Tumor Transforming Gene Binding Factor Research Articles

Abstract 3616: Investigating proto-oncogene PBF-induced cell migration and invasion

Abstract The proto-oncogene pituitary tumor transforming gene binding factor (PTTG1IP/PBF) is overexpressed in multiple tumors and associated with tumor progression. PBF mediates several tumorigenic processes, including cell motility, whereby it potently induces cancer cell migration and invasion. PBF is phosphorylated by Src kinase at Y174 and mutation of this residue (Y174A) renders PBF unable to induce cell invasion, suggesting that phosphorylation mediates the stimulation of cancer cell motility by PBF. This study aimed to further elucidate the mechanism of PBF-induced cell motility. PBF-Y174 is also the key residue in a YXXΦ endocytosis motif. Both PBF phosphorylation and endocytosis were found to be essential for PBF-induced cell motility as mutants which disrupted either process were unable to stimulate thyroid and breast cancer cell migration and invasion. To elucidate molecular events downstream of PBF overexpression, phosphoproteomic and RNA-Seq analyses of normal thyroid cells (Nthy-ori 3-1) with stable PBF overexpression were performed and showed an enrichment in molecules involved in cell adhesion and cytoskeleton organisation. These findings prompted further investigation into a physiological role for PBF in cell motility. We utilised a novel Pbf knockout (Pbf−/−) mouse model generated through CRISPR/Cas9-mediated deletion of Pbf exon 4 in C57BL/6N mice to determine the impact of Pbf deletion on cellular mechanisms. Mouse embryonic fibroblasts (MEFs) were isolated at embryonic day 13.5 and used as primary cultures. Pbf−/- MEFs showed a significant reduction in migration and invasion compared with wild-type (Pbf+/+) MEFs. Interestingly, the loss of one functional copy of Pbf in heterozygote MEFs (Pbf+/−) resulted in an intermediate decrease in motility suggesting a gene-dosage effect. Initial immunofluorescent studies of Pbf−/− MEFs suggest alterations in focal adhesions (FAs). In Pbf+/+ MEFs focal adhesion kinase (FAK) and paxillin staining highlighted FA structures that were normally elongated and aligned with actin stress fibers. In contrast, Pbf−/- MEFs demonstrated a significant reduction in FAK and paxillin staining with smaller, punctate and more radially distributed FAs. These studies demonstrate a physiological role for PBF in cell adhesion and migration and further elucidate the mechanism by which PBF induces cell motility in tumor progression. Citation Format: Merve Kocbiyik, Selvambigai Manivannan, Ling Zha, Katie Brookes, Martin L Read, Chris J McCabe, Vicki E Smith. Investigating proto-oncogene PBF-induced cell migration and invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3616.

Pituitary tumor transforming gene binding factor (PBF) is required for androgen-induced prostate cancer proliferation and invasion.

Pituitary tumor transforming gene binding factor (PBF) is a proto-oncogene that plays a role in many cancers; however, its involvement in prostate cancer (PCa) remains unclear. Here, we examined PBF expression in clinical specimens and investigated its regulation and function in human PCa cell lines. Immunohistochemical staining of patient tissues revealed higher PBF expression in PCa than in benign prostatic hyperplasia or adjacent normal prostate specimens. In LNCaP and 22Rv1 cells, PBF expression was upregulated by androgen treatment in a manner partially blocked by the androgen receptor (AR) antagonist bicalutamide. We identified a novel androgen response element in the PBF gene promoter and demonstrated its functional relevance using luciferase reporter assays. Androgen treatment of LNCaP cells induced binding between the endogenous AR and the androgen response element in PBF, as measured by chromatin immunoprecipitation assays. Finally, RNA interference of PBF expression significantly reduced androgen-induced LNCaP cell growth and invasion. Thus, PBF is a novel AR target gene and plays a role in androgen-induced proliferation and metastatic functions in PCa cells.

Pituitary tumor transforming gene binding factor (PBF): a novel modulator of iodide uptake and target for Src phosphorylation in breast cancer

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Characterising the functional significance of the first reported mutations in the pituitary tumor-transforming gene binding factor

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract P6-03-13: Inhibition of Src increases radioiodide uptake in breast cancer cells by inhibiting phosphorylation of pituitary tumor transforming gene binding factor (PBF)

