Interferon gamma-induced human guanylate binding protein 1 inhibits mammary tumor growth in mice

Abstract

Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.

Figure 1

Characterization of TS/A mammary carcinoma cells inducibly expressing hGBP-1. (A) Western Blot analysis of TS/A cell clones 13–18 harboring the Tet on/off regulatory system and the vector pUHD10.3-GBP-Hyg, mediating tightly regulated, inducible expression of hGBP-1 in response to doxycycline (+ dox). As a control for analysis of equal amounts of protein, a Coomassie-stained SDS-polyacrylamide gel with the respective samples is depicted (lower panel). (B) Analysis of proliferation characteristics of pUHD10.3-GBP–transfected cells (cell population, gray bars; cell clone, white bars) in comparison to pUHD10.3-Hyg–transfected cells (black bars). Cell numbers (mean values ± SD) from three independent experiments are shown in % relative to nontreated (− dox) samples. (C) Soft agar assay. Nontransfected TS/A cells and TS/A cells transfected with pUHD10.3-Hyg or the hGBP-1–encoding vector (pUHD10.3-GBP) were analyzed for their ability to form colonies in soft agar. Expression of hGBP-1 was induced by addition of 1 (gray bars) or 2.5 (black bars) μg/mL doxycycline and the number of colonies (mean values ± SD) obtained by counting 10 different optical fields compared with nontreated samples (white bars).

Figure 2

Effect of hGBP-1 expression on tumor growth in a syngeneic mammary carcinoma mouse model. (A) Tumor growth was monitored in Balb/c mice injected with 1 × 10⁶ TS/A cells transfected with either pUHD10.3-GBP (▴, ○) or the parental vector pUHD10.3-Hyg (▾, ▪). At d 7 after injection, animals were divided into two groups (n = 20) given either doxycycline (+ dox) or saline (− dox). Mean tumor volumes ( ± SEM) of individual animals are indicated. Animals that did not develop a tumor at the end of the experiment or died due to unrelated reasons were excluded. Statistical significance (P values) was determined applying a Student t test comparing the tumor doubling times of doxycycline-treated versus nontreated animals. (B) hGBP-1–specific immunohistochemical analysis of tumors derived from pUHD-GBP–transfected TS/A cells treated with doxycycline (a, b) or saline (c). No unspecific staining was observed in tumors from doxycycline-treated animals that had been injected with TS/A cells harboring the control vector pUHD (d).

Figure 3

Evaluation of effects of hGBP-1 expression on tumor cell proliferation. (A) Immunohistochemistry. Consecutive sections of hGBP-1–expressing (TS/A-GBP + dox) and nonexpressing (TS/A-Hyg + dox) tumors were stained for expression of hGBP-1 (α-hGBP-1, brown cytoplasmic staining) and murine Ki-67 (α-mKi-67, red nuclear staining), a cellular proliferation marker. Using the Zeiss AxioVision analysis software, sections were divided into different areas correlating with a high level (a, b, d, e, g, h, i) or low level (c, f) hGBP-1 expression. (B) The number of Ki-67–positive TS/A cells was calculated and expressed as mean values per 100.000 μm². Statistical analysis revealed significant differences (P = 0.017) in the number of Ki-67–positive cells (mean values ± SD) in hGBP-1–expressing (total GBP⁺, n = 11) compared with nonexpressing tumors (total GBP⁻, n = 10). Highly significant differences (P < 0.0001, one-way ANOVA) were observed when Ki-67–positive cell numbers were correlated with hGBP-1 expression levels.

Figure 4

Quantification of tumor infiltration. Sections of TS/A-pUHD10.3-GBP–derived tumors from four different animals that had been treated with doxycycline (TS/A-GBP + dox) or saline (TS/A-GBP − dox) were analyzed by immunohistochemistry for the presence of CD3⁺ T cells or B220⁺ B cells. In both experiments, 10 different optical fields were evaluated. Results are indicated as mean values ± SD. No statistically significant differences were observed. Examples of successful staining are shown (inserts).

Figure 5

Analysis of tumor angiogenesis. (A) Immunohistochemistry. Consecutive sections of hGBP-1–expressing (TS/A-GBP + dox) and nonexpressing (TS/A-Hyg + dox, TS/A-GBP − dox) tumors were stained for the presence of hGBP-1 (α-hGBP-1, brown cytoplasmic staining) and CD31 (α-mCD31), a specific marker of endothelial cells (red staining). (B) Biochemical determination of hemoglobin contents. The amount of hemoglobin, normalized to total protein amounts, was determined in frozen tissue (n = 18) of hGBP-1–expressing (TS/A-GBP⁺) and nonexpressing (TS/A-GBP⁻) tumors. Statistical significance (P = 0.0032) of the encountered differences was shown by applying an unpaired Student t test.

Figure 6

Effect of hGBP-1 expression on VEGF-A synthesis. (A) The Tet-regulated hGBP-1–expressing TS/A cell clones 14 and 18, which were used for generation of subcutaneous tumors in Balb/c mice, were analyzed for VEGF-A synthesis applying a mouse-specific ELISA. As a control, cells harboring the parental vector pUHD were used. Cells (duplicates) were cultured for 96 h in the presence of doxycycline (1 and 2.5 μg/mL), and the respective cell culture supernatants were analyzed. Obtained values (mean ± SD) were normalized to cell numbers (1 × 10⁶) and set in relation to values obtained with nontreated cells (0 μg/mL dox). Corresponding cell extracts were analyzed for hGBP-1 expression by Western blotting (insert). (B) Quantitative Western blot analysis of VEGF-A in TS/A cell–derived tumor samples. Frozen tissue samples (n = 6) of tumors grown in mice that had received doxycycline for induction of hGBP-1 expression (TS/A-GBP⁺) or were injected with saline (TS/A-GBP⁻) were subjected to Western blot analysis to detect murine VEGF-A protein. GAPDH was stained as an internal control to monitor the amount of loaded protein. Quantification of band intensities was performed with the ImageQuant software. Background corrected single values were normalized to measurements of GAPDH-specific bands and summarized, and the mean values (± SEM) were compared for statistically significant differences by applying an unpaired Student t test. The calculated P value is indicated.