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Pituitary Tumor-Transforming Gene: Physiology and Implications for Tumorigenesis

Department of Medicine, Cedars-Sinai Medical Center, University of California School of Medicine, Los Angeles, California 90048

Correspondence: Address all correspondence and requests for reprints to: Shlomo Melmed, Academic Affairs, Room 2015, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu

	Abstract

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
Pituitary tumor-transforming gene-1 (PTTG1) is overexpressed in a variety of endocrine-related tumors, especially pituitary, thyroid, breast, ovarian, and uterine tumors, as well as nonendocrine-related cancers involving the central nervous, pulmonary, and gastrointestinal systems. Forced PTTG1 expression induces cell transformation in vitro and tumor formation in nude mice. In some tumors, high PTTG1 levels correlate with invasiveness, and PTTG1 has been identified as a key signature gene associated with tumor metastasis. Increasing evidence supports a multifunctional role of PTTG1 in cell physiology and tumorigenesis. Physiological PTTG1 properties include securin activity, DNA damage/repair regulation and involvement in organ development and metabolism. Tumorigenic mechanisms for PTTG1 action involve cell transformation and aneuploidy, apoptosis, and tumorigenic microenvironment feedback. This paper reviews recent advances in our understanding of PTTG1 structure and regulation and addresses known mechanisms of PTTG1 action. Recent knowledge gained from PTTG1-null mouse models and transgenic animals and their potential application to subcellular therapeutic targeting PTTG1 are discussed.

I. Introduction

II. PTTG1 Gene Structure and Regulation

A. PTTG1 gene structure

B. PTTG1 mRNA expression profile

C. Regulation of gene expression

III. PTTG Protein

A. PTTG1 protein structure

B. PTTG1 protein subcellular localization and complex formation

C. PTTG1 protein posttranscriptional modification

IV. PTTG Family Members

A. Cloning and characterization of PTTG2 and -3

B. PTTG family member expression profile

V. PTTG1: Physiological Functions

A. Securin function/replication

B. DNA damage/repair

C. Interaction partners and transactivation activity

VI. PTTG1: Tumorigenic Mechanisms

A. PTTG1 and cell proliferation

B. Cell transformation and aneuploidy

C. Apoptosis

D. Tumorigenic microenvironment

VII. Pttg1-Null Mouse Model

VIII. PTTG1 and Cancer

A. Endocrine-related cancer

B. Nonendocrine-related cancer

C. Potential therapeutic applications

IX. Summary

	I. Introduction

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
PITUITARY TUMOR-transforming gene (PTTG) was isolated from rat pituitary tumor cells in 1997 and identified as a pituitary-derived transforming gene (1). Structural homology led to the identification of PTTG1 protein as a vertebrate securin critical in regulating sister chromatid separation during mitosis (2). PTTG1 overexpression has been reported in a variety of endocrine-related tumors, especially pituitary, thyroid, breast, ovarian, and uterine tumors, as well as nonendocrine-related cancers involving the central nervous system, pulmonary system, and gastrointestinal system (3, 4, 5, 6, 7, 8, 9, 10). PTTG1 levels correlate with tumor invasiveness (10, 11), and PTTG1 has been identified as a key signature gene associated with tumor metastasis (12). PTTG1 functions in cell replication (2), DNA damage/repair (13), organ development, and metabolism (14, 15, 16). Partially elucidated mechanisms of PTTG1 action include protein-protein regulation, transactivation activity, and paracrine/autocrine regulation.

In this review, we describe the structure and regulation of the PTTG1 gene and protein and address the known PTTG1 physiological functions. We review current information on PTTG1-mediated tumorigenic mechanisms, including cell transformation and aneuploidy, apoptosis, and tumorigenic microenvironment feedback. We describe the knowledge gained from Pttg1-null mouse models and transgenic animals and review the known impact of PTTG1 on endocrine and nonendocrine-related neoplasms, focusing on potential subcellular therapeutic applications for these tumors.

	II. PTTG1 Gene Structure and Regulation

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
A. PTTG1 gene structure
Pttg1 was isolated from rat GH-secreting pituitary cell lines by differential mRNA display PCR (1), yielding two pituitary-specific mRNAs, one of which was a differentially expressed, 396-bp mRNA that showed no homology to current GenBank entries. Using this PCR fragment to probe a rat pituitary tumor cDNA library enabled the cloning and characterization of the full-length (974 bp) rat Pttg1 cDNA clone (GenBank accession no. U73030) (1). Rat Pttg1 gene is composed of five exons and four introns (17), sized about 0.8, 1.8, 1.1, and 0.6 kb, respectively (17) (Fig. 1). Northern analysis revealed two mRNA isoforms of this gene; a 1.3-kb isoform strongly expressed in pituitary tumor cells, whereas rat testis and embryonic liver expressed a truncated Pttg1 mRNA isoform of approximately 1 kb (1). In both pituitary and testicular cells, the transcription start site for rat Pttg1 was mapped to a thymidine residue 44 bp upstream from the ATG initiation codon (17), suggesting that the different testis transcript size is not a result of alternative promotion. A minimal 745 bp of the 5'-flanking sequences (Fig. 2) are required for Pttg1 transcriptional activation in rat testicular cell lines, and the sequence between –509 and –624 bp was identified as the core enhancer sequence critical for Pttg1 transcriptional activation (17).

View larger version (8K): [in this window] [in a new window]   FIG. 1. Schematic illustration of mammalian PTTG1 gene structure. Exons are indicated as boxes (translated sequences in black and untranslated regions in white), whereas the connecting lines represent the introns. Exon and intron sizes (bp) are indicated. To simplify comparison between species, the translation initiation sites (ATG) have been set at +1. Major TSS are indicated relative to the ATG. The lower panel represents a scale from –1000 to +7000 bp. This combined information was obtained from original published sequences (1 17 18 23 25 ) and published sequences in the University of California, Santa Cruz, genome browser (http://genome.ucsc.edu/). It should be noted that, due to discrepancies in the information available from the aforementioned sources, the depicted sizes should be viewed as a guide.

View larger version (47K): [in this window] [in a new window]   FIG. 2. Sequence alignment of human, mouse, and rat PTTG1 5'-flanking region. 5'-Flanking region sequences of human (h), mouse (m), and rat (r) PTTG1 were obtained from GenBank (accession no. F200719, AF060887/AF071209, and AF021802, respectively). Nucleotide sequence alignment was obtained with MegaAlign (DNAStar, Madison, WI) by the clustal method. Differing nucleotides are highlighted.

The hPTTG1 gene is located on chromosome 5 [5q33 (18, 22), 5q35.1 (23)] spans about 10 kb and is composed of five exons and four introns. The exons are sized about 130, 185, 94, 160, and 140 bp, respectively (18, 23). The four introns are about 0.3, 1.2, 2.4, and 0.69 kb, respectively (18, 23). Recently, the existence of an additional exon (159 bp) was reported within the 5' untranslated region, followed by a 147-bp intron (24) (Fig. 1). Northern analysis using a specific hPTTG1 probe reveals a single approximately 1.3-kb transcript (18, 20); however in human fetal liver the detected size is approximately 0.8 kb (18). The major transcription start site localizes at an adenine residue at 37 bases, and a second transcription initiation site (TSS) 317 bp upstream of the ATG translation start site (23, 24). Serial deletions of an active approximately 1.3 kb upstream region showed sequences –126 to +34 bp sufficient for hPTTG1 promoter activity and that the sequence between nucleotide –706 and –407 contains an enhancer element (23).

By testing several pairs of degenerate primers designed from the rat Pttg1 cDNA sequence, one pair produced a prominent RT-PCR band in murine cell lines (25). Cloning and sequencing revealed that PCR products comprised identical sequences and contained an entire coding region (GenBank accession no. AF069051), which at the cDNA level showed 88 and 78% identity to rat and human PTTG1 cDNA, respectively. The entire murine Pttg1 gene spans about 7 kb and comprises five exons of 391, 179, 94, 150, and 131 bp, respectively, and four introns of 0.9, 2.1, 2, and 0.8 kb, respectively (25) (Fig. 1). As determined by Northern analysis, the major mRNA transcript is sized approximately 1 kb; in several samples, such as whole embryo, heart, and F9 and AtT20 cells, an approximately 1.7 kb band was detected, whereas in whole embryo and embryonic F9 cells, a third, approximately 3.0 kb band was also detected, indicating the existence of alternatively spliced variants (25). Indeed, a murine Pttg1 cDNA variant has been identified (GenBank accession no. AF071209), which encodes a shorter Pttg1 with differences in the C-terminal tail and altered activity (26). A single transcription start site was localized 303 bp upstream of the Pttg1 ATG translation start site (25), and serial deletions of an active 4.3 kb upstream region showed sequences –313 to –150 bp to be critical for promoter activity. Three elements (A-C) within this region contribute to promoter activity, with element A (–313/–293) and particularly subregion –305 to –293 bp, most critical for murine Pttg1 promoter activity (25).

B. PTTG1 mRNA expression profile
Initially, studies of rat Pttg1 mRNA expression using Northern blotting revealed detectable levels exclusively in adult testis and embryonic liver (1). Subsequently, examination of rat Pttg1 mRNA in testicular cell lines, showed high levels of Leydig, Sertoli, as well as germ (GC2) cell expression (17). In murine tissues, however, detectable mRNA levels (by Northern blot) were reported in thymus, spleen, testis, and ovary, as well as intact embryo, but not in adult liver, heart, lung, brain, and kidney (25). The discrepancy between rat and mouse tissue is likely due to differences in Northern blot sensitivity.

In normal adult human tissues (Fig. 3), abundant PTTG1 mRNA expression is evident in the testis (18, 19, 20). Strong expression is also observed in thymus, and weak colon, small intestine, placenta, and spleen expression are seen (18, 19). In some cases, weak signals have been reported in brain, pancreas (18), and lung (19), whereas expression has not been detected in leukocytes, heart, liver, skeletal muscle, kidney (18, 19), or ovary (18).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Tissue distribution of hPTTG1 mRNA, as determined by Northern blot. a, Normal human adult tissues. b, Normal human fetal tissues. c, Human cancer cells, left to right: promyelocytic leukemia HL-60; HeLa cell S3; chronic myelogenous leukemia K-562; lymphoblastic leukemia MOLT-4; Burkitt’s lymphoma Raji; colorectal adenocarcinoma SW480; lung carcinoma A549; melanoma G361. d, Pituitary tumors: NF, nonfunctioning tumor; PRL, PRL-secreting tumor; ACTH, ACTH-secreting tumor. [Reproduced with permission from Zhang et al. (18 ). Copyright 1999, The Endocrine Society.]

PTTG1 is abundantly expressed in several human cancer cell lines, including hematopoetic cell lines [promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenous leukemia K-562 (18, 21), lymphoblastic leukemia MOLT-4, and Burkitt’s lymphoma Raji (18)], colorectal adenocarcinoma SW480 (18, 21), lung carcinoma (A549), melanoma (G361) (18), hepatoma (HepG2) (21), breast tumor (MCF-7), endometrium (HEC-101), and ovarian tumor cell lines (CaO4, PA1, SKOV3, and VOI101) (20).

In addition to high PTTG1 mRNA levels reported in most pituitary tumors (3, 27, 28, 29), PTTG1 overexpression has been confirmed in colorectal (10) and esophageal (30) cancer, and hepatocellular carcinoma (31), testicular, ovarian, and breast tumors (6), in uterine leiomyomas (7), thyroid (11) and lung cancer (32).

C. Regulation of gene expression
Estrogen has been shown to be an important regulator of pituitary Pttg1 mRNA expression (33, 34, 35) (Fig. 4). Estrogen treatment of rat pituitary somato-lactotroph GH3 cells dose-dependently induced Pttg1 mRNA expression at 12 h, peaking at 24 h (33). In vivo, estrogen administration to Fischer 344 rats by osmotic minipump increased Pttg1 mRNA levels within 24 h, peaked at 48 h, and remained elevated throughout the 14-wk treatment period (33). In vivo estrogen-induced Pttg1 was blocked by antiestrogen coinfusion (34).

View larger version (56K): [in this window] [in a new window]   FIG. 4. Estrogen and Pttg1. a, Representative normal rat pituitary (NI) and rat pituitary tumor (E2). b, Serum PRL and pituitary wet weight. OVx, Ovariectomized controls. *, P < 0.05; **, P < 0.01. c, Northern blot analysis of pituitary tissue extracts derived from estrogen-treated rats. ß-Actin was used as loading control. M, marker lane. d and e, Reticulin fiber staining of rat anterior pituitary tissue at 24 h (d) and 1 wk (e) (broken circle) after initiation of estrogen treatment. f and g, Reticulin stain (arrows) (f) and hematoxylin and eosin stain (g) of rat anterior pituitary tissue 4 wk after commencement of estrogen infusion. Widespread vacuolation, vascular lakes (g, arrow), nuclear pleomorphism, and frequent mitosis (g, arrowhead) are visible. h, FGF-2 immunoreactivity after 4 wk of estrogen treatment. Original magnification, x200. [Reproduced with permission from S. Melmed (130 ). Copyright 2003, The American Society for Clinical Investigation.]