Abstract Although not detectable in normal breast tissue, the sodium iodide symporter (NIS) has been found to be expressed in 70-80% of breast cancers. However, the majority of NIS is intracellular, leaving only 20-30% functional at the plasma membrane. Whilst radioiodine therapy has been proposed as a potential treatment for breast cancer, effective therapy would require increased levels of membranous NIS localisation in tumours. Previous work revealed that overexpression of pituitary tumor transforming gene binding factor (PBF) in thyroid cells leads to the redistribution of NIS from the plasma membrane into intracellular vesicles, thereby reducing radioiodide uptake, a process modulated by Src phosphorylation of PBF. Here we show that PBF and NIS have a consistent relationship in breast cancer, with phosphorylation of PBF at residue Y174 being critical for the association. Immunofluorescent microscopy revealed co-localisation between NIS and PBF in co-transfected MDA-MB-231, MCF-7 and T47D cells, with increased intracellular staining for NIS compared to cells transfected with NIS alone. Phosphorylated PBF was also observed to co-localise with NIS in T47D cells. Treatment with PP1, a Src inhibitor which modulates the phosphorylation of PBF, led to increased NIS plasma membrane staining and less intracellular co-localisation with PBF. Functional studies in MCF-7 and MDA-MB-231 cells demonstrated that PBF significantly repressed radioiodide uptake in cells expressing exogenous NIS (25% and 30% reduction respectively; n=3, p<0.05). Treatment with PP1 restored the ability of MCF-7 and MDA-MB-231 cells to uptake I-125 (1.24 and 1.69 fold increase respectively; n=3, p<0.05). Combined all-trans retinoic acid (ATRA) and dexamethasone treatment has previously been shown to enhance NIS expression and radioiodide uptake in MCF-7 cells. Importantly, in the face of NIS-induction via ATRA and dexamethasone, PBF retained its ability to repress iodide uptake (31% reduction; n=3, p<0.05). This repression was overcome by PP1 treatment, which restored radioiodide uptake to vector only transfected levels (1.22-fold increase; p=ns, n=3). In keeping with PP1 data, the Src inhibitors saracatinib and dasatinib inhibited the phosphorylation of PBF in MCF-7 and MDA-MB-231 cells. Radioiodide uptake studies revealed that 1nM dasatinib was capable of restoring the ability of MDA-MB-231 cells to uptake I-125 (2 fold increase; n=3, p<0.05) when expressing exogenous NIS and PBF compared to vehicle only treated cells. 10nM saracatinib was also capable of restoring I-125 uptake but with less potency than dasatinib. Inhibition of focal adhesion kinase (FAK) with PF573228 failed to ameliorate PBF’s repression on I-125 uptake, suggesting that FAK is not responsible for PBF phosphorylation. Taken together, these data suggest that PBF alters the subcellular location of NIS and potently represses its activity in breast cancer cells. Further, tyrosine phosphorylation of PBF by Src modulates the ability of breast carcinoma cell-lines to take up radioiodide, with important implications for adapting NIS as a potential therapy in breast cancer. Citation Format: Vikki L Poole, Martin L Read, Rachel J Watkins, Bhavika Modasia, Waraporn Imruetaicharoenchoke, Kristien Boelaert, Vicki E Smith, Christopher J McCabe. Inhibition of Src increases radioiodide uptake in breast cancer cells by inhibiting phosphorylation of pituitary tumor transforming gene binding factor (PBF) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-13.

The first reported mutations in the pituitary tumor-transforming gene binding factor

The first reported mutations in the pituitary tumor-transforming gene binding factor

Manipulation of PBF/PTTG1IP Phosphorylation Status; a Potential New Therapeutic Strategy for Improving Radioiodine Uptake in Thyroid and Other Tumors

Context:The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of 131I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient.Objective:Pituitary tumor–transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors.Results:Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells.Conclusions:We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.

Abstract 638: PBF overexpression causes increased p53 ubiquitination and degradation via MDM2.

