TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors

The gene structural organization of human TRIP-Br2 was analyzed by NCBI Entrez Gene, NCBI AceView and BLAST/ClustalW http://​www.​ncbi.​nlm.​nih.​gov/​. The PSORT II analysis software http://​psort.​nibb.​ac.​jp was used to predict the subcellular localization of TRIP-Br2 proteins. The GNF SymAtlas v 1.2.4 (Novartis, http://​symatlas.​gnf.​org/​SymAtlas/​) human microarray database was interrogated to determine the in silico gene expression profiling of TRIP-Br2 across all human tissues. The NCBI symbol SERTAD2 was used in the query of the GNF SymAtlas database. The median (med) was calculated based on expression of TRIP-Br2 across all human tissues; med × 3: 3-fold more than the median; med × 10: 10-fold more than the median. In silico TRIP-Br2 expression, (χ), across all human tissues was scored via the following scheme: +: (χ) ≤ median; ++: median < (χ) ≤ med × 3, +++: med × 3 < (χ) ≤ med × 10, ++++: med × 10 < (χ).

NIH3T3 mouse primary fibroblasts, WI38 human primary lung fibroblasts, U2OS human osteosarcoma cells, PC3 human prostate adenocarcinoma cells, 769-P human renal adenocarcinoma cells, HCT-116 human colorectal carcinoma cells, HepG2 human hepatocellular carcinoma cells and MCF-7 human breast carcinoma cells were purchased from American Type Culture Collection (Manassas, VA). All cell lines were cultured in DMEM supplemented with 10% FBS and maintained at 37°C in a 5% CO₂ environment. Rabbit anti-TRIP-Br2 polyclonal antibodies were generated as previously described [23] and used in Western blot, immunocytochemical and immunohistochemical analyses. All other antibodies used in Western blot analyses were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). They include anti-HA (sc-805), anti-cyclin E (sc-481) and anti-β-tubulin (sc-5274). The use of expression plasmids pcDNA3.1 (Invitrogen, Carlsbad, CA) and pcDNA3.1-TRIP-Br1-HA have been previously described [7]. The nucleotide sequence of human TRIP-Br2 (hTRIP-Br2) was obtained from NCBI PubMed (GenBank™ accession no. BC101639) and used as the template in the design of hTRIP-Br2-specific primers for the construction of C-terminal HA-tagged hTRIP-Br2 expression plasmid (Additional File 1).

NIH3T3 fibroblasts were transfected with the empty vector pcDNA3.1 as a control or with the expression vectors pcDNA3.1-TRIP-Br1-HA or pcDNA3.1-TRIP-Br2-HA using FuGENE 6 Transfection Reagent (Roche Diagnostics Co., Mannheim, Germany) in accordance with the manufacturer's instructions. Stable clones were selected using Geneticin (Invitrogen, Carlsbad, CA) at a concentration of 750 μg/ml. Expression levels of the carboxyl terminal HA-tagged TRIP-Br1 and TRIP-Br2 in each respective clone were determined by Western blot analysis.

NIH3T3^vector-only, NIH3T3^TRIP-Br1-HA and NIH3T3^TRIP-Br2-HA fibroblasts were cultured in 96-well plates (for BrdU) or 100 mm culture dishes (for flow cytometry) in DMEM supplemented with 0.2% FBS and were maintained for 72 h at 37°C in a 5% CO₂ environment. BrdU incorporation was monitored using a cell proliferation/colorimetric ELISA assay according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany). Flow cytometry was performed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) at a wavelength of 488 nm.

Soft agar assays were used to assess anchorage-independent growth of NIH3T3 cells as previously described [24]. For tumor induction assays, athymic nude mice (nu/nu) purchased from Charles River Laboratories, Inc. (Wilmington, MA) were kept under SPF conditions and used under protocol #06-231, which was approved by the Harvard Institutional Animal Care and Use Committee (IACUC) and the Harvard Committee on Microbiological Safety (COMS). 5 × 10⁶ NIH3T3^vector-only or NIH3T3^TRIP-Br2-HA fibroblasts were injected subcutaneously into 6-week-old athymic nude mice (n = 4 for each group). On day 13 post-injection, the mice were examined for tumor formation. Tumor dimensions were measured every 2 days from day 13 until day 25 post-injection, at the end of which time both groups were sacrificed and all tumors were harvested for histological, immunohistochemical and Western blot analyses. The experiment was repeated by injection of new NIH3T3^vector-only or NIH3T3^TRIP-Br2-HA clones into new groups of 6-week-old athymic nude mice (n = 4). The penetrance of tumor induction from subcutaneous injection of NIH3T3^vector-only or NIH3T3^TRIP-Br2-HA into these athymic nude mice was 0% and 100%, respectively. Tumor ellipsoid volume was estimated using the formulae previously described [25].