Insulin and IGF-I activate PTTG1 mRNA transcription in breast (MCF-7) tumor cells and malignant astrocytes, mediated in both cases by phosphotidylinositol 3-kinase (PI3K) and MAPK signaling (8, 38). In U87 astrocytoma cells, PTTG1 mRNA expression was also induced by the epidermal growth factor (EGF) receptor ligands, EGF and TGF, and hepatocyte growth factor (HGF) (37).

In most of these models it was unclear, however, whether induction of PTTG1 transcription occurred as a direct effect of the specific treatment agent or secondary to induced proliferation and associated activation of the cell cycle machinery. Using pituitary folliculostellate cells (TtT/GF), induction of Pttg1 mRNA levels by EGF required cells to progress through the cell cycle, because blockade of cells in early S phase suppressed EGF-mediated Pttg1 induction (39). PI3K, protein kinase C and MAPK pathways signal for EGF-induced Pttg1 expression in folliculostellate cells, and signaling suppression of Pttg1 was associated with inability of cells to proceed through S phase (39).

Indeed, PTTG1, as a mammalian securin protein (2), exhibits a cell cycle-dependent expression pattern (2) and is subject to ubiquitin-mediated degradation at the end of metaphase (2, 40) (see Section III.B). Pttg1 mRNA levels are also cell cycle-dependent; Pttg1 mRNA increases during S phase and peaks at the S-G₂ transition to decline thereafter (39, 41) (Fig. 5). Transcription factors Sp1 and, to a lesser extent, nuclear factor-Y are important for basal transcription activity of the PTTG1 promoter (24, 42), particularly in cancer cells. The nuclear factor-Y site appears to be essential for DNA damage-induced and p53-mediated inhibition of PTTG1 transcription (42). Although the ß-catenin/T cell factor (TCF) pathway has been implicated in regulation of hPTTG1 expression in esophageal and colorectal cancer (43, 44), mechanisms controlling cell cycle-dependent PTTG1 transcription and patterns of dysregulation in transformed cells remain to be elucidated.

View larger version (38K): [in this window] [in a new window]   FIG. 5. Cell cycle-dependent Pttg1 mRNA expression. a, Time-dependent effects on Pttg1 mRNA expression. Pituitary folliculostellate TtT/GF cells were serum-starved for 20 h and subsequently treated with EGF (5 nM) for indicated times. b, Effect of early S phase blockade on Pttg1 mRNA expression. TtT/GF cells were serum-starved for 20 h and subsequently treated with aphidicolin for 1 h before stimulation with EGF (5 nM). a and b, Pttg1 mRNA expression was determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PBF and PCNA, respectively (middle panels). The ratios of Pttg1 and PCNA mRNA vs. ß-actin mRNA were calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio or PCNA/ß-actin ratio of the control group was set as 1.0. Relative mRNA expression levels of other groups were normalized to these control groups. Relative Pttg1 or PCNA mRNA expression (mean ± SEM) of three independently performed experiments (upper panel). *, P < 0.05; **, P < 0.01; ***, P < 0.001. At the indicated times, cells were fixed, and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/G1 phase is depicted in black bars, cells in S phase in white bars, and cells in the G2/M phase of the cell cycle in gray bars (lower panel). [Reproduced with permission from Vlotides et al. (39 ). Copyright 1999, The Endocrine Society.]

	III. PTTG Protein

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

Figure 6 is a schematic representation of mammalian PTTG1 protein, comprising an N-terminal regulatory domain and a C-terminal functional domain (19, 45).

View larger version (6K): [in this window] [in a new window]   FIG. 6. Schematic illustration of mammalian PTTG1 protein. Mammalian PTTG1 protein comprises a mainly regulatory N-terminal domain (left panel) and functional C-terminal domain (right panel). Regulatory and functional domains, as well as SH3-binding sites and important serine amino acid residues are indicated. Positions of the elements are shown by amino acid residue numbers. Italics indicate information obtained from rodent Pttg1 protein data.

View larger version (32K): [in this window] [in a new window]   FIG. 7. Sequence alignment of vertebrate securins. The hSecurin (hSec) sequence is identical to that of hPTTG1. The sequences of mouse (mSec) and rat securin (rSec) were obtained from GenBank (accession no. AF069051 and U73030, respectively) (xSec, Xenopus securin). Amino acid sequence alignment was obtained with MegaAlign (DNAStar) by the clustal method. Identical or conserved residues are shaded in black. The conserved D box is boxed. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. [Reproduced with permission from Zou et al. (2 ). Copyright 1999, American Association for the Advancement of Science.]

C-terminal PXXP motifs are critical for hPTTG1 action (18, 46, 47). Similar to its human counterpart, mutations of C-terminal key amino acids (P¹³⁹, S¹⁵⁹) or in the PPXP (P¹⁵⁷-P-S-P¹⁶⁰) motif in murine Pttg1 disrupt its functionality (26). Interestingly, a murine Pttg1 188-amino acid variant containing a different C-terminal tail (GKGVRSNGCKDLVT) is devoid of primary functions of Pttg1, and postulated as an endogenous competitor to wild-type Pttg1 (26). Studies with rodent Pttg1 have identified DNA-binding (approximately from residue 60 to 118) (48) and transactivating domains (approximately from residue 119 to 164), important for Pttg1 transcriptional activity (49).

The N terminus contains a destruction (D) box (RKALGTVN; amino acid residues 61 to 68), which is conserved in human, rat, and mouse PTTG1, as well as a KEN or KDN box in human or mouse and rat, respectively (2), both involved in PTTG1 degradation (see Section III.C).

B. PTTG1 protein subcellular localization and complex formation
Reported differences in cytoplasmic vs. nuclear localization may be due to different cellular systems and techniques employed; although the physiological role of cytoplasmic PTTG1 expression remains unclear, nuclear localization of PTTG1 is consistent with the biological activity of PTTG1 as a transcriptional activator and securin protein.

hPTTG1 localizes both to the cytoplasm and nucleus (3, 19, 27, 41, 50, 51); however, the ratio of cytoplasmic vs. nuclear localization remains controversial. Endogenous Jurkat cell hPTTG1, as determined by subcellular fractionation, was reported to be mainly cytoplasmic (85%) (19). Predominantly cytoplasmic PTTG1 expression was observed by in situ hybridization (3) and immunohistochemistry (27) in a large series of human pituitary adenomas (27). Nuclear PTTG1 staining was detected in some tumor cells, whereas PTTG1 was not detected in normal pituitary tissue (27). In the same study, 14 of 18 breast and 9 of 10 lung adenocarcinomas showed mainly cytoplasmic immunoreactivity; however, in these more aggressive tumors, stained nuclei were also frequently detected (27). Similarly, predominantly cytoplasmic localization was reported in HCT116 cells transfected with enhanced green fluorescent protein (EGFP)-tagged PTTG1 (51). However, JEG-3 cells, with low constitutive PTTG1 expression, transfected with three different PTTG1 constructs (wild-type PTTG1, a FLAG epitope-tagged PTTG1, or a PTTG1-EGFP construct), all demonstrated predominantly nuclear PTTG1 localization during interphase (41). Interestingly, in some cells (<5%) PTTG1 also localized to the plasma membrane (41). Predominantly nuclear expression was observed regardless of the respective expression level of PTTG1-EGFP and was confirmed in a variety of other cell lines (3T3 murine fibroblast, rat GH3 and mouse AtT20 pituitary tumor, SKOV-3 human ovarian cancer, COS-7 monkey kidney cells). In the same study, live imaging of PTTG1-EGFP during mitosis revealed colocalization with microtubule asters in prophase and prometaphase, aggregation into distinct granules during anaphase, and diminished telophase expression (41). Mu et al. (52) reported cell type-dependent PTTG1 subcellular distribution, which was predominantly nuclear in HeLa, Cos-7, and DU145 cells, but diffuse nuclear and cytoplasmic localization in A549, DLD-1, and NIH3T3 cells.

Although the PTTG1 molecule does not possess an obvious nuclear localization signal (NLS), it may enter the nucleus due to its small molecular size. However, PTTG-binding factor (PBF) was identified, which facilitates PTTG1 nuclear translocation (50). PBF is a 22-kD protein ubiquitously expressed in various tissues and contains a bipartite NLS between amino acids 149 and 166 at the C terminus (50). COS-7 cells transfected with a hemagglutinin-tagged full-length PBF construct exhibited mainly nuclear expression, whereas a NLS deletion mutant yielded a predominantly perinuclear and cytoplasmic pattern (50). In vitro glutathione-S-transferase fusion protein binding assays and immunoprecipitation studies showed that PTTG1 specifically interacts with PBF and that the region between the C-terminal amino acids 123 and 154 of PTTG1, as well as the C-terminal region of PBF, are essential for this interaction (50). The mainly cytoplasmic PTTG1-EGFP localization (overexpression of PTTG1 alone) became predominantly nuclear with simultaneous PBF overexpression (50). Furthermore, PBF was shown to be important for PTTG1 transcriptional activity (50). However, in HCT116 cells transfected with PTTG1-EGFP, which was primarily cytoplasmic, cotransfection with hemagglutinin-tagged PBF did not result in different subcellular localization, and colocalization of PTTG1 and PBF was mainly cytoplasmic (51). In addition to the PTTG1 interaction with PBF, activation of the MAPK cascade was proposed as another mechanism for PTTG1 nuclear translocation and required the presence of MAPK phosphorylation and MAPK kinase 1 (MEK1) interaction sites (49).

C. PTTG1 protein posttranscriptional modification
1. PTTG1 phosphorylation.
PTTG1 sometimes migrates as a doublet, possibly due to cell cycle-dependent phosphorylation (1, 2, 13, 53) particularly during mitosis (53) (Fig. 8). Cdc2 is responsible for PTTG1 phosphorylation, mainly at S¹⁶⁵, which is also conserved in rat and mouse Pttg1 (53). Because this site is located between the two C-terminal proline-rich motifs, important for PTTG1 transcriptional activity, PTTG1 phosphorylation may be required for its functional activity (53). In rat Pttg1, S¹⁶² is phosphorylated by MAPK, and this site plays a critical role in Pttg1 transactivation function (49). Furthermore, at least six potential motifs (S/T-Q or P-S/T) for DNA-dependent protein kinase (DNA-PK) phosphorylation have been identified in hPTTG1, and in vitro, the catalytic subunit of the DNA-PK complex does indeed phosphorylate hPTTG1. The exact location as well as significance of DNA-PK-mediated phosphorylation remains to be elucidated, because it had no demonstrable effect on hPTTG1-Ku-70 association (13).

View larger version (15K): [in this window] [in a new window]   FIG. 8. PTTG1 phosphorylation during mitosis. HeLa cell PTTG1 protein expression determined by Western blot arrested at the indicated cell cycle phases. Shown is one representative Western blot (lower panel). The level of hPTTG1 was adjusted relative to the amount of an unrelated noncycling protein that was detected with a polyclonal serum (data not shown). The highest level, observed in M phase, was set at 100%. Shown are relative values of three independent experiments (upper panel). [Adapted by permission from Macmillan Publishers Ltd.: Oncogene (53 ). Copyright 2000.]

PTTG1 degradation ensues as a result of ubiquitination by anaphase-promoting complex (APC) (40), a ubiquitin ligase complex that contains several different subunits and acts to ubiquitinate several cell cycle proteins (57). In contrast to budding yeast pds1, hPTTG1 degradation is catalyzed by both fzy (fizzy/cdc20) and fzr (fizzy-related/cdh1/hct1) proteins (40). hPTTG1 degradation is both D box and KEN box-dependent (40), in contrast to Xenopus securin, where D box mutations alone prevent degradation (2). Mutations of the KEN or D box alone did not, or only partially, inhibited hPTTG1 degradation, respectively (40), whereas double mutations of the KEN box and the D box abolished PTTG1 degradation (40).

