Interferon-Induced Transmembrane Protein 1 (IFITM1) Promotes Distant Metastasis of Small Cell Lung Cancer

Abstract

Small cell lung cancer (SCLC) is a severe malignancy associated with early and widespread metastasis. To study SCLC metastasis, we previously developed an orthotopic transplantation model using the human SCLC cell line DMS273. In the model, metastatic foci were found in distant tissues such as bone and the adrenal gland, similarly as observed in patients with SCLC. In this study, we evaluated the differentially expressed genes between orthotopic and metastatic tumors in the model. We isolated tumor cells from orthotopic and metastatic sites, and the tumor cell RNA was analyzed using DNA microarray analysis. We found that 19 genes in metastatic tumors were upregulated by more than 4-fold compared with their expression in orthotopic tumors. One of these genes encodes a transmembrane protein, interferon (IFN)-induced transmembrane protein 1 (IFITM1), and immunohistochemical analysis confirmed the higher expression of the protein in metastatic sites than in orthotopic sites. IFITM1 was also detected in some SCLC cell lines and lung tumors from patients with SCLC. The overexpression of IFITM1 in DMS273 cells increased their metastatic formation in the orthotopic model and in an experimental metastasis model. Conversely, the silencing of IFITM1 suppressed metastatic formation by DMS273 cells. We also found that IFITM1 overexpression promoted the metastatic formation of NCI-H69 human SCLC cells. These results demonstrate that IFITM1 promotes distant metastasis in xenograft models of human SCLC.

Keywords: IFITM1; experimental metastasis model; metastasis; orthotopic transplantation model; small cell lung cancer.

Figure 1

Gene expression analysis of tumor cells in orthotopic and metastatic sites of the orthotopic small cell lung cancer metastasis model. (A) Cells from the orthotopic and metastatic sites of the orthotopic metastasis model developed using DMS273 cells were subjected to DNA microarray analysis. (B) Schematic representation of the isolation of tumor cells from orthotopic and metastatic sites in the mice. (C) A clustering analysis of the 43 differentially expressed genes (p < 0.01, fold change > 4) between the orthotopic and metastatic tumors.

Figure 2

Expression of interferon (IFN)-induced transmembrane protein 1 (IFITM1) in the orthotopic small cell lung cancer (SCLC) metastasis model. (A) Quantitative analysis of IFITM1 mRNA in tumor cells subjected to the DNA microarray analysis. In total, 1 µg of total RNA from the tumor cells was subjected to real-time RT-PCR. Data are expressed as the mean ± SD of triplicate experiments. (B) Representative immunohistochemistry images for IFITM1. Images of IFITM1 staining of an orthotopic tumor and three distant metastases of the orthotopic models using DMS273 cells are shown. The images were taken at ×400 magnification. Numbers represent the immunohistochemical scores. The estimated visual intensity of IFITM1 staining was graded on an arbitrary 4-point scale as follows: negative, 0; weakly positive, 1; positive, 2; and strongly positive, 3. (C) Summary of immunohistochemical analysis of IFITM1 expression in SCLC tumors. The immunohistochemical scores of the orthotopic tumors of eight mice and their corresponding distant metastases were calculated. * p < 0.05, Mann–Whitney U-test.

Figure 3

IFITM1 expression in human SCLC cell lines and lung tumor tissues from patients with SCLC. (A) Western blotting for IFITM1 in human SCLC cell lines. Whole-cell lysates (20 µg) were separated by 15% SDS-PAGE, and membranes were blotted with anti-IFITM1 (top panel) and anti-α-tubulin antibodies (bottom panel, loading control). (B) Western blotting for IFITM1 in IFN-treated DMS273-GFP cells and H69ZN cells (a ZsGreen-labeled subline of NCI-H69 cells). Whole-cell lysates (20 µg) were separated by 15% SDS-PAGE. (C) Representative immunohistochemistry images of IFITM1 staining in lung tumor tissues from patients with SCLC on cancer tissue arrays. The images were taken at ×200 magnification. (D) Summary of the cancer tissue array analysis. The estimated visual intensity of IFITM1 immunostaining was graded on an arbitrary 3-point scale as follows: negative, 0; positive, 1; and strongly positive, 2. n.s.; not significant, Fisher’s exact test.

