Tumor suppressive protein gene associated with retinoid-interferon-induced mortality (GRIM)-19 inhibits src-induced oncogenic transformation at multiple levels

Abstract

Interferons (IFNs) inhibit the growth of infectious pathogens and tumor development. Although IFNs are potent tumor suppressors, they modestly inhibit the growth of some human solid tumors. Their weak activity against such tumors is augmented by co-treatment with differentiation-inducing agents such as retinoids. Previous studies from our laboratory identified a novel gene product, gene associated with retinoid-interferon-induced mortality (GRIM)-19, as an IFN/all-trans retinoic acid-induced growth suppressor. However, the mechanisms of its growth suppressive actions are unclear. The src-family of tyrosine kinases is important regulators of various cell growth responses. Mutational activation of src causes cellular transformation by altering transcription and cytoskeletal properties. In this study, we show that GRIM-19 suppresses src-induced cellular transformation in vitro and in vivo by down-regulating the expression of a number of signal transducer and activator of transcription-3 (STAT3)-dependent cellular genes. In addition, GRIM-19 inhibited the src-induced cell motility and metastasis by suppressing the tyrosyl phosphorylation of focal adhesion kinase, paxillin, E-cadherin, and gamma-catenin. Effects of GRIM-19 on src-induced cellular transformation are reversible in the presence of specific short hairpin RNA, indicating its direct effect on transformation. GRIM-19-mediated inhibition of the src-induced tyrosyl phosphorylation of cellular proteins, such as focal adhesion kinase and paxillin, seems to occur independently of the STAT3 protein. GRIM-19 had no significant effect on the cellular transformation induced by other oncogenes such as myc and Ha-ras. Thus, GRIM-19 not only blocks src-induced gene expression through STAT3 but also the activation of cell adhesion molecules.

Figure 1

Effect of GRIM-19 on src-induced cellular transformation. 3Y1 cells were electroporated using the Nucleofector technology (Amaxa, Inc.) with the indicated expression vectors, as recommend by the manufacturer, and used for soft-agar growth assays in the presence of continuous selection with G-418 (750 μg/ml). A: Photomicrographs of representative fields from various transfections are shown. B: Quantification of colony formation. Bars: mean ± SE of triplicates. The Western blots below this graph show the expression levels of Src, GRIM-19, and actin proteins in these cells. A portion of the cells used for colony formation assays (A) was plated in parallel, without soft agar, and selected with G-418 for 3 weeks. An equal quantity of protein (45 μg) from each transfectant was used for the Western blot analyses. C: Effect of GRIM-19 on myc-induced cellular transformation. The blots below it show the expression of the transfected gene products in the transfectants. Total protein (60 μg) was used for Western blot analysis with the indicated antibodies. Different antibodies were used for detecting c-myc (N-262, rabbit polyclonal antibody; Santa Cruz Biotechnology) and myc-tagged GRIM-19 (mouse monoclonal antibody, which has a very low affinity for endogenous rodent myc protein; Cell Signaling Technology). D: GRIM-19 suppresses soft agar colony formation by cells pretransformed v-src. A v-src-expressing cell line was transfected with various plasmids along with a pcDNA3.1 zeo. Stable cell lines were isolated after selecting for 3 weeks with G-418 (750 μg/ml) and Zeocin (250 μg/ml). An equal number of cells (2000 per well in a 12-well plate) was used for soft agar assays. Six replicates were used for each transfectant, and experiments were repeated at least three times. Mean ± SE colony numbers were plotted. E: Western blot (WB) analysis of the expression of transfected genes in various cells. Total protein (60 μg) was used. Western blotting with actin-specific antibodies was used as a loading control for these blots. Original magnifications, ×100.

Figure 2

Effect of GRIM-19 on src-dependent cell motility. A: Injury-induced migration of cells into the wounded area was monitored. White line indicates the edge of injured site. Note the rapid migration of src*- and v-src-expressing cells, but not the controls, into the wounded area. B: Invasion assay was performed using Transwell chambers. Serum (10%) was used as an attractant in the lower chamber. Cells (n = 25,000) were seeded in the upper chamber of insert and incubated for 24 hours, migrated cells were fixed and stained with crystal violet, and the bound dye was eluted and quantified at 595 nm. C: Growth of various cell lines as measured by a colorimetric assay. D: Western blot analysis of src expression and its activating phosphorylation (pY⁴¹⁶) in the transfectants. Approximately 75 μg of total protein was used for Western blotting. Original magnifications, ×100.

Figure 3

Effect of GRIM-19 on the growth of Src-expressing cells in vivo. Cell lines (in 3Y1 background) expressing various genes were subcutaneously transplanted into athymic nude mice (n = 10), and tumor growth was monitored. Untransfected 3Y1 cells were used as an additional control in these experiments. The growth of these cells was similar to the pCDNA3.1-transfected cells (data not shown). Insets show the immunohistochemical analyses of the tumors grown in nude mice. Tumors were excised and fixed, and an immunohistochemical analysis was performed with Ki-67-specific antibodies. Original magnifications, ×40.