	IV. PTTG Family Members

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
A. Cloning and characterization of PTTG2 and -3
Evidence for the existence of more than one hPTTG isoform was reported in 1999 (22); PCR of genomic DNA led to the identification of an intronless homolog of hPTTG1, mapped to chromosome 4p12 and termed hPTTG2 (22). Sequence analysis (GenBank accession no. AF116538) revealed 94% nucleotide homology to hPTTG1 cDNA and 91% identity at the amino acid level (22). A subsequent study of hPTTG family members confirmed the existence of hPTTG2 (GenBank accession no. AF200719), which mapped to chromosome 4p15.1 by fluorescence in situ hybridization (58). hPTTG2 contains a 606-bp coding sequence, encoding a 202-amino acid protein, with a 91% identity to hPTTG1 at the amino acid level (58). hPTTG2 also contains two conserved motifs at the C terminus (58).

The observation of multiple polymorphisms in a region of hPTTG2 in the normal thymus suggested the existence of yet a third PTTG isoform (22). Indeed, Chen et al. (58) confirmed the existence of a second intronless homolog of hPTTG1, which was termed hPTTG3 and mapped to chromosome 8q13.1. hPTTG3 also contains a 606-bp coding sequence and encodes a 202-amino acid protein with 89 and 84% identity to hPTTG1 and hPTTG2, respectively (58). Similar to hPTTG1 and -2, hPTTG3 contains two conserved proline-rich motifs at the C terminus (58).

B. PTTG family member expression profile
With the use of specific probes, which did not appear to cross-hybridize between the two family members, Northern analysis revealed low-level hPTTG2 mRNA expression in spleen, prostate, testis, ovary, small intestine, and colon, but not in the thymus or peripheral blood leukocytes (58). With the use of more sensitive RT-PCR, hPTTG2 mRNA expression was detected in pituitary and pituitary tumors (22), liver and liver tumors (58), testis and testicular tumor, and ovary and ovarian tumor cell lines (22, 58) (Table 1).

	V. PTTG1: Physiological Functions

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
A. Securin function/replication
During metaphase, replicated paired sister chromatids are held together by the cohesin complex, a process essential for chromosome biorientation on mitotic spindles (59). This multisubunit "cohesin" requires activity of four proteins, i.e., Smc1, Smc3, Scc1, and Scc3 (extensively reviewed by Nasmyth in Ref. 60 and Yanagida in Ref. 61), and several variants of a large cohesin ring model entrapping sister chromatids have been proposed (60, 61, 62, 63, 64). In contrast to yeast, two distinct processes induce cohesin dissociation from chromosomes during mitosis in mammalian cells. The "prophase pathway" begins during prophase and involves phosphorylation of Scc3-like subunits by the Polo-like kinase PLK1 (65, 66). The second process takes place at the onset of anaphase and involves cleavage of Scc1 by a protease termed separase (as in yeast) (67). Separases are, in most species, large proteins (150–230 kDa), initially described as Esp1 in budding yeast (68) and Cut1 in fission yeast (69). A C-terminal (ca. 50 kDa) "separase domain" is highly conserved and harbors the proteolytic site (70, 71), whereas the N-terminal regions are not obviously conserved between species and likely harbor sites important for cellular trafficking of the protein and its interaction with securin (72, 73, 74, 75).

During most of the cell cycle, securin binds separase and thus inhibits its proteolytic activity (67, 74, 76, 77), in addition to inhibition of vertebrate but not yeast separase by Cdk1/cyclin B protein kinase-dependent phosphorylation (78, 79). At the metaphase to anaphase transition, once chromosome biorientation is complete, securin is targeted for proteasomal degradation by the ubiquitin ligase APC (see Section III.C); securin destruction releases tonic separase inhibition, which in turn mediates degradation of the cohesin complex (80), and equal separation of sister chromatids proceeds to diploid daughter cells. Interestingly, experiments in yeast have shown both an activating and inhibitory role of securin on separase, suggesting a dual mechanism of separase regulation (76). Binding of securin to separase (both N- and C-terminal binding sites) not only blocks interaction of separase with its substrates, but also prevents the N terminus from interacting with and possibly inducing an activating conformational change at the C-terminal active site, thus inhibiting separase activity (76). On the other hand, securin is important for nuclear accumulation of (inactive) separase and ensures that separase gains full proteolytic activity in anaphase after its own destruction (76).

In addition to PTTG1 securin function, recent reports indicate a role for PTTG1 in regulation of the G₁/S phase transition (81). PTTG1 interacts with the transcription factor Sp1, and a PTTG1-Sp1 complex colocalizes on the cyclin D3 (CCND3) promoter in JEG-3 and HCT116 cells. Suppression of Sp1 or PTTG1 [small interfering RNA (siRNA)] resulted in attenuated G₁/S transition in JEG3 cells, with increased G₁ and decreased S phase (81). Cotransfection of Sp1 siRNA and PTTG1 plasmid or PTTG1 siRNA and Sp1 plasmid reversed the effect of the other, indicating a coordinate role of PTTG1 and Sp1 on modulation of G₁/S phase transition (81). Sp1 transfection induced CCND3 mRNA and protein expression, whereas cotransfection of Sp1 and PTTG1 siRNA neutralized CCND3 induction. Furthermore, using p21 –/– HCT116 cells, PTTG1-mediated G₁/S phase transition was shown to be p21-independent (81).

B. DNA damage/repair
In budding yeast, securin is required to prevent anaphase in response to spindle and DNA damage (82, 83). Securin (Pds1) is directly phosphorylated by Chk1 (83), a conserved kinase critical in the DNA checkpoint pathways (84). Because DNA damage had no effect on APC activity (83), it was suggested that Chk1-dependent securin phosphorylation may increase its stability and thus block cell cycle progression. In fission yeast, securin (Cut2) is essential for proper repair of DNA damaged by UV, x-ray, and -ray irradiation, possibly by removal of local cohesin in interphase cells by the securin/separase complex (85).

In mammalian cells, hPTTG1 binds to Ku-70, the regulatory subunit of the DNA-PK, an enzyme involved in repairing DNA double-strand breaks (13). DNA-PKs phosphorylate hPTTG1 in vitro, and the hPTTG1-Ku association is disrupted by genome damaging events, such as DNA double-strand breaks, implicating a role for hPTTG1 in the linkage of DNA damage-response pathways with sister chromatid segregation, delaying the onset of mitosis while DNA repair proceeds (13). hPTTG1 is required for cell proliferation arrest after UV treatment (86). However, unlike in yeast cells, x-rays and UV light rapidly reduce hPTTG1 protein levels in mammalian cells, mediated by specific protein synthesis inhibition as well as proteasome-dependent degradation (86). Treatment of human cancer cell lines with DNA-damaging drugs doxorubicin and bleomycin induces p53-dependent suppression of hPTTG1 expression (42). hPTTG1 overexpression induced genetic instability in colorectal cancer cells, mediated at least in part by inhibition of DNA damage repair activity and repressed Ku heterodimer function (87). Thus, PTTG1-mediated inhibition of Ku-70 activity may be critical in DNA-damage repair regulation and induction of genetic instability in this human cancer model.

C. Interaction partners and transactivation activity
With the use of the yeast two-hybrid system in testicular cells, Pttg1 was shown to specifically interact with a novel human homolog of the bacterial heat-shock protein DnaJ (HSJ2) and with the ribosomal large subunit protein S10 (88). Pttg1 may therefore be targeted to the ribosome through interaction with S10 and association with HSJ2 required for subsequent dissociation of the Pttg1-S10 complex (88). However, this hypothesis and its potential significance in testicular physiology has not yet been further explored (for overview of major PTTG1 interaction partners, see Table 2).

Pttg1 contains a consensus MAPK phosphorylation site (P-X-S/T-P) within the transactivation domain and is phosphorylated by MAPK at S¹⁶² in vitro, a site critical for Pttg1 transactivation function (49). Furthermore, Pttg1 directly interacts with MEK1 through the N-terminal SH3 domain-binding motif (located between amino acids 51 and 54), and this interaction is required to mediate effects of MAPK on Pttg1 transactivation activity (49).

By generating cell lines with tightly regulated inducible Pttg1 expression and profiling using DNA arrays (investigation of 84 genes), five genes showed increased expression (>2-fold) after Pttg1 induction. These included c-myc oncogene, protein kinase C ß-1, MEK1, MEK3, and heat shock protein HSP70 (48). Detailed examination of the effect of Pttg1 on c-myc expression revealed that Pttg1 activates c-myc transcription in transfected cells and that Pttg1 binds to the c-myc promoter near the TSS in a protein complex containing the upstream stimulatory factor (USF1) (48). In this study, a DNA binding region was identified from amino acid residue 60 to 118, and Pttg1 DNA binding is necessary for its transcriptional activity (48).

A well-established target of PTTG1 is the mitogenic and angiogenic factor fibroblast growth factor (FGF)-2. Both PTTG1 and FGF-2 are overexpressed in pituitary (3, 29, 33), thyroid (11), ovarian tumors (6), and uterine leiomyoma (7). In vitro experiments have shown FGF-2 mRNA induction by PTTG1 overexpression in several cell types, including NIH3T3 cells (18, 90), primary thyroid cells (47), human fetal neuronal NT2 cells, JEG-3 and MCF-7 cells (46). In primary thyroid and PTTG1-null cell lines, transactivation of FGF-2 by hPTTG1 was not affected by PTTG1 phosphorylation but was dependent on the integrity of C-terminal PXXP motifs (47). Furthermore, direct transcriptional control of FGF-2 by PTTG1 has been reported (50). COS-7 cells transiently transfected with the FGF-2 promoter linked to luciferase were cotransfected with PTTG1 and/or PBF (50). Coexpression of PBF and PTTG1 significantly enhanced reporter gene activity, indicating the requirement of PBF for activation of FGF-2 transcription by PTTG1 (50).

In addition to FGF-2 regulation, hPTTG1, and particularly its C terminus (amino acids 147–202), regulates pituitary hormone expression (91). Pituitary lacto-somatotroph GH3 cells transfected with wild-type PTTG1 C-terminus expression vector decreased (10-fold) prolactin (PRL) mRNA expression and PRL hormone secretion, compared with cells overexpressing a mutated PTTG1 C-terminus SH3-binding motif (91). In contrast to PRL, GH mRNA expression and GH secretion in GH3 cells was enhanced (4-fold) (91). Direct effects of PTTG1 C terminus on PRL gene transcription were confirmed, because cells overexpressing wild-type PTTG1 C terminus demonstrated approximately 10-fold decreased PRL promoter-driven luciferase activity compared with mutant transfectants (91).

Recently, ChIP-on-chip technology was used to survey PTTG1 action on 20,000 genes (81). Of 700 gene promoters significantly enriched by immunoprecipitation with PTTG1 antibody, about 400 were identified and categorized into three major functional groups, involved in cell cycle, metabolic control, or signal transduction pathways (81). The use of an algorithm designed to specifically unravel transcriptional patterns yielded by the ChIP-on-chip results revealed enriched transcription factor binding sites in PTTG1-targeted genes, with a significant overrepresentation of Sp1 (81). PTTG1 was shown to physically interact with the Sp1 complex, and the PTTG1-Sp1 complex was shown to regulate CCND3 expression and G₁/S phase transition (see Section V.A). These results support a role for PTTG1 in transcriptional regulation of genes involved in a variety of cellular processes.

	VI. PTTG1: Tumorigenic Mechanisms

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
A. PTTG1 and cell proliferation
The effect of PTTG1 on cell proliferation as tested in transfection studies remains unclear. Because it is an oncogenic protein, one would expect a pro-proliferative effect; on the other hand, as a securin protein, which normally inhibits cell division unless degraded, high PTTG1 expression levels would be expected to inhibit cell proliferation. Indeed, results using cell lines overexpressing PTTG1 have been inconclusive. Stable transfection of NIH3T3 cells with rat Pttg1 (1) or HeLa and A549 with hPTTG1 (52) inhibited cell proliferation, as determined by nonradioactive cell proliferation assays. Cell cycle analysis of JEG-3, MG-63, or H1299 cells overexpressing EGFP-tagged or pcDNA3-PTTG1 revealed blockade of the cell cycle and accumulation of cells in G₂M (41, 92, 93). On the other hand, NIH3T3 cells (20) and human embryonic kidney (HEK) 293 cells (94) stably transfected with hPTTG1 exhibited increased cell proliferation rates compared with control vector-transfected cells. In HeLa S3 cells engineered to exhibit tetracycline-regulated PTTG1 expression, induction of PTTG1 also stimulated cell growth (48).

The observed differences could be ascribed to different cellular systems tested, varying PTTG1 expression levels depending on transfection efficiency or other factors that may interfere with PTTG1 physiology, such as the ability of a particular cell to translocate to the nucleus or degrade high levels of exogenous PTTG1 expression.