Figure 4

Effect of IFITM1 overexpression on the metastatic formation of DMS273-GFP cells in nude mice. (A) Western blotting for IFITM1 in IFITM1-overexpressing DMS273-GFP cells. Whole-cell lysates (20 µg) were separated by 15% SDS-PAGE, and membranes were blotted with anti-IFITM1 (top panel) and anti-α-tubulin antibodies (bottom panel, loading control). (B) In vitro growth rate of IFITM1-overexpressing cells as determined using the 3-(4,5-dimethylthiazol-2-yl)- 2,5 diphenyltetrazolium bromide (MTT) assay. The growth rate was calculated as the ratio of the absorbance of cultured cells to that of cells on day 0. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. (C–E) Orthotopic tumor growth and metastatic formation in the orthotopic model using DMS273-GFP vector control cells (n = 19) or IFITM1-overexpressing cells (n = 20). In total, 2.5 × 10⁵ cells were transplanted into the left lung of each nude mouse. One hundred micrograms of anti-asialo GM₁ were injected into mice intravenously before and after tumor inoculation (a total of four injections). Mice were sacrificed when they became moribund, and orthotopic and metastatic tumors were assessed. (C) Representative fluorescence images of the orthotopic tumors and adrenal gland metastases of mice transplanted with IFITM1-overexpressing cells. Bar, 2 mm. (D) Distant metastatic tumor formation. The dotted lines represent the means of the number of metastasis-positive organs per mouse. Percentages show the incidence of distant metastasis. * p < 0.05, Fisher’s exact test. (E) Orthotopic tumor formation. Results are expressed as the mean + SD. (F,G) Metastatic colony formation in lungs in experimental metastasis models generated using DMS273-GFP vector control (n = 17) or IFITM1-overexpressing cells (n = 14). In total, 1 × 10⁶ cells were injected into the tail vein of each nude mouse. At 7 weeks post-inoculation, lungs were dissected, and metastatic foci were counted. (F) Representative fluorescence images of the lung metastases of both groups. Bar, 2 mm. (G) Percentages show the incidence of distant metastasis. ** p < 0.01, Mann–Whitney U-test. (H) Subcutaneous tumor growth of DMS273-GFP vector control and IFITM1-overexpressing cells. Cells (1 × 10⁶) were inoculated subcutaneously in nude mice (n = 6). The tumor volumes are shown as the mean ± SD.

Figure 5

Effect of IFITM1 silencing on the metastatic formation of DMS273-GFP cells in nude mice. (A) Quantitative analysis of IFITM1 mRNA in silenced cells. One microgram of total RNA extracted from the cells was subjected to real-time RT-PCR. Data are expressed as the mean + SD of a triplicate experiment. (B) Western blotting for IFITM1. The cells were treated with 10 ng/mL interferon β (IFNβ) for 24 h and then lysed. Whole-cell lysates (20 µg) were separated by 15% SDS-PAGE, and membranes were blotted with anti-IFITM1 (top panel) and anti-α-tubulin antibodies (bottom panel, loading control). Relative IFITM1 expression was calculated from the signal intensity and normalized to α-tubulin levels. Silencing efficiency was calculated as the percentage of the relative expression level in shIFITM1 cells to that in shLacZ cells. (C) The in vitro growth rate of the silenced cells was determined using the MTT assay. The growth rate was calculated as the ratio of the absorbance of cultured cells to that of cells on day 0. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. (D–F) Orthotopic tumor growth and metastatic formation in mice with orthotopic tumors generated using DMS273-GFP control shRNA (shLacZ) (n = 10) or IFITM1-silenced (shIFITM1) cells (n = 9). In total, 2.5 × 10⁵ cells were transplanted into the left lung of each nude mouse. Mice were sacrificed when they became moribund, and orthotopic and metastatic tumors were assessed. (D) Representative fluorescence images of the orthotopic tumor and bone metastases of mice transplanted with control shRNA (shLacZ) cells. Bar, 2 mm. (E) Distant metastatic tumor formation. The dotted lines represent the means of the number of metastasis-positive organs per mouse. Percentages show the incidence of distant metastasis in each group. * p < 0.05, Mann–Whitney U-test. (F) Orthotopic tumor formation. Results are expressed as the mean + SD. (G–H) Metastatic colony formation in the lungs of experimental metastasis models generated using DMS273-GFP control shRNA (shLacZ) (n = 22) or IFITM1-silenced (shIFITM1) cells (n = 20). In total, 1 × 10⁶ cells were injected into the tail vein of each nude mouse. At 7–8 weeks post-inoculation, lungs were dissected, and metastatic foci were counted. (G) Representative fluorescence images of the lung metastases of both groups. Bar, 2 mm. (H) Percentages show the incidence of distant metastasis. * p < 0.05, Mann–Whitney U-test. # p < 0.05, Fisher’s exact test.

Figure 6

Effect of IFITM1 overexpression on the metastatic formation of NCI-H69 cells in nude mice. (A) Western blotting of IFITM1 expression in H69ZN cells. Whole-cell lysates (20 µg) were separated by 15% SDS-PAGE, and membranes were blotted with anti-IFITM1 (top panel) and anti-α-tubulin antibodies (bottom panel, loading control). (B) Representative fluorescence images of metastases in the lungs and ovaries of mice inoculated with IFITM1-overexpressing cells. Bar, 2 mm. (C) Metastatic tumor formation in the experimental metastatic models generated using H69ZN vector control (n = 20) and IFITM1-overexpressing cells (n = 20). In total, 4 × 10⁶ cells were injected into the tail vein of each nude mouse. At 6 weeks post-inoculation, mice were sacrificed, and metastatic tumors were assessed. Percentages show the incidence of distant metastasis of each group. ** p < 0.01, Mann–Whitney U-test. (D) Organ distribution of metastases in the experimental metastatic models generated using H69ZN vector control and IFITM1-overexpressing cells. Data represent the percentage of metastasis-positive mice. ** p < 0.01, Fisher’s exact test.