Abstract The pituitary tumor-transforming gene-binding factor (PBF) is a relatively uncharacterised proto-oncogene, which is overexpressed in thyroid tumours. PBF elicits tumor growth in nude mice, whilst thyroid targeted transgenic overexpression in the PBF-Tg mouse induces hyperplasia and macrofollicular lesions, accompanied by induction of the E2 ubiquitin ligase Rad6. Our previous unpublished data showed that PBF binds to p53, and reduces stimulation of downstream target genes by competitive binding. Further, half-life studies of p53 showed reduced p53 stability when PBF was overexpressed in K1 and TPC-1 thyroid papillary cancer cell lines, and ubiquitination assays confirmed this was due to increased ubiquitination and subsequent degradation by the proteasome. Now, GST pull-down assays demonstrate direct binding between PBF and MDM2, the principal negative regulator of p53. The competitive inhibitor of p53-MDM2 binding, Nutlin-3, revealed that the increased degradation of p53 observed when PBF was overexpressed was mediated by MDM2. No change in p53-MDM2 binding stringency was detected when PBF expression was ameliorated by siRNA treatment, and MDM2 subcellular localisation was unchanged by PBF overexpression. However, co-immunoprecipitation assays revealed that PBF specifically interacts with Rad6, which has previously been shown to regulate p53 ubiquitination. Further, PBF-Tg mice demonstrated significantly induced genetic instability at 6 weeks of age, as determined by FISSR-PCR. Thus we propose that aberrantly expressed PBF functionally inactivates p53 via a complex interplay between Rad6 and MDM2, thus promoting genetic instability and tumorigenesis. Our studies, carried out in a panel of thyroid and colorectal cells lines, suggest this may be pertinent not only to thyroid cancer, but to colorectal and other tumour types. Citation Format: Gavin Ryan, Robert Seed, Martin Read, Jim Fong, Andrew Turnell, Vicki Smith, Jayne Franklyn, Christopher McCabe, Kristien Boelaert. PBF overexpression causes increased p53 ubiquitination and degradation via MDM2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 638. doi:10.1158/1538-7445.AM2013-638

PTTG-Binding Factor (PBF) Is a Novel Regulator of the Thyroid Hormone Transporter MCT8

Within the basolateral membrane of thyroid follicular epithelial cells, two transporter proteins are central to thyroid hormone (TH) biosynthesis and secretion. The sodium iodide symporter (NIS) delivers iodide from the bloodstream into the thyroid, and after TH biosynthesis, monocarboxylate transporter 8 (MCT8) mediates TH secretion from the thyroid gland. Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is a protooncogene that is up-regulated in thyroid cancer and that binds NIS and modulates its subcellular localization and function. We now show that PBF binds MCT8 in vitro, eliciting a marked shift in MCT8 subcellular localization and resulting in a significant reduction in the amount of MCT8 at the plasma membrane as determined by cell surface biotinylation assays. Colocalization and interaction between PBF and Mct8 was also observed in vivo in a mouse model of thyroid-specific PBF overexpression driven by a bovine thyroglobulin (Tg) promoter (PBF-Tg). Thyroidal Mct8 mRNA and protein expression levels were similar to wild-type mice. Critically, however, PBF-Tg mice demonstrated significantly enhanced thyroidal TH accumulation and reduced TH secretion upon TSH stimulation. Importantly, Mct8-knockout mice share this phenotype. These data show that PBF binds and alters the subcellular localization of MCT8 in vitro, with PBF overexpression leading to an accumulation of TH within the thyroid in vivo. Overall, these studies identify PBF as the first protein to interact with the critical TH transporter MCT8 and modulate its function in vivo. Furthermore, alongside NIS repression, PBF may thus represent a new regulator of TH biosynthesis and secretion.

Functional variable number of tandem repeats variation in the promoter of proto‐oncogene PTTG1IP is associated with risk of estrogen receptor‐positive breast cancer

Genetic polymorphisms in the signalling pathway of estrogen receptor (ER) could modify the risk of breast cancer. A variable number of tandem repeats (VNTR) polymorphism in the promoter of PTTG1IP, pituitary tumor transforming gene binding factor targeted by estrogen receptor α (ERα) in endocrine neoplasia, has been shown to be functional, but its relevance to cancer etiology was unknown. We investigated its association with breast cancer risk by genotyping in 658 patients and 866 controls and further analysed its differential interaction with ERα. We found nine types of alleles ranging from 2 to 9 and 11 repeats that form 29 distinct genotypes and 11 different biallelic repeat numbers. Subjects who carry the six-repeats allele (odds ratio [OR], 1.45; 95% confidence interval [CI], 1.17-1.79), long alleles (≥6 repeats) (OR, 1.55; 95% CI, 1.17-2.05) or a high dose of biallelic repeats (OR, 1.38; 95% CI, 1.07-1.77) were at significantly increased risk of cancer. In stratification analysis, these associations consistently manifested in ER-positive breast cancer: in ER positive, PR-positive subtype, genotypes with the six-repeats allele (OR, 1.42; 95% CI, 1.06-1.90), long alleles (OR, 1.77; 95% CI, 1.17-2.67) or a high dose of biallelic repeats (OR, 1.67; 95% CI, 1.19-2.33) were associated with cancer risk; in ER positive, HER2-negative subtype, they were susceptible factors with the ORs being 1.46 (95% CI, 1.06-2.02), 2.06 (95% CI, 1.28-3.32) and 1.85 (95% CI, 1.26-2.71), respectively. Furthermore, functional analysis revealed that an increase in the number of tandem repeats enhances the binding affinity of ERα. The present study provides the first epidemiological evidence that functional regulatory variants of PTTG1IP were associated with the risk of ER-positive breast cancer, further supporting its relevance as one proto-oncogene in breast cancer.