Targeting endogenous PTTG1 expression might constitute a more physiological approach for understanding PTTG1 regulation; however, very low levels of PTTG1 may also inhibit cell cycle progression due to the dual effect of PTTG1 on separase regulation and the requirement of PTTG1 abundance for nuclear accumulation and separase activation (76). Inhibition of cell proliferation by hPTTG1 siRNA has been reported in U87 glioma cells (37), HeLa S3 (95), and SH-J1 hepatoma cells (31).

The impact of transfected PTTG1 expression levels on cell proliferation was demonstrated in NT-2 cells. Relatively low PTTG1 up-regulation (1.7-fold) stimulated cell proliferation, whereas high PTTG1 up-regulation (6-fold) inhibited cell turnover (96).

Interestingly, the phosphorylation status of PTTG1 is also involved in pro- or antiproliferative effect of PTTG1 overexpression. The use of stable NIH3T3 cells overexpressing mutants preventing Pttg1 phosphorylation (Phos–) or those mimicking constitutive phosphorylation of Pttg1 (phos+) revealed that cells expressing Phos– demonstrated increased, and Phos+ decreased, proliferation compared with wild-type Pttg1 (47). Thus, the ability of a particular cell system for posttranslational modification (e.g., phosphorylation) of exogenous PTTG1 may influence the outcome of PTTG1 overexpression on cell proliferation.

The significance of PTTG1 regulation of cell cycle control gene promoters and the impact on cell proliferation is currently under active investigation.

B. Cell transformation and aneuploidy
In contrast to the effect of PTTG1 overexpression on cell proliferation, the ability of PTTG1 to transform cells is well established both in vitro and in vivo. As assessed by anchorage-independent growth in soft agar, PTTG1-transfected NIH3T3 and HEK293 cells formed much larger colonies compared with control vector-transfected cells (1, 18, 47, 94). In HeLa S3 cells engineered to exhibit tetracycline-regulated PTTG1 expression, induction of PTTG1 with tetracycline withdrawal led to 2-fold increased colony formation (48). Consistent with these results, H1299 lung carcinoma cells targeted with hPTTG1 siRNA formed smaller colonies in soft agar (9). In vivo, sc injection of PTTG1-transfected NIH3T3 or HEK293 cells into nude mice led to formation of large tumors within 2 to 4 wk (1, 18, 20, 94).

Several lines of evidence suggest that an important transforming mechanism underlying PTTG1 overexpression is the induction of chromosomal instability and aneuploidy.

p53-deficient MG-63 osteosarcoma cells transiently or stably transfected with PTTG1-EGFP were observed for signs of aneuploidy, such as the presence of micronuclei, macronuclei, or chromosomal bridges (92). Indeed, 34% of transiently transfected cells (1% in control cells) and 10% of stably transfected cells (5% in control cells) showed signs of aneuploidy (92). Live imaging of single human lung cancer H1299 cells, which are p53 deficient and exhibit undetectable levels of endogenous PTTG1 expression, transfected with EGFP-tagged PTTG1, revealed that high PTTG1-EGFP levels blocked progression of mitosis to anaphase (97). In all PTTG1-EGFP-expressing cells that underwent apparent normal mitosis, PTTG1-EGFP was degraded about 1 min before anaphase onset, consistent with the securin function of PTTG1 (97). Cells that failed to degrade PTTG1-EGFP demonstrated asymmetrical cytokinesis without chromosome segregation, or chromosome decondensation without cytokinesis, resulting in the appearance of macronuclei (97). Most cells expressing a nondegradable PTTG1 mutant exhibited asymmetrical cytokinesis without chromosome segregation, whereas few cells had decondensed chromosomes, and both resulted in formation of macronuclei (97). Thus, this study performed in single live human cells, demonstrated that PTTG1 accumulation (overexpression or failure of degradation) inhibits mitosis progression and chromosome segregation, but does not directly affect cytokinesis, resulting in aneuploidy.

The importance of hPTTG1 for chromosomal stability was confirmed in human colorectal cancer HCT116 cells (98), a cell line with a stable karyotype and intact DNA damage and mitotic spindle checkpoints (99, 100). hPTTG1 inactivation by homologous recombination in HCT116 cells resulted in frequent chromosome loss, linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts at chromosomal segregation (98). Abnormal mitosis due to lack of PTTG1 activity was associated with biochemical defects in separin activation and reduced cohesin subunit Scc1 cleavage efficiency (98).

Interestingly, with the use of a similar technique to inactivate hPTTG1 in HCT116 cells, chromosome loss was observed to be transient (101). Although approximately 60% of metaphase spreads indeed showed loss of at least one chromosome during the first three passages, the rate of chromosome loss progressively decreased, and by passage 12 karyotyping of HCT116 cells was identical to parental cells (101). However, PTTG1 loss-associated reduction of separase activity and cleavage of the cohesin subunit Scc1 persisted. hPTTG1-null HCT116 cells were chromosomally stable and mitosis was normal, suggesting additional compensatory mechanisms for chromosome segregation in human cells (101).

C. Apoptosis
In a study of placental JEG-3 cells, most cells overexpressing hPTTG1 underwent apoptosis (41). With the use of MCF-7 breast cancer cells expressing wild-type p53 and p53-deficient MG-63 osteosarcoma cells, Yu et al. (92) demonstrated that PTTG1 overexpression may cause both p53-dependent and p53-independent apoptosis. PTTG1 induced p53 in MCF-7 cells (92, 102) and its translocation to the nucleus (92). Furthermore, induction of p53 promoter activity by PTTG1 was shown to be mediated through regulation of c-myc, which then interacted with the p53 promoter (102). PTTG1 overexpression also stimulated Bax (encodes a pro-apoptotic member of the BCL2 gene family), a known downstream target of p53 (102). The nature of the p53-independent mechanism remains unclear. Thus, PTTG1-induced apoptosis may be a protective mechanism for clearing cells becoming aneuploid. When both apoptotic systems fail, PTTG1-induced aneuploid cells may continue to divide, thereby supporting the transformed phenotype.

In contrast, Bernal et al. (93) reported that hPTTG1 specifically interacts with p53 in vitro and in vivo and that this interaction blocks specific binding of p53 to DNA and inhibits its transcriptional activity (93). Interestingly, Bax promoter activity was diminished by the p53-securin interaction, leading to decreased apoptosis. In hPTTG1-deficient human tumor cells, both apoptotic and transactivating functions of p53 were potentiated (93). In addition, overexpressed PTTG1 in hepatoma cell lines attenuated p53 induction of apoptosis (31). Thus, these results propose a tumorigenic mechanism for PTTG1, because inhibition of p53-mediated apoptosis by high securin expression could explain the survival of tumor cells harboring functional p53 (93, 103).

The discrepancy between findings in these studies may depend on transfection systems used, because the amount of PTTG1 protein expressed in the study of Bernal et al. (93) was relatively low compared with expression levels in other studies (92, 102). Thus, induction of p53 promoter activity and associated apoptosis induction may require a threshold intracellular PTTG1 protein level.

D. Tumorigenic microenvironment
The microenvironment surrounding transformed cells is permissive for accelerated growth, which may be altered by paracrine/autocrine feedback mechanisms (104, 105, 106). In this context, an important element of tumor growth is angiogenesis. In colon cancers, PTTG1 expression and tumor vascularity correlate strongly (10), and in vivo pituitary FGF-2 and vascular endothelial growth factor (VEGF)-A induction coincides with estrogen-stimulated PTTG1 expression and angiogenesis (33). Induction of angiogenesis by PTTG1 was demonstrated in vitro by activation of proliferation, migration, and tube formation of human umbilical vein endothelial cells treated with conditioned medium derived from NIH3T3 cells overexpressing hPTTG1 (90). In vivo, concentrated hPTTG1-conditioned medium induced chick chorioallantoic membrane spoke-wheel-like appearances, and in both cases, FGF-2 antibody suppressed PTTG1-mediated angiogenic induction (90). Indeed, overexpression of PTTG1 induces FGF-2 mRNA and/or protein in NIH3T3 (18, 47, 90), HEK293 (94), NT-2 (46, 96), JEG-3, MCF-7 (46), and uterine leiomyoma cells (7). In some cases, PTTG1 overexpression also induces VEGF-A, another potent angiogenic growth factor, in uterine leiomyoma cells (7), FTC133 thyroid cells (107), NT-2, JEG-3, and MCF-7 cells (46), and induces VEGF-A as well as IL-8 in HEK293 cells (94). A recent study with angiogenesis-specific cDNA arrays after PTTG1 transfection in thyroid cells revealed regulation of multiple downstream angiogenic genes, including inhibitor of DNA binding-3 (ID3) and thrombospondin-1 (TSP-1) (108). Although the regulatory mechanism of PTTG1-mediated growth factor induction, including that of VEGF-A, has not yet been elucidated, PTTG1 in complex with its binding factor PBF has been shown to act as a direct transcriptional activator of FGF-2 (50) (see Section V.C).

Thus, overexpressed PTTG1 may act as a paracrine/autocrine activator, while overexpressed, enhancing expression of growth factors that in turn further sustain tumor growth and contribute to the tumorigenic microenvironment. The stimulatory effect of growth factors such as FGF-2 or EGF on PTTG1 expression per se (see Section II.C) further enhances this positive autofeedback mechanism.

	VII. Pttg1-Null Mouse Model

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
Although loss of yeast Pds1p or Drosophila securin pimples is lethal (74, 109), disruption of murine Pttg1 by homologous recombination resulted in a viable and fertile phenotype with tissue-specific defects, including pituitary, testis, and pancreatic ß-cell hypoplasia (14, 15). Pttg1 deficiency did not enhance intrauterine mortality, but Pttg1 –/– mice demonstrated female subfertility with a reduced average litter size compared with Pttg1 –/+ or Pttg1 +/+ (14). Pttg1 –/– mice exhibited testicular and splenic hypoplasia, thymic hyperplasia, and thrombocytopenia (14). Testicular hypoplasia was more pronounced in sexually mature mice, whereas splenic hypoplasia was observed after weaning and continued for up to 8 months of observation (14). Thymic hyperplasia was apparent approximately 4 wk after birth and persisted for up to 8 months, but it was more pronounced at an early age (14). Furthermore, the distribution of CD4+CD8+, CD4+CD8–, and CD4–CD8– thymocytes differed in Pttg1 –/– mice, and hematological analysis revealed the presence of thrombocytopenia, despite normal bone marrow megakaryocyte numbers (14).

Although Pttg1 –/– and Pttg1 +/+ mouse embryonic fibroblasts (MEFs) exhibited similar doubling times (30 h), their cell cycle parameters differed markedly, because Pttg1 –/– MEFs had a shortened G₁ and prolonged G₂M phase, an effect partially reversed by introduction of Pttg1 into Pttg1 –/– MEFs via retroviral transfection (14). Pttg1 –/– MEFs demonstrated damaged nuclei and aberrant chromosome morphology, such as quadric-radials, tri-radials and breaks, binucleated or multinucleated nuclei, and enhanced signs of aneuploidy and premature centromere division. These abnormalities in Pttg1 –/– MEFs were not lethal for these cells (14).

This Pttg1 –/– mouse also revealed an important role for Pttg1 in pancreatic ß-cell function (15). Pttg1 disruption impaired glucose homeostasis and led to male-selective diabetes developing during late adulthood (15). Development of diabetes was associated with nonautoimmune islet damage, insulinopenia, and reversed -/ß-cell ratio (15). Reduction of islet ß-cell mass was evident before diabetes manifestation, and islet cell proliferation, as determined by 5-bromo-2'-deoxyuridine incorporation, was reduced. Pttg1 –/– ß-cells had pleiotropic nuclei, suggesting defects in cell division (15). The sex steroid milieu determined diabetes rescue in gonadectomized and estrogen-treated Pttg1 –/– male mice (110). Although gonadectomy and estradiol therapy had no direct observable effect on hypoplastic ß-cell morphology, altered sex steroid milieu prevented diabetes development by increasing insulin sensitivity, possibly mediated by serum adiponectin elevation (110).

The involvement of Pttg1 in diabetes is further supported by a recent application of a novel network-based approach elucidating genetic networks underlying complex traits in an extensive murine whole brain expression study (16). Application of these techniques identified and experimentally validated gene expression traits predicted to respond to a strong expression quantitative trait locus for Pttg1, and Pttg1 brain expression was linked to metabolic traits, including obesity and diabetes (16).