Total RNA was isolated from serum-deprived NIH3T3^vector-only, NIH3T3^TRIP-Br1-HA and NIH3T3^TRIP-Br2-HA fibroblasts using the TRIZOL^® Reagent (Invitrogen, Carlsbad, CA). Total RNA (3 μg) was reverse transcribed using the ABI High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Polymerase Chain Reactions (PCR) were performed on 1 μl cDNA samples in the presence of 10 mM deoxyribonucleotide triphosphates (dNTPs) and 10 μM of specific primer pairs in a total reaction volume of 20 μl. PCR was performed as follows: 20 cycles of denaturation (94°C, 30 sec), annealing (51°C, 30 sec) and extension (72°C, 1 minute) with a 2-minute initial denaturation step at 94°C and a 3-minute terminal polishing step at 72°C. The primer sequences used for RT-PCR are available upon request.

Subcellular fractionation of the cells was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology, Inc., Rockford, IL) according to the manufacturer's instructions. Proteins from whole-cell lysates were resolved using standard denaturing polyacrylamide gel electrophoresis and immunostained as described previously [7].

Multiple TMA slides were obtained from the Department of Pathology TMA Program at the National University of Singapore, in compliance with Institutional Review Board approval (IRB 05-017). These tumor TMAs were constructed as previously described [26‐29] and represented samples from the following human tumor types that occur in a broad range of organs: prostate carcinoma, squamous cell lung carcinoma, lung adenocarcinoma, breast carcinoma, gastrointestinal stromal tumor, ovarian cystadenocarcinoma, colorectal carcinoma, basal cell carcinoma, renal cell carcinoma, osteosarcoma, hepatocellular carcinoma. Antigens were retrieved from the tissues using a microwave histoprocessor (Milestone, Shelton, CT) and DAKO pH 6.0 citrate buffer (DAKO, Via Real Carpinteria, CA). Immunohistochemical staining was performed on paraffin-embedded tissue sections using the DAKO Envision kit (DAKO) and the rabbit anti-TRIP-Br2 antibody or its pre-immune serum control at a concentration of 1:300. Staining was visualized using a Leica DM LB2 microscope. The intensity of TRIP-Br2 expression by immunostaining in the tumor TMAs was scored independently by three research pathologists in a double-blinded manner. For immunocytochemistry, cells were grown to 80% confluence on coverslips, washed three times with PBS, fixed in pre-chilled 4% paraformaldehyde for 20 minutes, and permeabilized in 0.1% Triton-X for 10 minutes. Primary immunostaining with rabbit anti-TRIP-Br2 antibody (1:4000) was performed at room temperature for 1 h. Pre-immune rabbit serum was used as a negative control for the primary immunostaining of cells. Secondary immunostaining with goat anti-rabbit-FITC antibodies (sc-2012, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was performed at room temperature for 1 h, following 3 washes with PBS at the end of primary immunostaining. Cellular DNA was subsequently counterstained with DAPI. Staining was visualized and photographed using a Nikon Eclipse E1000 fluorescence microscope.

5 × 10⁴ HCT-116 cells were plated in 12-well plates and transfected with Cy3-labeled oligomer, scrambled siRNA (negative control) or three different TRIP-Br2-specific siRNAs at the dose of 4 picomoles (pmol) or 40 pmol (in 1 ml of DMEM supplemented with 10% FBS) respectively (TriFECTa™ kit, IDT, Coralville, IA) using Lipofactamine™ Transfection Reagent (Invitrogen, Carlsbad, CA), in accordance with the manufacturer's instructions. Twenty-four hours post-transfection, these cells were cultured in serum-free DMEM and maintained at 37°C in a 5% CO₂ environment for 72 h. HCT-116 cells that were not subjected to transfection reagent treatment were included as controls. Cells in colony forming assays were stained with 0.4% Giemsa stain as previously described [23]. The dye in these cells was subsequently eluted with 1% SDS and quantitated using a spectrophotometer at a wavelength of 595 nm. A standard curve was plotted using OD readings taken from dye-eluted HCT-116 cells that were plated at pre-determined cell densities.

