Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells
© Hamid et al; licensee BioMed Central Ltd. 2005
Received: 18 November 2004
Accepted: 13 January 2005
Published: 13 January 2005
Pituitary tumor transforming gene1 (PTTG1) is a novel oncogene that is expressed in most tumors. It encodes a protein that is primarily involved in the regulation of sister chromatid separation during cell division. The oncogenic potential of PTTG1 has been well characterized in the mouse, particularly mouse fibroblast (NIH3T3) cells, in which it induces cell proliferation, promotes tumor formation and angiogenesis. Human tumorigenesis is a complex and a multistep process often requiring concordant expression of a number of genes. Also due to differences between rodent and human cell biology it is difficult to extrapolate results from mouse models to humans. To determine if PTTG1 functions similarly as an oncogene in humans, we have characterized its effects on human embryonic kidney (HEK293) cells.
We report that introduction of human PTTG1 into HEK293 cells through transfection with PTTG1 cDNA resulted in increased cell proliferation, anchorage-independent growth in soft agar, and formation of tumors after subcutaneous injection of nu/nu mice. Pathologic analysis revealed that these tumors were poorly differentiated. Both analysis of HEK293 cells transiently transfected with PTTG1 cDNA and analysis of tumors developed on injection of HEK293 cells that had been stably transfected with PTTG1 cDNA indicated significantly higher levels of secretion and expression of bFGF, VEGF and IL-8 compared to HEK293 cells transfected with pcDNA3.1 vector or uninvolved tissues collected from the mice. Mutation of the proline-rich motifs at the C-terminal of PTTG1 abolished its oncogenic properties. Mice injected with this mutated PTTG1 either did not form tumors or formed very small tumors. Taken together our results suggest that PTTG1 is a human oncogene that possesses the ability to promote tumorigenesis in human cells at least in part through the regulation of expression or secretion of bFGF, VEGF and IL-8.
Our results demonstrate that PTTG1 is a potent human oncogene and has the ability to induce cellular transformation of human cells. Overexpression of PTTG1 in HEK293 cells leads to an increase in the secretion and expression of bFGF, VEGF and IL-8. Mutation of C-terminal proline-rich motifs abrogates the oncogenic function of PTTG1. To our knowledge, this is the first study demonstrating the importance of PTTG1 in human tumorigenesis.
Pituitary tumor transforming gene 1 (PTTG1), a recently characterized oncogene, was initially identified on analysis of a rat pituitary tumor ; subsequently, a human homologue of PTTG1 was cloned by us and others [2–4]. Three members (PTTG1, PTTG2 and PTTG3) of the PTTG family, which exhibit differential expression in normal and tumor cells have been reported , although only PTTG1 has been studied in detail. PTTG1 is located on chromosome 5q35.1 , a locus associated with recurrent lung cancer and myelogenous leukemias . Moreover, it has been shown to be expressed highly in various tumors, and cell lines derived from such tumors, including tumors of the pituitary, thyroid, colon, ovary, testicles, and breast [8–12]. In normal tissues, its expression is low or undetectable except in testis [4, 1]. Recent studies have indicated that elevated expression of PTTG1 in some tumors may serve as a prognostic marker for tumor invasiveness and metastasis . A clue to its function was gained from its structural similarity with the yeast securin, which led to its identification as a human securin  and suggested that it may play a role in regulation of sister chromatid separation. It appears, however, to have multiple effects in cells with enhanced expression being associated with an increase in the expression of the c-myc oncogene , an increase in the expression of p53 [16, 17], an increase in the secretion and expression of basic growth factor (bFGF)  and an increase in the secretion and expression of vascular endothelial growth factor [VEGF) [18, 19].
To date, the evidence for the oncogenic function of PTTG1 has been obtained by overexpression of PTTG1 in mouse fibroblast cells (NIH3T3) followed by assessment of its ability to induce cellular transformation in vitro (colony formation in soft agar) and tumor formation in nude mice [2, 4]. Due to the biological differences between human and rodent cells, however, care must be taken in extrapolating results obtained using rodent cells to human cells. There are now a number of examples in which it has been demonstrated that although overexpression of an oncogene can induce transformation of primary rodent cells , it fails to induce transformation of the same cell type derived from humans. Usually this failure is attributable to the requirement for co-expression of another gene or oncogenic cooperation of another gene [21–26].
