Downregulation of tissue factor by RNA interference in human melanoma LOX‐L cells reduces pulmonary metastasis in nude mice

Abstract

Tissue factor (TF), a transmembrane protein, initiates the blood coagulation cascade by complexing with its serine protease ligand factor VII(a) (FVIIa), ultimately leading to thrombin generation, fibrin deposition and platelet aggregation. TF is aberrantly expressed in many solid tumors and tumor cell lines 1-4 with higher expression levels in malignant tumor cells compared to their nonmalignant counterpart. Subsequent results suggested that TF participates in the tumor growth and dissemination of various cancers. 5 Clinically, TF expression strongly correlates with tumor progression. 6 -9 TF can promote tumor progression via coagulation-dependent or -independent mechanisms. 10,11 Activation of coagulation may trap tumor cells in a fibrin-platelet clot and allow local tumor growth, which is critically important for tumor dissemination and metastasis. 12,13 Moreover, several products of the coagulation system, including thrombin and fibrin, can stimulate angiogenesis, 13-15 which is necessary for tumor growth and metastasis. Independent of the downstream coagulation factors, FVIIa binding to TF can promote tumor-associated angiogenesis by upregulating angiogenic factors VEGF and IL-8. 16 -18 TF/FVIIa signaling resulting from the proteolytic activity of the complex is required for the upregulation of these angiogenic factors 19 -21 and for TF-dependent metastasis. 22 TF/FVIIa signaling has received widespread interest 10,[23][24][25][26] after the discovery of TF/FVIIa-induced calcium signals. 27 FVIIa/TF signaling, independent of the coagulation cascade, results in the upregulation of cytokines, growth factors 22,24 and induction of antiapoptosis, 28 which may contribute to tumor growth. Blocking TF function by administration of TF antibody or inactivated FVIIa has resulted in the reduced metastasis in murine metastasis models. 29,30 We have in this study used RNA interference specifically to knock down TF expression and investigated the effects on the metastatic capacity of human malignant melanoma cells.RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. This phenomenon has been developed into a powerful technology to elucidate mammalian gene function by delivery of small interfering RNA (siRNA), duplexes of 19-21-nt RNA with 2-nt 3Ј overhangs, into cells. 31 Variable transfection efficiency and the transient nature of silencing by exogenously delivered siRNA limit the applicability of chemically synthesized siRNA in functional genomic studies. 32 Stable RNAi can be achieved in mammalian cells through RNA polymerase III-dependent expression of so-called short hairpin RNAs (shRNA) from integrated DNA constructs in stably transfected cells. 33,34 The transcribed shRNA is thought to be processed into siRNA-like molecules intracellularly. Studies from different cell types and against various genes have demonstrated the efficacy of endogenously expressed shRNA. 33,[35][36][37] In order to explore the potential role and possible mechanism o...

Ex vivo and In vivo Delivery of Anti-Tissue Factor Short Interfering RNA Inhibits Mouse Pulmonary Metastasis of B16 Melanoma Cells

“…The moderate effects on localized growth are consistent with in vitro proliferation experiments (Fig. 1D), and our previous data with human cells exhibiting stable tissue factor knockdown indicated a very marginal effect of tissue factor knockdown on proliferation compared with the effects on colony formation and in vivo metastasis (18).…”

“…Formation of a complex between tissue factor and activated FVII (FVIIa) triggers a substantial activation of cell signaling, leading to up-regulation of the expression of >25 immediate early genes (26). Analysis of the global effects on gene expression of a human melanoma cell line exhibiting stable depletion of tissue factor expression through short hairpin RNA -mediated RNA interference (27) identified 44 and 228 genes that were up-regulated and down-regulated, respectively (18). Genes involved in cell growth, invasion, and angiogenesis were down-regulated in tissue factor -deficient cells, whereas up-regulated genes included those with known functions in apoptosis.…”

Tissue Factor and Cancer Stem Cells

“…TF downregulation by shRNA induces upregulation of vimentin, a marker of mesenchymal differentiation Wang et al 95 MSCs indicates mesenchymal stem cells; HSCs, hemopoietic stem cells; PAR-1/2, protease-activated receptor 1/2; NSCs, neural stem cells: ESCs, embryonic stem cells; TF, tissue factor. plified by several findings: First, we found that not all tumorigenic cells with properties of CSCs/TICs express TF on their surfaces.…”

Downregulation of voltage-dependent anion channel-1 expression by RNA interference prevents cancer cell growth in vivo

“…These target molecules include growth factors and receptors, anti-apoptotic proteins, as well as downstream signal transduction proteins, all crucial to tumor cell growth and survival. [39][40][41][42][43][44] In contrast, siRNA-hVDAC1 is expected to inhibit the development of a cancer tumor by suppressing VDAC1 expression. Thus, targeting hVDAC1 expression holds great promise for the field of cancer therapy.…”