Survival curves for various patient cohorts were estimated according to the method of Kaplan and Meier, and curves were compared using the generalized Wilcoxon's test. The log-rank test was used to assess the strength of association between survival time and single variables corresponding to factors thought to be prognostic for survival.

The TRIP-Br2 gene locus is approximately 22.3 kb long and is localized at the poorly-characterized chromosome 2p14 region of the human genome (position 64734550 bp to 64712250 bp; reverse strand) (Figure 1A). The precursor of TRIP-Br2 mRNA is approximately 5556 bp in length and consists of two exons that are separated by a long intron (Figure 1B). The intron encodes a splice donor (GT) and a splice acceptor (AG) at either end, respectively. The 297 bp-long 5' untranslated region (UTR) resides in exon 1 of TRIP-Br2. It contains an in-frame stop signal that is 6 bp prior to the 945 bp-long coding sequence of TRIP-Br2, which is localized in exon 2. The 3' UTR of TRIP-Br2 spans a region of approximately 4314 bp, followed by a standard AATAAA polyadenylation signal. Due to the lack of other splice donor-acceptor sites, transcription of the human TRIP-Br2 gene is predicted to yield only one mRNA transcript that encodes a 314 amino acid protein. We studied the primary protein sequence of human TRIP-Br2 by BLAST/ClustalW analyses and found that TRIP-Br2 is highly conserved in widely divergent species, such as chimpanzee (99%), rhesus monkey (97%), rat (86.9%), mouse (88.3%), chicken (81.4%) and zebrafish (67.1%) (Figure 1C). Furthermore, based on the PSORT II analysis, the subcellular localization of TRIP-Br2 protein is predicted to be predominantly in the nucleus (69%), with scant presence in the mitochondria (17%), in the cytoplasm (4%), in the vacuoles (4%) or in vesicles of the secretory system (4%).

In order to investigate the biological significance of TRIP-Br2 in humans, we first performed in silico gene expression profiling of TRIP-Br2 using a comprehensive web-based human microarray database, GNF SymAtlas v 1.2.4 (Novartis, http://​symatlas.​gnf.​org/​SymAtlas/​). As compared to other tissues/cell types, TRIP-Br2 is highly expressed in bone marrow, the thymus, the tonsil and smooth muscle. It is also highly expressed in lymphohematopoietic cell lineages, particularly in BDCA4+ dendritic cells, CD34+ cells (bone marrow hematopoietic stem cells), CD71+ early erythroid cells, B lymphoblasts, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells and CD33+ myeloid cells (Table 1). As these cell types are highly proliferative, we postulated that TRIP-Br2 plays an important role in cellular proliferation and/or tumor progression. This is supported in part by our previous observation that ablation of TRIP-Br2 resulted in cellular proliferation arrest in primary cells, which was associated with downregulation of a subset of E2F-responsive genes such as CYCLIN E [17].

To validate the protooncogenic role of TRIP-Br2 in cell cycle regulation and tumorigenesis, we stably overexpressed C-terminal HA-tagged-TRIP-Br2 in NIH3T3 fibroblasts (NIH3T3^TRIP-Br2-HA; Figure 2A). Although TRIP-Br1 overexpression has been recently shown to transform NIH3T3 fibroblasts, the underlying molecular mechanism of cellular transformation by TRIP-Br1 remains elusive. Thus, we also stably overexpressed carboxyl-terminal HA-tagged-TRIP-Br1 in NIH3T3 fibroblasts (NIH3T3^TRIP-Br1-HA) and investigated the mechanism(s) by which TRIP-Br1 and TRIP-Br2 facilitate cellular transformation. Overexpression of TRIP-Br1-HA or TRIP-Br2-HA in NIH3T3 fibroblasts conferred the ability to proliferate under low serum concentrations, possibly by enhancing DNA synthesis (Figure 2B). Flow cytometric DNA analysis revealed significantly higher proportions of NIH3T3^TRIP-Br1-HA and NIH3T3^TRIP-Br2-HA fibroblasts in S phase of the cell cycle as compared to the NIH3T3^vector-only control, despite serum deprivation (Figure 2C). As TRIP-Br proteins have been shown to regulate E2F/DP-mediated transcriptional activities [7], we screened these serum-deprived NIH3T3 fibroblasts for a panel of E2F-responsive cell cycle regulators that govern cell cycle progression. Elevated levels of a subset of these E2F-responsive cell cycle regulators comprised of CYCLIN E (CCNE), CYCLIN A2 (CCNA2), CDC6 and DHFR, were found in serum-deprived fibroblasts that stably overexpress TRIP-Br proteins (Figure 2D, upper panel). Notably, in serum-deprived NIH3T3 cells that stably overexpress TRIP-Br1-HA or TRIP-Br2-HA, we observed a concomitant increase in cyclin E expression (Figure 2D, lower panel). This is consistent with our previous observation that cyclin E was downregulated following ablation of TRIP-Br1 or TRIP-Br2. Hence, our data suggest that CYCLIN E may be a TRIP-Br1- and TRIP-Br2-coregulated gene.

