997; 111: 1710-7Abstract Full Text Full Text PDF PubMed Scopus (4538) Google Scholar, 14Duque J.K. López‐Encuentra A. Porta R.R. Bronchogenic Carcinoma Cooperative Group of the Spanish Society of Pneumology and Thoracic Surgery. Survival of 2,991 patients with surgical lung cancer: the denominator effect in survival.Chest. 2005; 128: 2274-81Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. All patients gave signed informed consent for genetic studies as recommended by French legislation and in accordance with the current revision of the Helsinki declaration. Total RNA was extracted from the 57 NSCLC tumors as previously described [11Regina S. Rollin J. Blechet C. Iochmann S. Reverdiau P. Gruel Y. Tissue factor expression in non‐small cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K‐ras mutation.J Thorac Oncol. 2008; 3: 689-97Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Regina S. Valentin J.B. Lachot S. Lemarie E. Rollin J. Gruel Y. Increased tissue factor expression is associated with reduced survival in non‐small cell lung cancer and with mutations of TP53 and PTEN.Clin Chem. 2009; 55: 1834-42Crossref PubMed Scopus (67) Google Scholar]. Specific asTF transcripts were assessed by a real‐time PCR method using the Taqman strategy adapted from Szotowski et al. [5Szotowski B. Goldin‐Lang P. Antoniak S. Bogdanov V.Y. Pathirana D. Pauschinger M. Dörner A. Kuehl U. Coupland S. Nemerson Y. Hummel M. Poller W. Hetzer R. Schultheiss H.P. Rauch U. Alterations in myocardial tissue factor expression and cellular localization in dilated cardiomyopathy.J Am Coll Cardiol. 2005; 45: 1081-9Crossref PubMed Scopus (72) Google Scholar]. We had previously measured all TF mRNA variants in these samples by PCR, including those specific for full‐length TF (flTF) that is the predominant form, using a sense primer targeting exon 2 and one antisense primer hybridizing exon 3 [11Regina S. Rollin J. Blechet C. Iochmann S. Reverdiau P. Gruel Y. Tissue factor expression in non‐small cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K‐ras mutation.J Thorac Oncol. 2008; 3: 689-97Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. The results specific for asTF were normalized with 18S RNA expression by calculating the 2−Δct equal to 2−(ct asTF−ct 18S) for each sample. The number copies of mRNA specific for asTF was highly variable from one tumor to another (median 5.32×10−5; range: 1.16×10−7 to 2×10−3), a variation that was similar to that previously observed with TF gene expression [11Regina S. Rollin J. Blechet C. Iochmann S. Reverdiau P. Gruel Y. Tissue factor expression in non‐small cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K‐ras mutation.J Thorac Oncol. 2008; 3: 689-97Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. However, the relative amount of asTF transcripts was very low, representing 0.39% of total TF mRNA measured in lung tumors (Fig. 1A). Moreover, no correlation was found between TF and asTF mRNA levels (Spearman's rank correlation test; P = 0.5). We recently showed that accumulation of mutations of several genes affecting the mTOR pathway dramatically increased TF gene expression in lung tumors [12Regina S. Valentin J.B. Lachot S. Lemarie E. Rollin J. Gruel Y. Increased tissue factor expression is associated with reduced survival in non‐small cell lung cancer and with mutations of TP53 and PTEN.Clin Chem. 2009; 55: 1834-42Crossref PubMed Scopus (67) Google Scholar]. However, in the present study, no association was found between levels of asTF and mutations of codon 12 KRAS, p53, PTEN or LKB1 (Table S1). This supports that the alternative splicing of TF in lung tumors is not influenced by these mutations. The mechanisms that are involved remain to be defined, but it has recently been shown that asTF expression in human endothelial cells may be specifically regulated by the PI3K/Akt pathway [15Eisenreich A. Malz R. Pepke W. Ayral Y. Poller W. Schultheiss H.P. Rauch U. Role of the phosphatidylinositol 3‐kinase/protein kinase B pathway in regulating alternative splicing of tissue factor mRNA in human endothelial cells.Circ J. 2009; 73: 1746-52Crossref PubMed Scopus (37) Google Scholar]. Levels of asTF transcripts in NSCLC tumors were also analyzed according to clinico‐pathological features, and no association with gender, age, stage, differentiation grade, TNM classification or histological type was evidenced. Interestingly, Kaplan–Meier curve analysis showed that patients with high tumor levels of asTF mRNA (i.e. above the median value) had a poorer prognosis than patients with low levels of transcripts (i.e. under the median value) (P = 0.01, HR 3.1; 95% CI: 1.2–6.5, Log‐rank test, Fig. 1B). A Cox's regression model including age, stage, TF and the number of asTF transcripts as variables also showed that asTF expression was an independent prognostic marker (RR = 3.08; 95% CI: 1.35–7.01; P = 0.0076). This study is therefore the first to demonstrate that measurement of asTF mRNA levels in tumors can be a useful tool to predict the survival of