GCIP functions as a tumor suppressor in non-small cell lung cancer by suppressing Id1-mediated tumor promotion.

Grap2 and cyclin D1 interacting protein (GCIP) has been recognized as a putative tumor suppressor, but the molecular mechanisms underlying its anti-tumor properties remain undefined. Here, we report that GCIP is frequently downregulated in non-small cell lung cancer (NSCLC) tissues. Binding assays indicated that inhibitor of DNA binding/differentiation 1 (Id1) interacts with GCIP in the nucleus. Ectopic GCIP expression in the highly invasive NSCLC cell line, H1299, inhibited proliferation, colony formation, invasion and migration, and increased susceptibility to anticancer drugs. Conversely, silencing GCIP expression in the minimally invasive NSCLS cell line, A549, increased proliferation, colony formation, invasion, and migration in vitro, and increased survival and resistance to anticancer drugs. GCIP also suppresses tumorigenicity of NSCLC cells in vivo and GCIP suppresses NSCLC progression is mediated in part by interfering with Id1 signaling, which was confirmed in conditionally induced stable cell lines. In addition, GCIP downregulates the expression of Id1, and GCIP and Id1 are inversely expressed in NSCLC cell lines and specimens. Taken together, these results suggest that GCIP is a potential tumor suppressor in NSCLC and that suppression of Id1-mediated oncogenic properties may be a key mechanism by which GCIP can potently suppress NSCLC tumor progression.

DNA sequencing. Stable cell line overexpressing Id1 was selected with changes of fresh medium containing G418 (600 μg/mL).

Lentivirus production and infection
Lentiviral vector carrying shRNA construct against GCIP and Id1 or full-length GCIP insert was prepared using a three-plasmid transfection method. Briefly, the pMD.G containing vesicular stomatitis virus glycoprotein, and pCMV-∆ 8.91carrying HIVbased packaging plasmid were co-transfected with lentiviral vector (control) or with the shRNA-, or GCIP-expressing lentiviral vector into 293T cells. The medium was changed to fresh DMEM containing 10 mg/ml BSA 24 hours post-transfection. The supernatant containing lentivirus was collected at 48 hour and 72 hour posttransfection, respectively. To infect A549 or H1299 cells, lentiviral supernatant was added directly to cells in the presence of 8 g/ml polybrene, respectively. Following infection, the cells were selected using 5 g/ml puromycin. A549/shG3 cells GCIPtargeting sequences were 5-CCACAATCATGAGGATGAT-3 and shG4 cells GCIPtargeting sequences were 5-GACTCAATGAGGCAGCTGT-3. H1299/G6 and G9 were from single clones using one plasmid.

Western blotting and antibodies
Cells were washed with cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer (Millipore, Billerica, MA, USA). Equal amounts of protein (50 g) were separated by 10-12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membrane (Millipore). The membranes were blocked with TBST buffer containing 5% non-fat milk, and incubated with indicated primary antibody.

Immunohistochemistry
Formalin-fixed paraffin-embedded tumor tissue sections (obtained from the National Cheng Kung University Hospital) were used to determine Id-1 protein expression in 72 NSCLC tissue samples. Slides were dewaxed, rehydrated, and placed in a container containing 1 liter of 0.01 M citrate buffer (pH 6.0); they were then microwaved at 700 W for 20 min, allowed to remain in the hot citrate buffer for 15 min, and cooled down in running cold water. The slides were washed in deionized water and incubated in 10% nonfat dry milk for 30 min at room temperature, washed in TBS, and incubated with 1 g/ml of anti Id-1 antibody overnight at 4°C. Control slides were incubated with rabbit immunoglobulin. The slides were washed in TBS and incubated with biotinylated swine anti-rabbit fragments (1: 400) for 30 min. After washing in TBS, endogenous peroxidase was blocked with 0.3% hydrogen peroxide and 0.1% sodium azide for 10 min. The slides were washed in TBS and incubated with 1:500 streptavidin-horseradish peroxidase for 30 min. After washing in TBS, peroxidase was visualized by incubating in 0.5 mg/ml diaminobenzidine-4-HCl and 0.03% hydrogen peroxide in TBS for 3 min. The slides were washed in TBS and water before mounting.