Next, we evaluated the oncogenic potential of TRIP-Br2 by assessing anchorage-independent growth of these NIH3T3^TRIP-Br2-HA fibroblasts in soft agar (Figure 3A). As many as 17.7% of the seeded NIH3T3^TRIP-Br2-HA fibroblasts formed colonies at 4 weeks post-plating, while NIH3T3^vector-only fibroblasts were incapable of anchorage-independent growth in soft agar. PC3 cells and NIH3T3^TRIP-Br1-HA fibroblasts were used as positive controls in this assay.

In addition, we validated the tumorigenic potential of TRIP-Br2 by an in vivo tumor formation assay. One inoculum (5 × 10⁶ cells) of either NIH3T3^vector-only or NIH3T3^TRIP-Br2-HA fibroblasts (from one representative clone each) was injected subcutaneously into the lower flanks of athymic nude mice (n = 4). This experiment was repeated at least twice by subcutaneous injection of a different clone of either NIH3T3^vector-only or NIH3T3^TRIP-Br2-HA fibroblasts into other groups of four athymic nude mice. Data from two mice from a representative experiment are shown in Figure 3B (Upper panel). All sites injected with NIH3T3^TRIP-Br2-HA fibroblasts developed a tumor, which was typically ~0.7 cm³ (data derived from one tumor induction assay, n = 4) at day 25 post-injection (Figure 2B, lower panel). Tumors derived from NIH3T3^TRIP-Br2-HA fibroblasts were histologically fibrosarcomas (Figure 3C). Western blot analyses of tumor extracts (Figure 3D, upper panel) as well as HA-immunostaining of paraffin-embedded tumor sections (Figure 3D, lower panel) indicated the presence of the transgene product TRIP-Br2-HA.

Given that overexpression of TRIP-Br2 alone was sufficient to transform NIH3T3 fibroblasts, we hypothesized that expression of TRIP-Br2 may be dysregulated and contribute to oncogenesis in human cancer. We screened normal and cancer cell lines for TRIP-Br2 expression using rabbit anti-TRIP-Br2 polyclonal antibodies and found that TRIP-Br2 was overexpressed in human cancer cell lines U2OS, PC3, 769-P, HCT-116, HepG and MCF-7 cells, but not in WI38 diploid fibroblasts (Figure 4A). The higher molecular weight endogenous species of TRIP-Br2 observed in Figure 4A (and 4C below) are specific bands that we have observed in only some human cancer cell lines, associated with the use of the rabbit polyclonal anti-TRIP-Br2 for immunoblot analysis [23].

We next sought to identify the cellular role(s) of TRIP-Br2 by investigating its localization in WI38 and U2OS cells. Using rabbit anti-TRIP-Br2 polyclonal antibodies, we first demonstrated by immunocytochemistry that TRIP-Br2 was predominantly localized to the nuclei of WI38 and U2OS cells (Figure 4B), with scant cytoplasmic expression. This is in agreement with our earlier PSORT II prediction of TRIP-Br2 subcellular localization and a recent observation made by Lai and coworkers [30]. As compared to WI38 cells, TRIP-Br2 was clearly overexpressed in the nuclei of U2OS cells. Our data from subcellular fractionation analysis is consistent with this observation. TRIP-Br2 was overexpressed and predominantly localized to the nuclear fractions of U2OS cells as well as other human cancer cell lines such as PC3, 769-P, HCT-116 and HepG2 (Figure 4C), suggesting that TRIP-Br2 expression and localization might be dysregulated in these cancer cells.