Mice bearing a single retinoblastoma (Rb) mutant allele develop pituitary tumors with almost complete penetrance (111, 112, 113). To examine the role of PTTG1 depletion on pituitary tumor development in Rb +/– animals, compound Rb X Pttg1 mutant mice were generated (114). This study confirmed spleen, pancreas, and testis hypoplasia in Pttg1-depleted mice and also showed decreased pituitary weights (114) in single Pttg1 mutants as well as Rb+/–Pttg1–/– mice (114). Hypoplastic pituitary glands exhibited decreased cell proliferation and induction of p21 expression in both Pttg1-depleted genotypes (114) (see Section VIII.A).

Mechanisms for the protective effect of Pttg1 deletion on pituitary tumorigenesis may include accelerated gland senescence (115), associated with elevated p21 levels.

Despite aberrant chromosome morphology observed in Pttg1 –/– cells, the nonlethal phenotype suggests the existence of additional compensatory mechanisms for sister chromatid separation in mammalian cells. Furthermore, tissue-specific phenotypic responses in Pttg1 –/– mice suggest a differential role of PTTG1 in different tissues and cell type-specific suppression of cell growth, with particular impact on slow-growing endocrine cells. The gender-specific effect of PTTG1 disruption on pancreatic ß-cells supports the role of additional factors, such as sex steroids, in PTTG1-target tissue regulation.

	VIII. PTTG1 and Cancer

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
A. Endocrine-related cancer
1. Pituitary.
Increased PTTG1 mRNA in pituitary tumor tissue has been reported in several studies (3, 18, 20, 27, 28, 29, 33, 34, 46).

Although Northern analysis and in situ hybridization initially failed to detect PTTG1 expression in the normal pituitary (1, 3, 18), PTTG1 is indeed expressed in normal pituitary tissue at low levels (3, 27) and displays an estrous cycle-dependent expression pattern (34).

Examination of PTTG1 mRNA expression in 54 pituitary tumors by RT-PCR revealed more than 50% increased expression in 21 of 30 nonfunctioning, all 13 GH, nine of 10 PRL, and one ACTH-secreting tumor, and in some cases, more than 10-fold increases were reported (3, 27) (Fig. 9A and Table 3). PTTG1 expression was higher in hormone-secreting tumors that had invaded the sphenoid bone compared with tumors confined to the pituitary fossa (3). McCabe et al. (29) examined PTTG1 mRNA expression in 10 normal and 111 tumorous pituitaries and reported an approximately 5-fold increase overall, and Western blot analysis showed that PTTG1 protein expression was consistent with the mRNA findings (Fig. 9B and Table 3). In this study, PBF mRNA expression was increased in all pituitary tumor subtypes (5.7-fold increase) but particularly in nonfunctioning pituitary tumors (29). A significant correlation between PTTG1 and PBF expression was observed in pituitary tumors, but not in normal pituitary glands (29).

View larger version (15K): [in this window] [in a new window]   FIG. 9. PTTG1 expression in pituitary tumor subtypes. A, Normalized PTTG1 vs. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression is shown as individual values for each tumor, and mean expression within each tumor subtype group is shown by the horizontal line. NFPA, Nonfunctioning pituitary adenoma (n = 18); somatotroph (n = 12); corticotroph (n = 5); lactotroph (n = 5). Differences between means were analyzed by ANOVA. [Reproduced with permission from Hunter et al. (28 ). Copyright 2003, Society of the European Journal of Endocrinology.] B, Representative Western blot analysis of PTTG1 protein expression in normal pituitary (NP) and nonfunctioning pituitary tumors (T). M, Size marker; +, JEG-3 cell extract (positive control). [Reproduced with permission from McCabe et al. (29 ). Copyright 2003, Blackwell Publishing.]

To explore further the role of PTTG1 on pituitary tumor development, mice heterozygous for Rb deletion, which develop pituitary tumors with high penetrance (111, 112, 113), were crossbred with Pttg1-null mice (114). Similar decreases in pituitary weight were observed in compound Rb+/–Pttg1–/– and single Pttg1–/– deficient animals (114). In the pretumorous pituitary gland, Pttg1 deletion resulted in decreased cell proliferation, as determined by 5-bromo-2'-deoxyuridine incorporation and Ki67 staining and increased expression of p21 mRNA and protein (114). Up-regulation of Pttg1 in the pretumorous Rb+/–Pttg1+/+ pituitary gland was confirmed and Pttg1 deletion was shown to suppress and delay pituitary tumor development in Rb+/– mice and prolong survival (114). On the other hand, pituitary-directed transgenic PTTG1 overexpression driven by the -subunit of glycoprotein hormone (GSU) promoter (GSU.PTTG1), led to early multipotential pituitary focal hyperplasia (117). Males presented with plurihormonal focal pituitary transgene expression with LH, TSH, and unexpectedly GH-cell focal hyperplasia and adenoma and pituitary enlargement confirmed by magnetic resonance imaging at 9–12 months (117). These morphological changes were associated with increased serum LH, GH, testosterone, and/or IGF-I levels and profound peripheral growth stimulatory effects on the prostate and urinary tract (117). In fact, urinary obstruction caused by prostate and seminal vesicle hyperplasia, with renal tract inflammation, was the cause of premature death in some animals (117). Confocal microscopy of monotransgenic GSU.PTTG1 and particularly bitransgenic GSU.PTTG1xRb+/– (GSU.PTTG1 mice crossbred with Rb+/– mice) pituitaries revealed nuclear enlargement and marked redistribution of chromatin, and electron microscopy showed enlarged gonadotrophs with prominent Golgi complex and numerous secretory granules (118). Magnetic resonance imaging studies revealed that pituitaries derived from GSU.PTTG1xRb+/– mice were the largest as early as 2 months of age, and the incidence of anterior (but not intermediate) lobe tumors increased 3.5-fold compared with Rb+/– mice (118). These results suggest that PTTG1 content correlates with pituitary gland plasticity and tumor formation potential, and PTTG1 overexpression facilitates pituitary tumor development (Fig. 10).

View larger version (13K): [in this window] [in a new window]   FIG. 10. Pituitary PTTG1 content correlates with gland plasticity and tumor formation potential. On the left side of the inverted triangle are listed mouse models with descending pituitary PTTG1 content, with or without the combination with tumorigenic Rb+/–. Horizontal bars represent observed effects of the different genotypes on pituitary trophic status, which correlates with pituitary tumorigenic potential (arrow). [Reproduced with permission from Donangelo et al. (118 ). Copyright 2006, The Endocrine Society.]

2. Thyroid.
Northern blot revealed increased PTTG1 mRNA levels in human thyroid tumors, compared with normal thyroid tissue (4). Increased PTTG1 expression was evident early in thyroid tumor development and was most abundantly expressed in a subset of thyroid hyperplasia, follicular adenomas, and follicular carcinomas, whereas only modest increases were observed in papillary cancers (4). FRTL5 thyroid cells overexpressing wild-type Pttg1 exhibited increased in vitro transforming activity compared with vector- or mutant-Pttg1 (mutation of potential C-terminal SH3-biding motif) transfectants (4). Furthermore, overexpression of Pttg1 in FRTL5 cells increased cell proliferation and was associated with a dedifferentiated phenotype as demonstrated by proliferating cell nuclear antigen (PCNA) elevation and decreased thyroglobulin expression (4). Stable Pttg1 overexpression also reduced iodide uptake in FRTL5 cells and to a lesser extent in normal human thyroid cells in vitro, associated with reduced expression of the sodium/iodide symporter (4).

Indeed, increased PTTG1 and FGF-2 mRNA and protein expression were observed in 27 differentiated thyroid cancers (DTC), followed by a nonsignificant increase in hyperplastic states, such as multinodular goiters (n = 25) and Graves’ disease (n = 13), whereas no increase was observed in normal thyroid tissue (n = 11) (11). PCNA levels did not differ between groups, demonstrating that PTTG1 expression was independent of proliferative activity in the samples examined (11). Interestingly, in this study, PTTG1 levels did not differ between papillary and follicular carcinomas (11). FGF-R1 mRNA, but not protein, was also increased in DTC (11). Multiple regression analysis confirmed independent associations of FGF-2 mRNA and lymph node involvement as well as distant metastasis, and association between PTTG1 mRNA and early tumor recurrence (11). Similarly, analysis of hPTTG1 expression by immunostaining on paraffin-embedded tissues from a large cohort of patients that underwent surgical resection for DTC (n = 60) revealed low and high PTTG1 expression in 35 and 65% of cases, respectively, whereas adjacent nonneoplastic tissue was largely unstained (120). PTTG1 expression was associated with the presence of nodal or distant metastasis, TNM stage, and decreased radioiodine uptake during follow-up, and expression levels of PTTG1 were confirmed as an independent prognostic factor for persistent disease (120).

An important mechanism for thyroid tumorigenesis may involve PTTG1-mediated induction of angiogenesis. FRTL5 thyroid cells overexpressing wild-type Pttg1 exhibited increased FGF-2 expression and decreased iodide uptake after FGF-2 treatment, suggesting involvement of autocrine/paracrine FGF-2 in PTTG1-mediated neoplastic phenotype in thyroid cells (4). In addition to FGF-2, elevated VEGF receptor KDR mRNA expression and KDR phosphorylation, as well as ID3 (important in VEGF-dependent angiogenesis) and VEGF mRNA expression in thyroid tumors were reported (107). Induction of both KDR and VEGF-A expression by PTTG1 overexpression in FTC133 cells suggests the presence of a PTTG1-regulated VEGF-KDR-ID3-dependent autocrine pathway in thyroid cells (107). Evidence for this pathway is supported by angiogenesis-specific cDNA array studies performed in PTTG1-transfected thyroid cells, showing regulation of downstream angiogenic genes in thyroid cancer, such as ID3 and TSP-1 (108). Pttg1 is overexpressed in a mouse model that spontaneously develops follicular thyroid carcinoma with metastasis (TR^ßPV/PV mouse), and crossbreeding of TR^ßPV/PV mice with Pttg1-null mice (Pttg1 –/–) revealed that Pttg1 functions as a contributor (decreased vascular invasion and lung metastasis), but not initiator (no difference in the incidence of hyperplasia and capsular invasion) of tumorigenesis (121). A mechanism proposed for regulation of thyroid growth was p21-mediated modulation of the Rb-E2F pathway, suggested by p21 elevation and decreased Rb phosphorylation in Pttg1-depleted TR^ßPV/PV mice. Pttg1-depleted TR^ßPV/PV mice also exhibited decreased FGF-2 and FGFR1 expression (121).

An additional mechanism for PTTG1-mediated thyroid tumorigenesis may be induction of genetic instability, as determined by fluorescent intersimple sequence repeat PCR (122). In vitro, transfection of FTC133 cells with PTTG1 dose-dependently increased genetic instability (122). Higher genomic instability index was observed particularly in follicular, but also in papillary thyroid tumors, compared with normal tissue, and the overall degree of genetic instability in thyroid cancers correlated with PTTG1 expression (122).

Recently, PBF was also shown to have transforming potential with respect to thyroid tumorigenesis (51). PBF mRNA expression was higher in differentiated thyroid carcinomas than in normal thyroid tissue and was independently associated with tumor recurrence (51). In vitro, PTTG1 overexpression up-regulated PBF mRNA expression, and stable overexpression of PBF in NIH3T3 cells resulted in significant colony formation. In vivo transformation was confirmed by stable sc overexpression of PBF in athymic nude mice (51). Thus, PTTG1-mediated PBF induction may further enhance the transforming activity of PTTG1.

3. Breast.
hPTTG1 is overexpressed in breast tumor cell lines (20, 38), and growth factor (insulin and IGF-I)-mediated induction of PTTG1 transcription has been reported in MCF-7 cells, which involved PI3K and MAPK signaling (38). Analysis of PTTG1 mRNA expression in 72 breast cancer tumor samples revealed a direct correlation between PTTG1 mRNA and lymph node infiltration, and PTTG1 overexpression correlated with a higher degree of tumor recurrence within a 5-yr observation period (5). Immunostaining of PTTG1 in 90 breast tumors and 18 normal breast tissues also showed highest PTTG1 expression in cells derived from metastatic breast tumors and in invasive ductal carcinoma as well as increased PTTG1 mRNA expression, particularly in invasive vs. noninvasive tumors (123). Thus, PTTG1 expression was proposed as a potential marker of breast cancer aggressiveness (5, 123).

4. Uterus.
Enhanced PTTG1 and FGF-2 mRNA and protein expression in leiomyomas vs. matched pairs of normal myometrium (n = 23) occurred independently of the menstrual cycle but were associated with increased cell proliferation, as evidenced by PCNA elevation (7). PTTG1 expression in cultured leiomyoma cells was not affected by estrogen and progesterone, but was induced by FGF-2, which also increased cell proliferation (7). Transient transfection of leiomyoma cells with full-length hPTTG1 cDNA decreased cyclins A and D1 and increased cyclin B1 expression 24 h after transfection, which suggested accumulation of cells at the G₂ phase (7). PTTG1 overexpression also induced FGF-2 and VEGF-A mRNA expression, suggesting the existence of growth factor-PTTG1 autofeedback regulation in leiomyoma cells (7).