Similarly, much of the evidence concerning the mechanisms by which PTTG1 may affect the phenotype of the cell has been obtained using transfected NIH3T3 cells. It is known that the secretion of growth factors and cytokines by tumor cells, and the cells that infiltrate and surround the tumor mass play an essential role in the regulation of tumor growth and metastasis . Both bFGF and VEGF have been implicated in tumorigenesis and the expression and secretion of these molecules has been demonstrated on transfection of NIH3T3 cells with PTTG1 cDNA [18, 19], but this has not been confirmed on transfection of human cells. The effects of expression of PTTG1 on another cytokine that is known to play a key role in tumorigenesis, interleukin-8, (IL-8), have not yet been analyzed.
The purposes of this study were, therefore, three-fold. Firstly, to determine whether PTTG1 can induce cellular transformation of normal human cells; secondly, to determine if PTTG1 is sufficient in itself to induce transformation; and, thirdly, to characterize the changes in secretion and expression of key metastatic, angiogenic and chemokine factors (bFGF, VEGF and IL-8) associated with PTTG1-mediated transformation in human cells. For these studies, we selected the human embryonic kidney (HEK293) cell line as our model. The transformation of these cells by human adenovirus type 5 prevents their senescence . These cells have been reported to have a moderate tumorigenic potential  and have been used as a cellular model for normal human cells to study the oncogenic potential of a number of genes [29–31]. Mice xenografted with these cells do not develop tumors even after three months of injection .
Generation of HEK293 cells stably expressing PTTG1 and mPTTG1
Stable transfection of PTTG1 induces cell proliferation and transformation of HEK293 cells overexpressing PTTG1
Overexpression of PTTG1 induces cellular transformation
PTTG1 induces tumor formation in nude mice injected with HEK293 cells stably expressing PTTG1 protein
Tumor formation induced by PTTG1 expressing HEK293 cells in nude mice
Animals with tumor
PTTG1 stimulates expression and secretion of bFGF, VEGF and IL-8
Oncogenic function of PTTG1 was established by its overexpression in mouse fibroblast cell line (NIH 3T3) followed by assessment of its ability to induce cellular transformation and tumor formation in nude mice [2, 4]. However, the differences in biology between the rodent cells and human cells have brought the validity of this model into question. There are a number of instances in which an oncogene has been shown to induce transformation in rodent cells but has failed to induce transformation of same types of cells obtained from humans. To test the ability of PTTG1 to induce transformation in human cells, we selected the human embryonic kidney-293 (HEK293) cell line as our model. Our data clearly demonstrate that over expression of PTTG1 in HEK293 results in an increase in cell proliferation induces cellular transformation in-vitro (increase in anchorage-independent growth) and promotes tumor formation in nude mice. Cells transfected with pcDNA3.1 vector did not form colonies in soft agar or develop into tumors on implantation in nude mice, confirming that HEK293 cells do not possess a tumorigenic phenotype. Furthermore our data suggest that overexpression of PTTG1 in these cells results in a greater propensity for tumor development, a shortened latency period and an enhanced growth rate compared with pcDNA3.1-transfected control HEK293 cells.
A second issue that we were able to address using the HEK293 cell model was the question of the ability of PTTG1 to induce transformation of normal human cells, i.e., whether it acts alone or in cooperation with another oncogene to achieve its tumorigenic function. It has been reported that a single oncogene may not be sufficient for induction of transformation but requires co-expression, or oncogenic cooperation, of another oncogene(s) to induce tumorigenesis in normal primary human cells [20, 21, 24, 25, 42, 43]. Our results clearly show that PTTG possess the ability to induce cellular transformation in vitro and promotes tumor formation in nude mice. Since, HEK293 cells are transformed with adenovirus type 5 thus making them different from the normal primary cells, therefore it remains unclear if PTTG is sufficient by itself to initiate the tumorigenesis of primary human cells. However, the data clearly demonstrate that PTTG1 overexpression in these cells accelerates their tumorigenic capacity in comparison to that of unmodified cells.
PTTG1 contains several-proline rich motifs (PXXP); two of these that are located in the C-terminal domain have been reported to be potential binding sites for SH3-domians . In our study we confirm that mutation of these C-terminal proline-rich motifs abrogates the tumorigenicity of PTTG1 in human cells. Such loss of tumorigenicity on mutation of PTTG1 could be due to a loss of expression. Our western blot analysis of the stable cell lines (HEKmPTTG1-2 and HEKmPTTG1-4) that constitutively express mutated PTTG1 protein showed high levels of expression of mPTTG1 protein (Fig. 1), suggesting that the loss of tumorigenic function of mPTTG1 protein is not due to loss of expression but due to the loss of its ability to induce cellular transformation. These results are consistent with other investigators for rodent cells  and confirm the importance of C-terminal proline-rich motifs to mediate the oncogenic function of PTTG1.