In order to address an oncogenic role for TRIP-Br2 in human cancers, we assessed the immunohistochemical expression of TRIP-Br2 by comparing normal and cancer tissue sections on microarrays that were constructed from patient specimens of 10 different human tumor types. Tissue microarray (TMA) is a high-throughput method for the analysis of large numbers of formalin-fixed, paraffin-embedded (FFPE) materials with minimum cost and effort [31]. We found that TRIP-Br2 was overexpressed in prostate carcinoma (50.8%), squamous cell lung carcinoma (100%), lung adenocarcinoma (48.7%), ovarian cystadenocarcinoma (73.1%), colorectal carcinoma (64.9%), renal cell carcinoma (50%), osteosarcoma (100%) and hepatocellular carcinoma (72.4%). Notably, the frequency of TRIP-Br2 overexpression was lower in breast carcinoma (25%), basal cell carcinoma (16.7%) and gastrointestinal stromal tumor (15.6%). We also observed minor variations of TRIP-Br2 overexpression between different subtypes of ovarian carcinoma such as serous, mucinous and endometroid ovarian cystadenocarcinoma (data not shown). A representative TRIP-Br2-immunostained tumor specimen from each of the 10 tumor tissues and corresponding normal tissues examined by TMA are shown in Figure 5A and Additional File 2, respectively. The frequency of TRIP-Br2 upregulation in these human cancers is summarized in Additional Table S1 (see Additional File 1).

Next, we investigated the effect of TRIP-Br2 overexpression on the survival of hepatocellular carcinoma (HCC) patients to determine whether TRIP-Br2 overexpression is associated with poor prognosis. A patient cohort (n = 12) with full survival data was divided into two groups, survival ≤ 1 year (n = 8) and survival > 1 year (n = 4). These two groups were subsequently scored as "TRIP-Br2 overexpressors" versus "TRIP-Br2 non-overexpressors" in the corresponding tumor tissue biopsies represented on TMAs (Figure 5A). A patient was scored as a "TRIP-Br2 overexpressor" if the intensity of TRIP-Br2 immunostaining in tumor tissue was observed to be more intense than adjacent normal tissue. Six of eight HCC patients were TRIP-Br2 overexpressors and were found to have survived for ≤ 1 year, while three of four HCC patients were TRIP-Br2 non-overexpressors and were found to have survived for > 1 year. A survival analysis using the Kaplan Meier log rank test was performed, which showed that the mean survival of patients exhibiting tumor tissue TRIP-Br2 overexpression (9 months) was found to be significantly lower than the survival of HCC patients without evidence of TRIP-Br2 overexpression (16 months) (p = 0.0452) (Figure 5B). This observation is not only significant from a statistical viewpoint, but also clinically in the context of a cancer type with a particularly poor prognosis.

To validate the potential of TRIP-Br2 as a novel transcription-based chemotherapeutic target for human cancers, we performed siRNA knockdown of TRIP-Br2 expression in HCT-116 cells. Cy3-labeled oligomer transfection control (Cy3-O), scrambled siRNA non-specific control (Scr) or TRIP-Br2- specific siRNAs (DS1, DS2 or DS3) were transiently transfected into HCT-116 cells, respectively, at a low dose of 4 pmol or a high dose of 40 pmol (in one ml of DMEM supplemented with 10% FBS). Twenty-four hours post-transfection, these cells were serum-deprived for 72 h to investigate the role of TRIP-Br2 in cell-autonomous growth of HCT-116 cells. As shown in Figure 6A (Left panel), specific knockdown of TRIP-Br2 expression in HCT-116 cells (12-well plate) was only achieved by TRIP-Br2- specific siRNAs, DS1 and DS2, at the higher dose of 40 pmol. There were no changes in the transcript levels of other TRIP-Br gene family members upon treatment with TRIP-Br2- specific siRNAs, DS1 and DS2, as assessed by semi-quantitative RT-PCR (Figure 6A, right panel).Western blot analyses further revealed that TRIP-Br2 protein expression was significantly knocked down by TRIP-Br2-specific siRNAs, DS1 and DS2 (Figure 6B). In addition, colony forming assays (Figure 6C) and cell count analyses (Figure 6D) showed that siRNA knockdown of TRIP-Br2 expression inhibited cell-autonomous growth of serum-deprived HCT-116 cells.