Immunoprecipitation
Cells that were transfected with plasmids as described above were washed with cold PBS and lysed in NP-40 buffer (NaCl, 150 mM, NP-40, 1.0%, Tris-Cl 50 mM, pH 8.0). Then 500 g of cell lysates were incubated with 2-5 g of the indicated antibodies for 10-14 h at 4°C. Fifty microliters of protein A-agarose (GE Healthcare Life Sciences, Piscataway, NJ, USA) was added and incubated; the immunecomplexes were resolved by SDS-PAGE followed by western blotting.

GST pull-down assay
BL-21 bacteria were transformed with pGEX-5X-3 (GST alone) or each GST fusion construct as described above and cultured in an incubator at 37 °C; 1 mM IPTG was added to the culture to induce expression of GST or each GST-fusion protein.
Suspensions were vortexed to resuspend the pellet, incubated on ice for 30 min, and sonicated for 5 min. The insoluble fraction was removed by centrifugation, and the supernatants incubated with glutathione-Sepharose beads (GE Healthcare Life Sciences) for 30 min at room temperature. For the pull-down assays, 30 l of the 50% GST or each GST-fusion protein bead slurry were incubated with recombinant human GCIP proteins from in vitro transcription-translation reactions (Thermo scientific). To detect protein that bound specifically, the beads were washed six times with lysis buffer, boiled in SDS-PAGE samples buffer, and subjected to SDS-PAGE and analyzed by immunoblotting.
Confocal scanning analysis of the cells was done with EZ-C1 confocal imaging system (Nikon).

Mammalian two-hybrid assay
HEK 293T cells were co-transfected with 2 g of pG5-luc, pSV--galactosidase, and each pBIND fusion construct as described above. In the case of a positive control experiment, we transfected cells with 2 g of pACT-GCIP, which contains the VP16 activation domain, and pBIND-P0 control vector of CheckMateTM mammalian twohybrid system (Promega) instead of pBIND vector. At 24 h after transfection, cells were washed three times with PBS and scraped in 400 l of reporter lysis buffer (Promega). The cells were subjected to a single freeze-thaw. After vortexing the cells, the lysates were centrifuged at 10,000 × g for 1 min at 4 °C, and the supernatant was assayed directly or stored at −70 °C. The -galactosidase activity was assayed using the -galactosidase assay system with reporter lysis buffer (Promega). Luciferase activity in 10 l of the cell extract was measured by an automated luminometer (Wallac-Berthold, Tokyo). Luciferase activity was normalized by -galactosidase activity, and the data thus normalized by -galactosidase activity are presented. Each pBIND construct was transfected into three separate dishes, and the results were confirmed in three independent experiments.

Generation of stable doxycycline inducible GCIP clones in A549/Id1 cell
A549/Id1 stable Cell at about 70% confluences was transfected with 2 g pCMV-Tet3G vector (Clontech) using LipofectAMINE Plus Reagent (Invitrogen) according to the manufacturer's protocol. After transfection, cells were plated in four 10-cmdiameter cell culture dishes. Replace medium with fresh complete medium plus G418 (150 g/ml) every four days. Appearance of G418 resistant clones was monitored using an inverted microscope. These clones were isolated using cloning cylinders, transferred to individual wells, and screened for screened for expression of transactivator using the Promega Luciferase Assay System (Promega) after transient transfections with pTRE3G-Luc plasmid.Experiments were performed in six-well plates. Then, clones with high expression were transfected with a linear hygromycin plasmid and pTRE3G-GCIP in which wild-type GCIP cDNA was incorporated downstream of Tet-regulated P TRE3G . Induction of GCIP was accomplished by addition of doxycycline. Two out of 24 hygromycin-resistant clones displayed tightly regulated induction of GCIP and were selected for further use.