Tissue factor is regulated by epidermal growth factor in normal and malignant human endometrial epithelial cells

Abstract

Tissue Factor (TF), the initiator of the extrinsic coagulation cascade, is overexpressed in a variety of cancers. TF is also expressed in normal human endometrium but little is known about its expression or regulation in endometrial cancer. We demonstrate herein that TF is expressed in the endometrial adenocarcinoma cell line Ishikawa. Furthermore, epidermal growth factor (EGF) induces a rapid and sustained increase in TF expression. Estradiol and progesterone had no effect on basal or EGF-induced TF expression in Ishikawa cells. In contrast to the pronounced and sustained upregulation at the protein level, EGF treatment elicited only a modest and transient increase in TF mRNA levels. This activity corresponded to the response observed from an exogenous TF promoter construct. However, the induction of TF was abrogated by cycloheximide as well as actinomycin-D, inhibitors or protein- and mRNA-synthesis, respectively, demonstrating that EGF mediates its effect through activation of the TF gene. Fractionation experiments showed that EGF increases TF presence in caveolin-I containing membrane fractions. Coagulation and invasion assays were used to explore the physiological implications of TF regulation. The results demonstrate that EGF-mediated induction of TF increases the procoagulant activity and invasive potential of Ishikawa cells. Furthermore, immunocytochemistry confirmed that TF is regulated by EGF in primary cultures of normal endometrial epithelial cells and malignant tumor cells. In conclusion, EGF-mediated upregulation of TF results in accumulation of this glycoprotein in caveolae-like membrane fractions and increased coagulative and invasive potential. Our results suggest that TF may play an integral role in endometrial carcinogenesis.

The relationship between tissue factor and cancer progression: insights from bench and bedside

“…Restoration of the tumor suppressor gene PTEN in these cells, which leads to inhibition of the PI3 kinase and MAPK pathways, down-regulates EGFR-dependent flTF expression. 9 Moreover, endometrial cancer cell lines display enhanced flTF levels in an EGF-dependent fashion, 10 and inhibition of EGFR signaling diminishes flTF expression in vulva carcinoma cells constitutively expressing the EGFRvIII mutant. 11 Recent studies in medulloblastoma cell lines indicate that Src family kinases stimulate an induction of flTF expression through both the scatter factor/hepatocyte growth factor (HGF) and as a result of a mutation in the c-MET oncogene, whereas flTF expression via the HGF/c-MET axis elicits an antiapoptotic response and provides resistance to chemotherapeutical agents.…”

Tissue Factor and Cancer

“…Recent reports indicate that expression of TF by cancer cells can also be regulated by hypoxia [90] , cell-cell contact [115] , growth factors [116] and hormones [117] . However, since these influences are transient in nature, they can only exert a moderate effect on TF in cancer cells and, therefore, cannot explain the exuberant and chronic expression of TF in a variety of tumours, or the recurrence and persistence of cancer-related coagulopathy [75] .…”

TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract

“…Primary culture of human endometrial epithelial cells Endometrial epithelial cell cultures were derived from tissue from pre-menopausal women undergoing routine hysterectomy or hysterectomy with bilateral salpingooophorectomy as described previously [16]. Briefly, immediately after hysterectomy, approximately 1-2 cm 3 of relevant tissue was removed from the endometrium at the endometrial cavity under sterile conditions and collected into warm saline serum.…”

“…DR4 sense: 5 -CCGCGGCCACACCCAGAAAGT-3 DR4 antisense: 5 -GTACATGGGAGGCAAGCAAACAAA-3 (generates a fragment of 415bp); DR5 sense: 5 GGGAGCCGCTCATG AGGAAGTTGG 3 DR5 antisense: 3 GGCAAGTCTCTC TCCCAGCGTCTC5 (generates a fragment of 181bp); DcR1 sense: 5 -GCCGGAA GTGTAGCAGGTG-3 DcR1 antisense: 5 -GAGATGTGTGGACCCCCA-3 (generates a fragment of 672bp); DcR2 sense: 5 -CCCCCGGCAGG ACGAAGTT-3 DcR2 antisense: 5 -CTCCTCCGCTGCTG GGGTTTT-3 (generates a fragment of 418bp) (BiosChile, Santiago, Chile) semi-quantitative PCR reactions were performed from cDNA generated from cell lines and primary cultured cells maintained under maintenance conditions (in the absence of TRAIL), using Taq polymerase (Invitrogen, NY, USA). As an internal control, primers amplifying a region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were used to test the integrity of the starting cDNA as previously published [16].…”