5. Ovary.
PTTG1 is overexpressed in several ovarian tumor cell lines (CaO4, PA1, SKOV3, and VOI101) (20). To determine differences in the primary structure of PTTG1 expressed in tumors, PTTG1 was cloned from several ovarian tumors by RT-PCR, but no loss of function mutations was observed (6). Increased PTTG1 mRNA expression was observed in epithelial, sex-cord, stromal cell, and germ cell tumors of the ovary and was also associated with increased expression of FGF-2 (6), whereas stable transfection of ovarian SKOV3 cancer cells with full-length antisense PTTG1 mRNA expression vector decreased FGF-2 protein as well as in vitro transforming activity (124).

B. Nonendocrine-related cancer
1. Central nervous system.
PTTG1 expression has been reported in murine fetal brain (125). In humans, PTTG1 expression is low in first and second trimester fetal cerebral cortex, whereas in adult cerebral cortex intense PTTG1 staining is observed in neuronal cell bodies associated with absent cell proliferation (96). FGF-2 demonstrated a similar expression pattern as PTTG1 and was induced by PTTG1 overexpression in fetal neural NT-2 cells (96). Low transient expression of PTTG1 increased cell proliferation, whereas high levels inhibited NT-2 cell proliferation, suggesting a potential role of PTTG1 in modulating both cell proliferation and FGF-2 expression in the developing human fetal brain (96).

PTTG1 is overexpressed in malignant astrocytomas and astrocytoma cell lines (U87MG, U138MG, LN405) compared with normal astrocytes (8, 37). Several growth factors, such as EGF, TGF, HGF, IGF-I, and insulin induce PTTG1 expression in human glioma cell lines (8, 37). In addition, PTTG1 down-regulation by RNA silencing inhibited U87 cell proliferation by approximately 50% (37), suggesting a role of PTTG1 in astrocytoma proliferation and as a marker of brain malignancy.

Interestingly, microarray analysis and RT-PCR in cerebellar neurons from Wld^S (Wallerian degeneration slow) mutant mice (expression of an Nmnat-1/truncated-Ube4b chimeric gene), which exhibit a neuroprotective phenotype with delayed axotomy-induced Wallerian degeneration, revealed the largest and most consistent changes induced by Wld^S protein expression in the levels of Pttg1 (down-regulation) and erythroid differentiation regulator 1-like gene (edr1l-EST) (up-regulation) (126). In these samples, approximately 10-fold decreased Pttg1 mRNA expression was observed, which was confirmed by in vitro transfection studies in HEK293 and neuronal NSC34 cells, and the Nmnat-1 component of the Wld^S gene was shown to be of particular importance for Pttg1 down-regulation (126). Thus, reduced Pttg1 may be involved in the neuroprotective effect of Wld^S gene expression. Mechanisms for Wld^S-mediated Pttg1 regulation and the impact on proliferative activity are currently being studied.

2. Lung.
PTTG1 is overexpressed in human lung cancer cell lines, as well as small cell lung cancer (SCLC) and non-SCLC (NSCLC) (9, 32, 52, 127). One study demonstrated p21-mediated inhibition of cell proliferation and surprisingly also in vitro transforming activity by PTTG1 overexpression in human lung cancer A549 cells (52). Discrepancies of the effect of PTTG1 overexpression on in vitro cell proliferation are well documented (see Section VI.A), however, this is the only report, to our knowledge, of inhibition of in vitro transforming activity by PTTG1. On the other hand, transfection of human H1299 lung carcinoma cells with PTTG1 siRNA duplex reduced in vitro and in vivo transforming activity, and PTTG1 silencing was associated with suppression of Ki67 and FGF-2 expression in H1299 tumors (9).

A total of 78 NSCLC lesions were sorted for a wide range of total (gain plus loss) chromosomal imbalance numbers, and analysis of PTTG1 mRNA expression in lesions with highest and lowest total chromosomal imbalance numbers revealed increased PTTG1 expression compared with normal lung tissue (127).

A more detailed examination of PTTG1 expression in lung cancer revealed PTTG1 expression in 64% of SCLC and 97.8% of NSCLC samples (32). Interestingly, this study demonstrated a differential role of PTTG1 in these two lung cancer types. In SCLC, negative or low PTTG1 expression was associated with a shorter mean survival time and was a significant predictor for survival (32). In contrast, in NSCLC elevated PTTG1 expression was associated with a shorter mean survival and was associated with a more aggressive phenotype (32). Thus, the role of PTTG1 may differ depending on the histological subtype of lung cancer, which might explain PTTG1-mediated inhibition of in vitro transforming activity in A549 tumor cells.

3. Liver.
PTTG1 is involved in the liver regeneration process (128) and is overexpressed in hepatoma cell lines (SH-J1, SK-Hep1, and Huh-7) (31). Increased PTTG1 mRNA and protein expression were shown in a large series of hepatocellular carcinomas (31). Down-regulation of PTTG1 expression in SH-J1 hepatoma cells by transfection with siRNA inhibited cell growth and induced p53 and p21 activity and apoptosis (31). Huh-7 cells transfected with PTTG1 siRNA exhibited decreased growth potential in nude mice, and intratumor delivery of PTTG1 siRNA suppressed SH-J1 tumor growth in nude mice (31).

4. Esophagus.
PTTG1 expression was enhanced in esophageal cancer (30), correlated with ß-catenin localization in primary esophagus squamous cell carcinoma tissues, and was regulated by the ß-catenin/TCF pathway in esophagus squamous cell carcinoma cell lines (43). Furthermore, PTTG1 mRNA was higher in esophageal tumors with higher pathological stage (IV vs. 0-III) or more extensive lymph node metastasis (N4 vs. N0–3). High PTTG1 expression was also associated with reduced mean survival rates (30).

5. Colon.
PTTG1 overexpression was reported in all of 48 colon carcinomas and in all but one colorectal polyp compared with normal colonic tissue (10). Increased PTTG1 expression correlated with higher tumor grade (Duke classification stage C and D vs. A and B) and invasion of surrounding lymph nodes and associated with a higher degree of colorectal carcinoma and adenoma vascularity. PTTG1 mRNA overexpression was not the result of gene amplification, as determined by Southern blot, and sequence analysis of the PTTG1 coding region in normal colon, and colorectal tumor tissue was identical to the wild-type sequence (10). Recently, Hlubek et al. (44) confirmed PTTG1 overexpression in colorectal tumors by DNA microarray analysis and RT-PCR. Similar to esophageal cancer, high PTTG1 protein expression correlated with aberrant nuclear ß-catenin expression in colorectal carcinoma and adenoma tissue. ß-Catenin was shown to bind to a TCF-binding PTTG1 promoter site and to induce promoter activity, whereas down-regulation of endogenous ß-catenin by siRNA transfection suppressed PTTG1 expression in HCT116, SW480, and DLD1 colon cancer cells (44). A potential mechanism for hPTTG1-mediated induction of genetic instability in colorectal cancer cells may be inhibition of double-stranded DNA damage repair pathway activation (87).

C. Potential therapeutic applications
Gene therapy techniques specifically targeting endogenous PTTG1 expression in tumors may serve as a useful investigative therapeutic tool. Disrupted PTTG1 action by introduction of PTTG1 carboxy-terminal peptide with dominant negative activity altered pituitary tumor cell hormonal phenotype and reduced in vivo transforming activity (91). Full-length antisense PTTG1 mRNA expression vector in ovarian carcinoma SK-OV-3 cells decreased PTTG1 expression and colony formation (124). siRNA-based techniques efficiently suppress endogenous PTTG1 and inhibit cell proliferation in human glioma (37, 129) and cervical cancer cells lines (95). siRNA-mediated PTTG1 suppression was associated with changes in PTTG1-target gene expression, such as decreased FGF-2 (124) and ID3 (108), and increased TSP-1 (108) expression. Adenovirus-mediated transfer of siRNA against PTTG1 SH-J1 hepatoma cells resulted in p53 activation, followed by p21 induction and apoptosis (31). PTTG1 depletion also reduced colony formation of lung H1299 cells (9) and in vivo transforming activity of H1299 (9) and Huh-7 hepatoma cells (31). Furthermore, intratumor delivery of adenoviral vector encoding PTTG1-siRNA inhibited tumor growth in SH-J1 tumor xenografts in nude mice (31). Thus, gene-based introduction of PTTG1-siRNA or dominant negative PTTG1 carboxy-terminal peptide (91) emerges as a promising subcellular therapeutic alternative, and efforts are under way to target tumor PTTG1 expression specifically, while bypassing endogenous PTTG1 in normal cells.

Characterization of PTTG1 mRNA regulation and signaling may also provide the basis for pharmacological treatments. Characterization of PTTG1 induction and activation of related intracellular signaling pathways by growth factors, such as FGF, EGF, HGF, or estrogen in particular cell systems, may allow pharmacological receptor blockade or inhibition of key intracellular molecules involved in PTTG1 regulation.

	IX. Summary

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 
The understanding of PTTG1 structure, regulation, and function has gained considerably since its discovery 9 yr ago. PTTG1 is a multifunctional protein, fundamentally involved in cell cycle regulation, DNA damage and repair mechanisms, direct transcriptional activity, organ development, and metabolism. Most importantly, PTTG1 is highly oncogenic, and its expression strongly correlates with neoplastic transformation. Increasing lines of evidence suggest potential clinically significant applications of PTTG1 physiology. These include increased PTTG1 tumor expression and correlation with aggressive phenotype or survival rate, facilitating use of PTTG1 as a marker of malignancy, with an impact on tumor staging and subsequent therapeutic interventions. Targeting PTTG1 expression (see Section VIII.C), has emerged as a promising subcellular therapeutic goal for pituitary and other slow-growing endocrine tumors.

	Footnotes

 
This work was supported by a scholarship from the Deutsche Forschungsgemeinschaft (VL 55/1-1), by National Institutes of Health Grants CA 075979 (to S.M.) and DK064169, and by the Endocrine Fellows Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 26, 2007

Abbreviations: APC, Anaphase-promoting complex; CCND3, cyclin D3; D box, destruction box; DNA-PK, DNA-dependent protein kinase; DTC, differentiated thyroid cancer; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; FGF, fibroblast growth factor; GSU, -subunit of glycoprotein hormone; HEK, human embryonic kidney; HGF, hepatocyte growth factor; hPTTG, human PTTG; ID3, inhibitor of DNA binding-3; KDR/FIK-1, kinase insert domain containing receptor/fetal liver kinase-1; MEF, mouse embryonic fibroblast; MEK, MAPK kinase; NLS, nuclear localization signal; NSCLC, non-SCLC; PBF, PTTG-binding factor; PCNA, proliferating cell nuclear antigen; PI3K, phosphotidylinositol 3-kinase; PRL, prolactin; PTTG, pituitary tumor-transforming gene; Rb, retinoblastoma; SCLC, small cell lung cancer; SH, Src-homology; siRNA, small interfering RNA; TCF, T cell factor; TSP-1, thrombospondin-1; TSS, transcription initiation site(s); VEGF, vascular endothelial growth factor; Wld^S, Wallerian degeneration slow.