The molecular mechanisms by which PTTG1 achieves its tumorigenic function remain unclear. PTTG1 has been reported to induce expression of the c-myc oncogene , bFGF  and VEGF . bFGF is a broad spectrum and pleiotropic mitogen for growth and differentiation affecting various mammalian cells and organ systems and a large number of cells lines [45, 46]; besides stimulating wound healing, tissue repair and hematopoiesis , bFGF induces cell migration and proliferation  and acts as an agiogenic factor that induces migration, proliferation and differentiation of endothelial cells . In addition, it has been reported to modulate the invasion of tumor cells through surrounding tissue to form new capillary cord structures by regulating the activities of extracellular molecules including collagenase, proteinases and integrins . Regulation of secretion and expression of bFGF by PTTG1 in NIH 3T3 cells has been shown . Consistent with these reports, our results demonstrate a significant increase in secretion and expression of bFGF in HEK293 cells on transient transfection with PTTG1 cDNA (Fig. 6) as well as in tumors developed by injection of stable cell lines that constitutively express PTTG1 both at protein and mRNAs levels (Figs. 6, 7, 8).
VEGF is a potent stimulant of the vascularization of tumors and is one of the most specific markers of tumor vasculature observed to date [50, 51]. VEGF is a multifunctional cytokine acting as a potent permeability agent, an endothelial cell chemotactic agent, an endothelial cell survival factor and an endothelial cell proliferation factor . The expression and secretion of VEGF has been shown to be a crucial rate-limiting step during tumor progression . Our results demonstrate a significant increase in secretion and expression of VEGF in HEK293 cells on transfection with PTTG1 and also from tumors excised from animals injected with HEK293 cells that stably express PTTG1 (Figs. 6, 7, 8).
A direct correlation between high IL-8 expression and metastases in melanoma , ovarian cancer , prostrate cancer  and pancreatic cancer  has been reported. To determine if overexpression of PTTG1 induces change in secretion and expression of IL-8, we measured its levels in HEK293 cells on transfection with PTTG1 cDNA and in tumors developed on injection of HEK293 cells transfected with PTTG1. Our results demonstrate for the first time that overexpression of PTTG1 induces IL-8 expression in vitro and also in tumors in vivo (Figs. 6, 7, 8).
Generation of cell lines constitutively expressing PTTG1
The human embryonic kidney cell line (HEK293), which had been transfected by exposing these cells to sheared fragments of adenovirus type 5 DNA  was purchased from ATCC (American Type Culture Collection; Rockville, MD) and cultured according to the instructions provided. The cells were transfected with pcDNA3.1 vector, pcDNA3.1-PTTG1 or pcDNA3.1-mPTTG1 to generate stable clones that constitutively express human wild-type PTTG1 or mutated PTTG1 (mPTTG1) protein as described previously . The mPTTG1, which carries a double amino acid change within the SH3 binding domain of PTTG1 (P163 to A163, P170 to A170 and P172 to A172, and P173 L173), was generated by site-directed mutagenesis using the Quick-change mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Mutation of these amino acids has been reported to abrogate the tumorigenic function of PTTG1 and to block the secretion and expression of bFGF in mouse NIH3T3 cells . The primers used for this site-directed mutagenesis were 5'-GATGCTCTCCGCACTCTGGGAATCCAATCTG-3' and 5'-TTCACAAGTTGAGGGGCGCCCAGCTGAAACAG-3'. The transfected cells were then selected in neomycin G418 (500 μg/ml) and the clones that expressed high levels of PTTG1 protein or mPTTG1 protein were selected. One clone from pcDNA3.1 transfected cells (HEKpcDNA3.1) two clones from pcDNA3.1-PTTG1 transfected cells (HEKPTTG1-1 and HEKPTTG1-3) and two clones from pcDNA3.1-mPTTG1 transfected cells (HEKmPTTG1-2 and HEKmPTTG1-4) were propagated into cell lines.
Cell proliferation assay
Cell proliferation was assayed using the CellTiter 96 non-radioactive cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer's instructions and as described previously . Briefly, cells growing in log phase were trypsinized and seeded in 96-well plates (5,000 cells/well in a final volume of 100 μl) in replicates of 4 and incubated at 37°C in 5% CO2 and 95% air. After incubation for 24 h, 48 h, 72 h or 96 h, 20 μl of dye solution from the kit was added to each well and incubated at 37°C for an additional 2 h. The quantity of formazon product was measured by its absorbance at 490 nm using a 96-well plate reader (Molecular Devices, Sunnyvale, CA). Each experiment was repeated at least three times.