Figure 4

GRIM-19 represses src-dependent expression of STAT3-inducible genes. A: Endogenous gene expression was quantified using qRT-PCR, and transcript abundance was measured after normalizing to an internal control, rpl32. Baseline level of each transcript (pCXN2 vector-transfected cells) was subtracted from the experimental samples. B: Effect of GRIM-19 on src-induced expression of the Luciferase reporters driven by the STAT3-responsive gene promoters. The indicated luciferase reporters and a β-galactosidase reporter (0.2 μg each) were transfected into the indicated stable cell lines. Luciferase activity was normalized to that of β-galactosidase and plotted. Bars, relative luciferase activity (RLA) ± SE RLA was calculated and plotted after comparing to the data obtained with pCXN2–3Y1 cells. C: Effect of GRIM-19 on the phosphorylation of STAT3. Western blot analyses were performed with the indicated antibodies. D: Lack of an inhibitory effect of GRIM-19 on IL-6-induced phosphorylation of STAT3. The indicated 3Y1 cell lines were treated with IL-6 (100 ng/ml) along with soluble IL-6 receptor (100 ng/ml) for 30 minutes (R&D Systems, Minneapolis, MN). Rodent fibroblasts express an extremely low level of the ligand binding chain of the IL-6 receptor. Hence, we treated these cells with the soluble receptor, during stimulation with IL-6.

Figure 5

Effects of GRIM-19- and STAT3-specific shRNAs on Src-dependent gene expression. A and D: shRNA-mediated knockdown of GRIM-19 and STAT3 expression in cells. Approximately 150 μg of total protein was used for Western blot analysis. B: Effect of STAT3-specific shRNA on v-src-induced expression of the indicated reporter genes. Various plasmids were co-transfected into cells along with the luciferase reporters, and the promoter activity was measured as RLA. C: Down-regulation of GRIM-19 by specific shRNA. V-src/GRIM-19 cells were infected with specific lentiviral vectors, and the loss of GRIM-19 expression was monitored using Western blot analyses of the cell extracts (120 μg) with myc-tag-specific antibodies. D: v-src/GRIM-19 cells were infected with lentiviral vectors carrying the indicated shRNAs (overnight) and then transfected with the indicated reporters as indicated in Materials and Methods. E and F: Effects of STAT3 (E)- and GRIM-19 (F)-specific shRNA on the expression of endogenous STAT3-regulated mRNAs. A qRT-PCR analysis was performed as in Figure 4. Vector- and v-src-alone-transfected 3Y1 cell lines were used as negative and positive controls. For E and F, v-src and vsrc/GRIM-19-expressing cell lines were infected with the indicated lentiviral vectors, carrying specific shRNA, for determining the effects on gene expression. G and H: GRIM-19-specific shRNA reverses the inhibitory effect of GRIM-19 on soft agar colony formation in v-src/GRIM-19 cells. Cells were infected with lentiviral vectors coding for the indicated shRNAs and then used for soft agar colony formation assays. I: Interaction between GRIM-19 and STAT3. Immunoprecipitation and Western blot analyses were performed with the indicated nuclear extracts (250 μg). Immunoprecipitation and Western blot were performed with myc-tag- and STAT3-specific antibodies, respectively. Histone H1 antibodies were used as an internal control.

Figure 6

Effect of GRIM-19 on src-induced phosphorylation of cellular proteins. A: Effect of GRIM-19 on src-induced tyrosine phosphorylation of cellular proteins. Total protein (60 μg) was used for a Western blot analysis with phosphotyrosine-specific antibodies. Specific phosphorylated bands are indicated. Common bands between Src*- and v-src-expressing cells are indicated with arrows. Arrowheads indicate the bands differentially phosphorylated between src* and v-src. A reprobing of the blot with tubulin-specific antibodies showed a comparable loading. B: Effect of GRIM-19 on src-induced tyrosyl phosphorylation of various cellular proteins. Immunoprecipitation and Western blot analyses performed with the indicated antibodies. Approximately 500 μg of total cellular protein from each transfectant was used. Blot regions corresponding to the appropriate bands are shown in each case. C: Loss of STAT3 does not affect the src-induced cellular protein tyrosyl phosphorylation profiles and the inhibitory effects of GRIM-19 on it. Cells were infected with lentiviral vectors coding for STAT3-specific shRNA (psh-S3) or a control vector (pLKO-1) as in Figure 5. Total cell lysates (70 μg) were used for Western blot analysis with the indicated antibodies. D: Effect of STAT3 knockdown on src-induced tyrosyl phosphorylation of paxillin and FAK and its inhibition by GRIM-19. Specific antibodies that detect the pY¹¹⁸ paxillin and pY⁵⁷⁶-FAK proteins were used for the Western analyses. Total cellular lysates (80 μg) were used in each case. Total paxillin and FAK levels were determined using native paxillin- and FAK-specific antibodies. Actin levels were determined for a comparable protein loading.