	References

Top Abstract I. Introduction II. PTTG1 Gene Structure... III. PTTG Protein IV. PTTG Family Members V. PTTG1: Physiological... VI. PTTG1: Tumorigenic... VII. Pttg1-Null Mouse Model VIII. PTTG1 and Cancer IX. Summary References

 

1. Pei L, Melmed S 1997 Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Mol Endocrinol 11:433–441
2. Zou H, McGarry TJ, Bernal T, Kirschner MW 1999 Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 285:418–422
3. Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant TR, Bronstein MD, Melmed S 1999 Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas. J Clin Endocrinol Metab 84:761–767
4. Heaney AP, Nelson V, Fernando M, Horwitz G 2001 Transforming events in thyroid tumorigenesis and their association with follicular lesions. J Clin Endocrinol Metab 86:5025–5032
5. Solbach C, Roller M, Fellbaum C, Nicoletti M, Kaufmann M 2004 PTTG mRNA expression in primary breast cancer: a prognostic marker for lymph node invasion and tumor recurrence. Breast 13:80–81
6. Puri R, Tousson A, Chen L, Kakar SS 2001 Molecular cloning of pituitary tumor transforming gene 1 from ovarian tumors and its expression in tumors. Cancer Lett 163:131–139
7. Tsai SJ, Lin SJ, Cheng YM, Chen HM, Wing LY 2005 Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas. J Clin Endocrinol Metab [Erratum (2005) 90:5233] 90:3715–3723
8. Chamaon K, Kirches E, Kanakis D, Braeuninger S, Dietzmann K, Mawrin C 2005 Regulation of the pituitary tumor transforming gene by insulin-like-growth factor-I and insulin differs between malignant and non-neoplastic astrocytes. Biochem Biophys Res Commun 331:86–92
9. Kakar SS, Malik MT 2006 Suppression of lung cancer with siRNA targeting PTTG. Int J Oncol 29:387–395
10. Heaney AP, Singson R, McCabe CJ, Nelson V, Nakashima M, Melmed S 2000 Expression of pituitary-tumour transforming gene in colorectal tumours. Lancet 355:716–719
11. Boelaert K, McCabe CJ, Tannahill LA, Gittoes NJ, Holder RL, Watkinson JC, Bradwell AR, Sheppard MC, Franklyn JA 2003 Pituitary tumor transforming gene and fibroblast growth factor-2 expression: potential prognostic indicators in differentiated thyroid cancer. J Clin Endocrinol Metab 88:2341–2347
12. Ramaswamy S, Ross KN, Lander ES, Golub TR 2003 A molecular signature of metastasis in primary solid tumors. Nat Genet 33:49–54
13. Romero F, Multon MC, Ramos-Morales F, Dominguez A, Bernal JA, Pintor-Toro JA, Tortolero M 2001 Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase. Nucleic Acids Res 29:1300–1307
14. Wang Z, Yu R, Melmed S 2001 Mice lacking pituitary tumor transforming gene show testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle progression, and premature centromere division. Mol Endocrinol 15:1870–1879
15. Wang Z, Moro E, Kovacs K, Yu R, Melmed S 2003 Pituitary tumor transforming gene-null male mice exhibit impaired pancreatic ß-cell proliferation and diabetes. Proc Natl Acad Sci USA 100:3428–3432
16. Lum PY, Chen Y, Zhu J, Lamb J, Melmed S, Wang S, Drake TA, Lusis AJ, Schadt EE 2006 Elucidating the murine brain transcriptional network in a segregating mouse population to identify core functional modules for obesity and diabetes. J Neurochem 97(Suppl 1):50–62
17. Pei L 1998 Genomic organization and identification of an enhancer element containing binding sites for multiple proteins in rat pituitary tumor-transforming gene. J Biol Chem 273:5219–5225
18. Zhang X, Horwitz GA, Prezant TR, Valentini A, Nakashima M, Bronstein MD, Melmed S 1999 Structure, expression, and function of human pituitary tumor-transforming gene (PTTG). Mol Endocrinol 13:156–166
19. Dominguez A, Ramos-Morales F, Romero F, Rios RM, Dreyfus F, Tortolero M, Pintor-Toro JA 1998 hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG. Oncogene 17:2187–2193
20. Kakar SS, Jennes L 1999 Molecular cloning and characterization of the tumor transforming gene (TUTR1): a novel gene in human tumorigenesis. Cytogenet Cell Genet 84:211–216
21. Lee IA, Seong C, Choe IS 1999 Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene. Biochem Mol Biol Int 47:891–897
22. Prezant TR, Kadioglu P, Melmed S 1999 An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family. J Clin Endocrinol Metab 84:1149–1152
23. Kakar SS 1999 Molecular cloning, genomic organization, and identification of the promoter for the human pituitary tumor transforming gene (PTTG). Gene 240:317–324
24. Clem AL, Hamid T, Kakar SS 2003 Characterization of the role of Sp1 and NF-Y in differential regulation of PTTG/securin expression in tumor cells. Gene 322:113–121
25. Wang Z, Melmed S 2000 Characterization of the murine pituitary tumor transforming gene (PTTG) and its promoter. Endocrinology 141:763–771
26. Wang Z, Melmed S 2000 Pituitary tumor transforming gene (PTTG) transforming and transactivation activity. J Biol Chem 275:7459–7461
27. Saez C, Japon MA, Ramos-Morales F, Romero F, Segura DI, Tortolero M, Pintor-Toro JA 1999 hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias. Oncogene 18:5473–5476
28. Hunter JA, Skelly RH, Aylwin SJ, Geddes JF, Evanson J, Besser GM, Monson JP, Burrin JM 2003 The relationship between pituitary tumour transforming gene (PTTG) expression and in vitro hormone and vascular endothelial growth factor (VEGF) secretion from human pituitary adenomas. Eur J Endocrinol 148:203–211
29. McCabe CJ, Khaira JS, Boelaert K, Heaney AP, Tannahill LA, Hussain S, Mitchell R, Olliff J, Sheppard MC, Franklyn JA, Gittoes NJ 2003 Expression of pituitary tumour transforming gene (PTTG) and fibroblast growth factor-2 (FGF-2) in human pituitary adenomas: relationships to clinical tumour behaviour. Clin Endocrinol (Oxf) 58:141–150
30. Shibata Y, Haruki N, Kuwabara Y, Nishiwaki T, Kato J, Shinoda N, Sato A, Kimura M, Koyama H, Toyama T, Ishiguro H, Kudo J, Terashita Y, Konishi S, Fujii Y 2002 Expression of PTTG (pituitary tumor transforming gene) in esophageal cancer. Jpn J Clin Oncol 32:233–237
31. Cho-Rok J, Yoo J, Jang YJ, Kim S, Chu IS, Yeom YI, Choi JY, Im DS 2006 Adenovirus-mediated transfer of siRNA against PTTG1 inhibits liver cancer cell growth in vitro and in vivo. Hepatology 43:1042–1052
32. Rehfeld N, Geddert H, Atamna A, Rohrbeck A, Garcia G, Kliszewski S, Neukirchen J, Bruns I, Steidl U, Fenk R, Gabbert HE, Kronenwett R, Haas R, Rohr UP 2006 The influence of the pituitary tumor transforming gene-1 (PTTG-1) on survival of patients with small cell lung cancer and non-small cell lung cancer. J Carcinog 5:4
33. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S 1999 Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 5:1317–1321
34. Heaney AP, Fernando M, Melmed S 2002 Functional role of estrogen in pituitary tumor pathogenesis. J Clin Invest 109:277–283
35. Yin H, Fujimoto N, Maruyama S, Asano K 2001 Strain difference in regulation of pituitary tumor transforming gene (PTTG) in estrogen-induced pituitary tumorigenesis in rats. Jpn J Cancer Res 92:1034–1040
36. Tfelt-Hansen J, Schwarz P, Terwilliger EF, Brown EM, Chattopadhyay N 2003 Calcium-sensing receptor induces messenger ribonucleic acid of human securin, pituitary tumor transforming gene, in rat testicular cancer. Endocrinology 144:5188–5193
37. Tfelt-Hansen J, Yano S, Bandyopadhyay S, Carroll R, Brown EM, Chattopadhyay N 2004 Expression of pituitary tumor transforming gene (PTTG) and its binding protein in human astrocytes and astrocytoma cells: function and regulation of PTTG in U87 astrocytoma cells. Endocrinology 145:4222–4231
38. Thompson 3rd AD, Kakar SS 2005 Insulin and IGF-1 regulate the expression of the pituitary tumor transforming gene (PTTG) in breast tumor cells. FEBS Lett 579:3195–3200
39. Vlotides G, Cruz-Soto M, Rubinek T, Eigler T, Auernhammer CJ, Melmed S 2006 Mechanisms for growth factor-induced pituitary tumor transforming gene-1 (PTTG1) expression in pituitary folliculostellate TtT/GF cells. Mol Endocrinol 20:3321–3335
40. Zur A, Brandeis M 2001 Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis. EMBO J 20:792–801
41. Yu R, Ren SG, Horwitz GA, Wang Z, Melmed S 2000 Pituitary tumor transforming gene (PTTG) regulates placental JEG-3 cell division and survival: evidence from live cell imaging. Mol Endocrinol 14:1137–1146
42. Zhou Y, Mehta KR, Choi AP, Scolavino S, Zhang X 2003 DNA damage-induced inhibition of securin expression is mediated by p53. J Biol Chem 278:462–470
43. Zhou C, Liu S, Zhou X, Xue L, Quan L, Lu N, Zhang G, Bai J, Wang Y, Liu Z, Zhan Q, Zhu H, Xu N 2005 Overexpression of human pituitary tumor transforming gene (hPTTG) is regulated by ß-catenin/TCF pathway in human esophageal squamous cell carcinoma. Int J Cancer 113:891–898
44. Hlubek F, Pfeiffer S, Budczies J, Spaderna S, Jung A, Kirchner T, Brabletz T 2006 Securin (hPTTG1) expression is regulated by ß-catenin/TCF in human colorectal carcinoma. Br J Cancer 94:1672–1677
45. Yu R, Melmed S 2001 Oncogene activation in pituitary tumors. Brain Pathol 11:328–341
46. McCabe CJ, Boelaert K, Tannahill LA, Heaney AP, Stratford AL, Khaira JS, Hussain S, Sheppard MC, Franklyn JA, Gittoes NJ 2002 Vascular endothelial growth factor, its receptor KDR/Flk-1, and pituitary tumor transforming gene in pituitary tumors. J Clin Endocrinol Metab 87:4238–4244
47. Boelaert K, Yu R, Tannahill LA, Stratford AL, Khanim FL, Eggo MC, Moore JS, Young LS, Gittoes NJ, Franklyn JA, Melmed S, McCabe CJ 2004 PTTG’s C-terminal PXXP motifs modulate critical cellular processes in vitro. J Mol Endocrinol 33:663–677
48. Pei L 2001 Identification of c-myc as a down-stream target for pituitary tumor-transforming gene. J Biol Chem 276:8484–8491
49. Pei L 2000 Activation of mitogen-activated protein kinase cascade regulates pituitary tumor-transforming gene transactivation function. J Biol Chem 275:31191–31198
50. Chien W, Pei L 2000 A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product. J Biol Chem 275:19422–19427
51. Stratford AL, Boelaert K, Tannahill LA, Kim DS, Warfield A, Eggo MC, Gittoes NJ, Young LS, Franklyn JA, McCabe CJ 2005 Pituitary tumor transforming gene binding factor: a novel transforming gene in thyroid tumorigenesis. J Clin Endocrinol Metab 90:4341–4349
52. Mu YM, Oba K, Yanase T, Ito T, Ashida K, Goto K, Morinaga H, Ikuyama S, Takayanagi R, Nawata H 2003 Human pituitary tumor transforming gene (hPTTG) inhibits human lung cancer A549 cell growth through activation of p21(WAF1/CIP1). Endocr J 50:771–781
53. Ramos-Morales F, Dominguez A, Romero F, Luna R, Multon MC, Pintor-Toro JA, Tortolero M 2000 Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product. Oncogene 19:403–409
54. Cohen-Fix O, Peters JM, Kirschner MW, Koshland D 1996 Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev 10:3081–3093
55. Funabiki H, Yamano H, Kumada K, Nagao K, Hunt T, Yanagida M 1996 Cut2 proteolysis required for sister-chromatid separation in fission yeast. Nature 381:438–441
56. Leismann O, Herzig A, Heidmann S, Lehner CF 2000 Degradation of Drosophila PIM regulates sister chromatid separation during mitosis. Genes Dev 14:2192–2205
57. Nakayama KI, Nakayama K 2006 Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 6:369–381
58. Chen L, Puri R, Lefkowitz EJ, Kakar SS 2000 Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization. Gene 248:41–50
59. Tanaka T, Fuchs J, Loidl J, Nasmyth K 2000 Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation. Nat Cell Biol 2:492–499
60. Nasmyth K 2005 How might cohesin hold sister chromatids together? Philos Trans R Soc Lond B Biol Sci 360:483–496
61. Yanagida M 2005 Basic mechanism of eukaryotic chromosome segregation. Philos Trans R Soc Lond B Biol Sci 360:609–621