Soft agar colony formation (anchorage-independent cell growth) assay
Anchorage-independent cell growth was determined by analyzing the formation of colonies in soft agar. Cells (104) from each cell line were suspended in 0.3% agar in DMEM containing 10% fetal bovine serum and plated on solidified agar (0.7%) in 35 mm dishes. After 14 days of culture, colonies formed were counted and photographed as described previously .
In vivo tumor growth assay
Cells growing in log phase were harvested by trypsinization and washed twice with PBS. The cells were resuspended in PBS to a final concentration of 5 × 106/ml. The cells (1 × 106 cells in 200 μl PBS/site) were injected subcutaneously (s.c.) into both flanks of 5- to 6-week old female nu/nu mice (4 mice/group) (Charles River Laboratory, Wilmington, MA). All procedures were carried out following the protocol approved by The University of Louisville Institutional Animal Care and Use Committee. Four weeks after injection, the mice were scarified, and the tumors and other tissues harvested. The skin and connective tissues were dissected from the tumors, and the tumor volume was calculated from measurements of length × width × height. The tissues were divided into two parts, one part being fixed in 10% buffered formalin and the other stored in liquid nitrogen. For histopathologic analysis, 5 μm sections were cut from paraffin-embedded tissues, and mounted on slides. Sections were stained with H&E , and processed for histopathologic evaluations.
Western blot analysis
Cells growing in log phase were lysed in chilled lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, and 1 mM NaF) supplemented with Complete Mini Protease Inhibitor tablets (Roche Molecular Biochemicals, Indianapolis, IN). Equal amounts of protein extracts (40 μg) were resolved on 12% SDS-PAGE gel, and transferred onto a nitrocellulose membrane (Amersham, Piscataway, NJ). Blots were probed with PTTG1 antiserum at a dilution of 1:1,500 as described previously . Immunoreactive proteins were visualized using the Enhanced Chemiluminescent Detection System (Amersham) according to the instructions provided.
ELISA analysis of bFGF, VEGF and IL-8
The levels of bFGF, VEGF and IL-8 in tissue culture supernatants and tissue homogenates were measured using commercially available ELISA kits from BD Biosciences (Minneapolis, MN). To measure bFGF, VEGF and IL-8 in the culture supernatants, HEK293 cells were transiently transfected with pcDNA3.1 or pcDNA3.1-PTTG1 cDNA using Fugene6 as the transfectant reagent as described previously . After 24 h of transfection, the medium was replaced with serum free DMEM medium. Twenty-four later, the medium was collected and concentrated 5-fold (1.0 ml to 200 μl) using a speedVac system (Savant, Holbrook, NY). To measure bFGF, VEGF and IL-8 in tumor and other tissues, tissues were homogenized in 50 mM Tris (pH 7.4), 0.25% Triton X-100, 5 mM EDTA and 0.1% NP40 supplemented with Complete Mini Protease Inhibitor tablets (Roche Molecular Biochemicals, Indianapolis, IN) using a polytron homogenizer. Homogenates were centrifuged to remove particulate matter and then diluted with the diluent provided in the ELISA kit. The concentration of bFGF, VEGF and IL-8 in a sample was determined by interpolation from a standard curve. All measurements were normalized to protein concentration and performed in triplicate.
Semiquantitative reverse transcriptase/polymerase chain reaction (RT/PCR)
Primer sequences and PCR conditions for the amplification of PTTG1, bFGF, VEGF, IL-8, m-PTTG1 and GAPDH.
95°C 5 min, 95°C 30 s, 54°C 30 s, 72°C 30 s. 28 cycles.
95°C 5 min, 95°C 30 s, 56°C 30 s, 72°C 30 s. 26 cycles.
95°C 5 min, 95°C 30 s, 56°C 30 s, 72°C 30 s. 28 cycles.
95°C 5 min, 95°C 30 s, 58°C 30 s, 72°C 30 s. 28 cycles.
95°C 5 min, 95°C 30 s, 54°C 30 s, 72°C 30 s. 26 cycles.
95°C 5 min, 95°C 30 s, 54°C 30 s, 72°C 30 s. 35 cycles.
In summary, our results demonstrate that PTTG1 is a potent human oncogene and has the ability to induce cellular transformation of human cells. Overexpression of PTTG1 in HEK293 cells leads to an increase in the secretion and expression of bFGF, VEGF and IL-8. Mutation of C-terminal proline-rich motifs abrogates the oncogenic function of PTTG1. To our knowledge, this is the first study demonstrating the importance of PTTG1 in human tumorigenesis.
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