The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in epithelial ovarian cancer cells

Abstract

Constitutive expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-alpha generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNF-alpha, constitutive production of TNF-alpha was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-alpha production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-alpha in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-alpha was achieved. Tumors derived from TNF-alpha knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-alpha knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-alpha by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits.

Figure 1

TNF-α release by ovarian cancer cell lines is associated with expression of other protein mediators. Proteins secreted by ovarian cancer cell lines were measured by ELISA after 48 h. Data are representative of three independent experiments done. Columns, mean of triplicate wells for TNF-α, CCL2, CXCL12, VEGF, IL-6, and MIF; bars, SD.

Figure 2

Appearance and location of tumor deposits in nude mice injected with ovarian cancer xenografts. A, gross patterns of peritoneal tumor spread of TOV112D, SKOV-3, TOV21G, and IGROV-1. B, location of tumor deposits in various organs as determined by histologic examination postmortem for TOV112D, SKOV-3, TOV21G, and IGROV-1.

Figure 3

In vitro effects of stable expression of shRNAto TNF-α. In all experiments, IGROV-Mock cells are compared with two independently isolated TNF-α shRNA clones RNAi TNF-α (I) and RNAi TNF-α (II). A, TNF-α protein secretion as measured by ELISA after 72 h. Columns, mean of triplicate wells; bars, SD. Silencing TNF-α expression in IGROV-1 cells also results in decreased protein secretion of CCL2, CXCL12, VEGF, IL-6, and MIF compared with IGROV-Mock as measured by ELISA after 48 h. Columns, mean of triplicate wells; bars, SD. B, proliferation of IGROV-1, IGROV-Mock, RNAi TNF-α (I), and RNAi TNF-α (II) cells in vitro as determined by WST-1 assay over a period of 4 d. Data are representative of three independent experiments. Points, mean for absorbance at 450 nm in triplicates; bars, SD. C, migration of IGROV-1 cells to CXCL12. IGROV-1 cells have low basal migration (black columns) but migrate toward CXCL12 (white columns). Results are representative of three experiments done Columns, mean of 10 determinations; bars, SD.

Figure 4

Effects of TNF-α knockdown on growth of cells in vivo. A, a, representative bioluminescence imaging in vivo of IGROV-Mock and RNAi TNF-α IGROV 42 d after i.p. injection. Bioluminescence is presented as a pseudocolor scale: red, highest photon flux; blue, lowest photon flux. b, quantification of bioluminescence from primary tumors (n = 6 each group) of images obtained on days 14, 28, and 42. B, tumor weight of dissectable tumors from each group (n = 5) are shown 6 wks after i.p. injection Columns, mean; bars, SD. C, dissemination of tumor deposits at 6 wks are presented as the percentage of mice with tumors at various anatomic sites. Because no differences between the RNAi TNF-α clones were seen, data from all mice injected with RNAi TNF-α cells were combined (n = 8). D, representative pictures of sections are shown from (a) IGROV-Mock and (b) RNAi TNF-α tumors (magnification, ×20). For detailed histologic analysis, images with higher magnification (magnification, ×40) from (c) IGROV-Mock and (d) RNAi TNF-α tumors are shown. Arrows, areas of apoptotic events.

Figure 5

TNF-α knockdown influences tumor angiogenesis. In vivo angiogenesis was evaluated in mice 42 d after tumor cell injection. A, confocal images (magnification, ×20) of representative sections from (I) IGROV-Mock, (II) RNAi TNF-α I, and (III) RNAi TNF-α II tumors, after injection of FITC-conjugated lectin and in (B)4′,6-diamidino-2-phenylindole–stained cell nuclei are shown as control. C, columns, mean vascular area in each group quantified in 10 randomly selected areas of tumor sections (n = 5); bars, SD. D, proliferation of primary mouse lung endothelial cells in vitro with conditioned medium of IGROV-1, IGROV-Mock, and RNAi TNF-α cells. Representative of two independent experiments. Points, mean values for absorbance at 450 nm in triplicates; bars, SD.