62. Gruber S, Haering CH, Nasmyth K 2003 Chromosomal cohesin forms a ring. Cell 112:765–777
63. Haering CH, Lowe J, Hochwagen A, Nasmyth K 2002 Molecular architecture of SMC proteins and the yeast cohesin complex. Mol Cell 9:773–788
64. Huang CE, Milutinovich M, Koshland D 2005 Rings, bracelet or snaps: fashionable alternatives for Smc complexes. Philos Trans R Soc Lond B Biol Sci 360:537–542
65. Sumara I, Vorlaufer E, Stukenberg PT, Kelm O, Redemann N, Nigg EA, Peters JM 2002 The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. Mol Cell 9:515–525
66. Hauf S, Roitinger E, Koch B, Dittrich CM, Mechtler K, Peters JM 2005 Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2. PLoS Biol 3:e69
67. Uhlmann F, Lottspeich F, Nasmyth K 1999 Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400:37–42
68. McGrew JT, Goetsch L, Byers B, Baum P 1992 Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae. Mol Biol Cell 3:1443–1454
69. Uzawa S, Samejima I, Hirano T, Tanaka K, Yanagida M 1990 The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene. Cell 62:913–925
70. Uhlmann F, Wernic D, Poupart MA, Koonin EV, Nasmyth K 2000 Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103:375–386
71. Aravind L, Koonin EV 2002 Classification of the caspase-hemoglobinase fold: detection of new families and implications for the origin of the eukaryotic separins. Proteins 46:355–367
72. Kumada K, Nakamura T, Nagao K, Funabiki H, Nakagawa T, Yanagida M 1998 Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis. Curr Biol 8:633–641
73. Funabiki H, Kumada K, Yanagida M 1996 Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes. EMBO J 15:6617–6628
74. Ciosk R, Zachariae W, Michaelis C, Shevchenko A, Mann M, Nasmyth K 1998 An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93:1067–1076
75. Jensen S, Segal M, Clarke DJ, Reed SI 2001 A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1. J Cell Biol 152:27–40
76. Hornig NC, Knowles PP, McDonald NQ, Uhlmann F 2002 The dual mechanism of separase regulation by securin. Curr Biol 12:973–982
77. Waizenegger I, Gimenez-Abian JF, Wernic D, Peters JM 2002 Regulation of human separase by securin binding and autocleavage. Curr Biol 12:1368–1378
78. Stemmann O, Zou H, Gerber SA, Gygi SP, Kirschner MW 2001 Dual inhibition of sister chromatid separation at metaphase. Cell 107:715–726
79. Nasmyth K 2005 How do so few control so many? Cell 120:739–746
80. Wirth KG, Wutz G, Kudo NR, Desdouets C, Zetterberg A, Taghybeeglu S, Seznec J, Ducos GM, Ricci R, Firnberg N, Peters JM, Nasmyth K 2006 Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression. J Cell Biol 172:847–860
81. Tong Y, Tan, Y, Zhou C, Melmed S, PTTG1 interacts with Sp1 to modulate G₁/S phase transition. Oncogene, in press
82. Yamamoto A, Guacci V, Koshland D 1996 Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J Cell Biol 133:99–110
83. Wang H, Liu D, Wang Y, Qin J, Elledge SJ 2001 Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function. Genes Dev 15:1361–1372
84. Sanchez Y, Bachant J, Wang H, Hu F, Liu D, Tetzlaff M, Elledge SJ 1999 Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 286:1166–1171
85. Nagao K, Adachi Y, Yanagida M 2004 Separase-mediated cleavage of cohesin at interphase is required for DNA repair. Nature 430:1044–1048
86. Romero F, Gil-Bernabe AM, Saez C, Japon MA, Pintor-Toro JA, Tortolero M 2004 Securin is a target of the UV response pathway in mammalian cells. Mol Cell Biol 24:2720–2733
87. Kim DS, Franklyn JA, Smith VE, Stratford AL, Pemberton HN, Warfield A, Watkinson JC, Ishmail T, Wakelam MJ, McCabe CJ, Securin induces genetic instability in colorectal cancer by inhibiting double-stranded DNA repair activity. Carcinogenesis, in press
88. Pei L 1999 Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells. J Biol Chem 274:3151–3158
89. Ptashne M 1988 How eukaryotic transcriptional activators work. Nature 335:683–689
90. Ishikawa H, Heaney AP, Yu R, Horwitz GA, Melmed S 2001 Human pituitary tumor-transforming gene induces angiogenesis. J Clin Endocrinol Metab 86:867–874
91. Horwitz GA, Miklovsky I, Heaney AP, Ren SG, Melmed S 2003 Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression. Mol Endocrinol 17:600–609
92. Yu R, Heaney AP, Lu W, Chen J, Melmed S 2000 Pituitary tumor transforming gene causes aneuploidy and p53-dependent and p53-independent apoptosis. J Biol Chem 275:36502–36505
93. Bernal JA, Luna R, Espina A, Lazaro I, Ramos-Morales F, Romero F, Arias C, Silva A, Tortolero M, Pintor-Toro JA 2002 Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis. Nat Genet 32:306–311
94. Hamid T, Malik MT, Kakar SS 2005 Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells. Mol Cancer 4:3
95. Solbach C, Roller M, Peters S, Nicoletti M, Kaufmann M, Knecht R 2005 Pituitary tumor-transforming gene (PTTG): a novel target for anti-tumor therapy. Anticancer Res 25:121–125
96. Boelaert K, Tannahill LA, Bulmer JN, Kachilele S, Chan SY, Kim D, Gittoes NJ, Franklyn JA, Kilby MD, McCabe CJ 2003 A potential role for PTTG/securin in the developing human fetal brain. FASEB J 17:1631–1639
97. Yu R, Lu W, Chen J, McCabe CJ, Melmed S 2003 Overexpressed pituitary tumor-transforming gene causes aneuploidy in live human cells. Endocrinology 144:4991–4998
98. Jallepalli PV, Waizenegger IC, Bunz F, Langer S, Speicher MR, Peters JM, Kinzler KW, Vogelstein B, Lengauer C 2001 Securin is required for chromosomal stability in human cells. Cell 105:445–457
99. Lengauer C, Kinzler KW, Vogelstein B 1997 Genetic instability in colorectal cancers. Nature 386:623–627
100. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B 1998 Requirement for p53 and p21 to sustain G₂ arrest after DNA damage. Science 282:1497–1501
101. Pfleghaar K, Heubes S, Cox J, Stemmann O, Speicher MR 2005 Securin is not required for chromosomal stability in human cells. PLoS Biol 3:e416
102. Hamid T, Kakar SS 2004 PTTG/securin activates expression of p53 and modulates its function. Mol Cancer 3:18
103. Rustgi AK 2002 Securin a new role for itself. Nat Genet 32:222–224
104. Bogdanos J, Karamanolakis D, Tenta R, Tsintavis A, Milathianakis C, Mitsiades C, Koutsilieris M 2003 Endocrine/paracrine/autocrine survival factor activity of bone microenvironment participates in the development of androgen ablation and chemotherapy refractoriness of prostate cancer metastasis in skeleton. Endocr Relat Cancer 10:279–289
105. Aguayo A, Giles F, Albitar M 2003 Vascularity, angiogenesis and angiogenic factors in leukemias and myelodysplastic syndromes. Leuk Lymphoma 44:213–222
106. Lazar-Molnar E, Hegyesi H, Toth S, Falus A 2000 Autocrine and paracrine regulation by cytokines and growth factors in melanoma. Cytokine 12:547–554
107. Kim DS, Buchanan MA, Stratford AL, Watkinson JC, Eggo MC, Franklyn JA, McCabe CJ 2006 PTTG promotes a novel VEGF-KDR-ID3 autocrine mitogenic pathway in thyroid cancer. Clin Otolaryngol 31:246
108. Kim DS, Franklyn JA, Stratford AL, Boelaert K, Watkinson JC, Eggo MC, McCabe CJ 2006 Pituitary tumor-transforming gene regulates multiple downstream angiogenic genes in thyroid cancer. J Clin Endocrinol Metab 91:1119–1128
109. Stratmann R, Lehner CF 1996 Separation of sister chromatids in mitosis requires the Drosophila pimples product, a protein degraded after the metaphase/anaphase transition. Cell 84:25–35
110. Fraenkel M, Caloyeras J, Ren SG, Melmed S 2006 Sex-steroid milieu determines diabetes rescue in pttg-null mice. J Endocrinol 189:519–528
111. Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA, Weinberg RA 1992 Effects of an Rb mutation in the mouse. Nature 359:295–300
112. Hu N, Gutsmann A, Herbert DC, Bradley A, Lee WH, Lee EY 1994 Heterozygous Rb-1 20/+mice are predisposed to tumors of the pituitary gland with a nearly complete penetrance. Oncogene 9:1021–1027
113. Classon M, Harlow E 2002 The retinoblastoma tumour suppressor in development and cancer. Nat Rev Cancer 2:910–917
114. Chesnokova V, Kovacs K, Castro AV, Zonis S, Melmed S 2005 Pituitary hypoplasia in Pttg–/– mice is protective for Rb+/– pituitary tumorigenesis. Mol Endocrinol 19:2371–2379
115. Chesnokova V, Zonis S, Kovacs K, Wawrowsky K, Melmed S, Mechanisms for pituitary senescence underlying pituitary cell proliferation. Program of the 88th Annual Meeting of The Endocrine Society, Boston, MA, 2006, p 129
116. Filippella M, Galland F, Kujas M, Young J, Faggiano A, Lombardi G, Colao A, Meduri G, Chanson P 2006 Pituitary tumour transforming gene (PTTG) expression correlates with the proliferative activity and recurrence status of pituitary adenomas: a clinical and immunohistochemical study. Clin Endocrinol (Oxf) 65:536–543
117. Abbud RA, Takumi I, Barker EM, Ren SG, Chen DY, Wawrowsky K, Melmed S 2005 Early multipotential pituitary focal hyperplasia in the -subunit of glycoprotein hormone-driven pituitary tumor-transforming gene transgenic mice. Mol Endocrinol 19:1383–1391
118. Donangelo I, Gutman S, Horvath E, Kovacs K, Wawrowsky K, Mount M, Melmed S 2006 PTTG over-expression facilitates pituitary tumor development. Endocrinology 147:4781–4791
119. Kanakis D, Kirches E, Mawrin C, Dietzmann K 2003 Promoter mutations are no major cause of PTTG overexpression in pituitary adenomas. Clin Endocrinol (Oxf) 58:151–155
120. Saez C, Martinez-Brocca MA, Castilla C, Soto A, Navarro E, Tortolero M, Pintor-Toro JA, Japon MA 2006 Prognostic significance of human pituitary tumor-transforming gene immunohistochemical expression in differentiated thyroid cancer. J Clin Endocrinol Metab 91:1404–1409
121. Kim CS, The pituitary tumor transforming gene (PTTG) is a contributor, but not an initiator of tumorigenesis in a mouse model of follicular thyroid carcinoma. Program of the 88th Annual Meeting of The Endocrine Society, Boston, MA, 2006, p 129
122. Kim D, Pemberton H, Stratford AL, Buelaert K, Watkinson JC, Lopes V, Franklyn JA, McCabe CJ 2005 Pituitary tumour transforming gene (PTTG) induces genetic instability in thyroid cells. Oncogene 24:4861–4866
123. Ogbagabriel S, Fernando M, Waldman FM, Bose S, Heaney AP 2005 Securin is overexpressed in breast cancer. Mod Pathol 18:985–990
124. Chen G, Li J, Li F, Li X, Zhou J, Lu Y, Ma D 2004 Inhibitory effects of anti-sense PTTG on malignant phenotype of human ovarian carcinoma cell line SK-OV-3. J Huazhong Univ Sci Technolog Med Sci 24:369–372
125. Tarabykin V, Britanova O, Fradkov A, Voss A, Katz LS, Lukyanov S, Gruss P 2000 Expression of PTTG and prc1 genes during telencephalic neurogenesis. Mech Dev 92:301–304
126. Gillingwater TH, Wishart TM, Chen PE, Haley JE, Robertson K, MacDonald SH, Middleton S, Wawrowski K, Shipston MJ, Melmed S, Wyllie DJ, Skehel PA, Coleman MP, Ribchester RR 2006 The neuroprotective WldS gene regulates expression of PTTG1 and erythroid differentiation regulator 1-like gene in mice and human cells. Hum Mol Genet 15:625–635
127. Honda S, Hayashi M, Kobayashi Y, Ishikawa Y, Nakagawa K, Tsuchiya E 2003 A role for the pituitary tumor-transforming gene in the genesis and progression of non-small cell lung carcinomas. Anticancer Res 23:3775–3782
128. Akino K, Akita S, Mizuguchi T, Takumi I, Yu R, Wang XY, Rozga J, Demetriou AA, Melmed S, Ohtsuru A, Yamashita S 2005 A novel molecular marker of pituitary tumor transforming gene involves in a rat liver regeneration. J Surg Res 129:142–146
129. Genkai N, Homma J, Sano M, Tanaka R, Yamanaka R 2006 Increased expression of pituitary tumor-transforming gene (PTTG)-1 is correlated with poor prognosis in glioma patients. Oncol Rep 15:1569–1574
130. Melmed S 2003 Mechanisms for pituitary tumorigenesis: the plastic pituitary. J Clin Invest 112:1603–1618

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