patients with lung cancer. The prognostic value of TF mRNA levels was also recently established in NSCLC [12Regina S. Valentin J.B. Lachot S. Lemarie E. Rollin J. Gruel Y. Increased tissue factor expression is associated with reduced survival in non‐small cell lung cancer and with mutations of TP53 and PTEN.Clin Chem. 2009; 55: 1834-42Crossref PubMed Scopus (67) Google Scholar]. We therefore evaluated the survival rates of patients according to both asTF and TF tumor expression levels. Three groups of patients were thus defined according to tumor mRNA levels of TF and asTF as follows: low asTF/low TF (n = 14); low asTF/high TF or high asTF/low TF (n = 20) and high asTF/high TF (n = 23). The best outcome was evidenced in patients for whom tumor levels of both asTF and TF transcripts were low. Survival was therefore significantly reduced when either asTF or TF expression in tumors was high but the poorest prognosis was recorded when tissue levels of both categories of TF mRNA were high (P = 0.018, Log‐rank test, Fig. 1C). Moreover, the survival time of patients with high asTF and high TF mRNA levels in tumors was significantly shorter than that of patients with low tumor levels of TF/asTF transcripts (P = 0.004; HR 4.1; 95% CI: 1.5–7.5, Log‐rank test). Our results therefore strongly suggest that evaluation of mRNA levels specific for both TF and asTF in tumors may be effective in evaluating the prognosis in NSCLC, but this hypothesis has to be confirmed in larger cohorts of patients. Whereas the role of full‐length TF in cancer has been well documented in recent years [2Rak J. Milsom C. Yu J. Tissue factor in cancer.Curr Opin Hematol. 2008; 15: 522-8Crossref PubMed Scopus (45) Google Scholar, 16Kasthuri R.S. Taubman M.B. Mackman N. Role of tissue factor in cancer.J Clin Oncol. 2009; 27: 4834-8Crossref PubMed Scopus (310) Google Scholar], the mechanisms by which asTF influences the progression of cancer remain completely unknown. Interestingly, its role in angiogenesis and in tumor growth has recently been suggested in pancreatic cancer [10Signaevsky M. Hobbs J. Doll J. Liu N. Soff G.A. Role of alternatively spliced tissue factor in pancreatic cancer growth and angiogenesis.Semin Thromb Hemost. 2008; 34: 161-9Crossref PubMed Scopus (25) Google Scholar], but no correlation was found in the present study between tumor mRNA levels of asTF in NSCLC and those of VEGF165 or VEGF189 that we had previously measured [11Regina S. Rollin J. Blechet C. Iochmann S. Reverdiau P. Gruel Y. Tissue factor expression in non‐small cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K‐ras mutation.J Thorac Oncol. 2008; 3: 689-97Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. On the other hand, we showed that TF gene expression was strongly correlated with that of the VEGF189 isoform [11Regina S. Rollin J. Blechet C. Iochmann S. Reverdiau P. Gruel Y. Tissue factor expression in non‐small cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K‐ras mutation.J Thorac Oncol. 2008; 3: 689-97Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. These results suggest that the regulation of angiogenesis by the various isoforms of TF probably involves different mechanisms. In this regard, flTF, in complex with FVIIa, influences angiogenesis by involving its cytoplasmic domain and through PAR‐2 activation [16Kasthuri R.S. Taubman M.B. Mackman N. Role of tissue factor in cancer.J Clin Oncol. 2009; 27: 4834-8Crossref PubMed Scopus (310) Google Scholar]. On the other hand, Van den Berg et al. has recently demonstrated that asTF enhances angiogenesis independently of activation of PAR‐2 but viaαvβ3 and α6β1 integrin ligation [17van den Berg Y.W. van den Hengel L.G. Myers H.R. Ayachi O. Jordanova E. Ruf W. Spek C.A. Reitsma P.H. Bogdanov V.Y. Versteeg H.H. Alternatively spliced tissue factor induces angiogenesis through integrin ligation.Proc Natl Acad Sci USA. 2009; 106: 19497-502Crossref PubMed Scopus (124) Google Scholar]. In conclusion, this study suggests that mRNA specific for alternatively spliced TF could be measured in lung tumors together with transcripts specific for full‐length TF to evaluate prognosis in patients with NSCLC. In addition, our results also support the hypothesis that asTF and flTF might exert various effects on tumor development, via specific pathways. The authors declare that they have no conflict of interest. We thank S. Guyétant, C. Bléchet (Department of Pathology) and P. Dumont (Department of Thoracic Surgery, Tours Hospital, France) for their help. We also thank D. Raine for editing the English. This study was supported by the Institut pour la Recherche sur la Thrombose et l'Hémostase. Table S1. Tumour expression of asTF alone orwith TF according to clinico‐pathological features of patients withNSCLC, status of codon 12 of the KRAS, TP53,STK11, and PTEN genes (WT or mutated). Please note: Wiley‐Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Download .doc (.08 MB) Help with doc files Supporting info item
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