Pituitary Tumor Transforming Gene Binding Factor: A New Gene in Breast Cancer

Pituitary tumor transforming gene (PTTG) binding factor (PBF; PTTG1IP) is a relatively uncharacterized oncoprotein whose function remains obscure. Because of the presence of putative estrogen response elements (ERE) in its promoter, we assessed PBF regulation by estrogen. PBF mRNA and protein expression were induced by both diethylstilbestrol and 17beta-estradiol in estrogen receptor alpha (ERalpha)-positive MCF-7 cells. Detailed analysis of the PBF promoter showed that the region -399 to -291 relative to the translational start site contains variable repeats of an 18-bp sequence housing a putative ERE half-site (gcccctcGGTCAcgcctc). Sequencing the PBF promoter from 122 normal subjects revealed that subjects may be homozygous or heterozygous for between 1 and 6 repeats of the ERE. Chromatin immunoprecipitation and oligonucleotide pull-down assays revealed ERalpha binding to the PBF promoter. PBF expression was low or absent in normal breast tissue but was highly expressed in breast cancers. Subjects with greater numbers of ERE repeats showed higher PBF mRNA expression, and PBF protein expression positively correlated with ERalpha status. Cell invasion assays revealed that PBF induces invasion through Matrigel, an action that could be abrogated both by siRNA treatment and specific mutation. Furthermore, PBF is a secreted protein, and loss of secretion prevents PBF inducing cell invasion. Given that PBF is a potent transforming gene, we propose that estrogen treatment in postmenopausal women may upregulate PBF expression, leading to PBF secretion and increased cell invasion. Furthermore, the number of ERE half-sites in the PBF promoter may significantly alter the response to estrogen treatment in individual subjects.

Abstract 493: A novel mechanism of colorectal tumorigenesis based on the functional inactivation of p53 by the pituitary tumor transforming gene binding factor (PBF)

Abstract PBF is the poorly characterized binding partner of the pituitary tumor transforming gene (PTTG). PTTG is a multifunctional proto-oncogene overexpressed in colorectal cancer, which regulates the function of the tumor suppressor protein p53. Recent data indicates that PBF can transform cells independently of PTTG and that subcutaneous expression of PBF elicits large tumors in nude mice. Given that PBF is both transforming in vitro and tumorigenic in vivo, our present studies aim to investigate whether PBF has a role in colorectal tumorigenesis. Initially we demonstrated that PBF represses p53-mediated gene regulation through Hdm2 promoter assays in p53-null H1299 cells. Transfection of p53 elicited a 30.7 ± 2.6-fold stimulation of promoter activity. However, co-transfection of PBF significantly repressed p53 transcriptional activity (16.3 ± 1.1-fold; p<0.0003). Exposure of HCT116 cells to gamma-irradiation resulted in significantly increased endogenous PBF protein expression from 4 hours after treatment and reached maximal levels after 24 hours, in keeping with the observed increase in p53 protein stability. Furthermore, co-immunoprecipitation assays revealed a direct interaction between PBF and p53 in vivo, with a significant increase in binding following gamma-irradiation. In immunofluorescence studies, HCT116 and COS-7 cells treated with gamma-irradiation displayed increased nuclear localisation of PBF compared to untreated controls, resulting in an enhanced degree of colocalisation of both PBF and p53 within the nucleus. Transient overexpression of PBF in HCT116 cells resulted in substantially elevated nuclear p53 protein expression from as early as 2 hours after treatment, with increased p53 stabilization persisting for 24 hours compared to mock-transfected controls. Finally, in a cohort of matched cancer versus normal colorectal tissue samples we observed a significant increase in PBF expression at both the mRNA (2.4-fold, n=24, p=0.009) and protein level in tumors, which correlated with a significant increase in p53 protein expression. Taken together, these results highlight a potential role for PBF within the DNA damage response, whereby PBF stabilizes in response to genotoxic stress, binds directly to p53 and inhibits p53 mediated responses. From these findings we propose that aberrant regulation of PBF leads to the functional inactivation of p53, and thus represents a potential novel mechanism for colorectal tumorigenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 493.

Pituitary Tumor Transforming Gene Binding Factor: A Novel Transforming Gene in Thyroid Tumorigenesis

There are currently no clear markers for the detection of differentiated thyroid cancer and its recurrence. Pituitary tumor transforming gene (PTTG) is a protooncogene implicated in the pathogenesis of multiple tumor types, which stimulates fibroblast growth factor-2 secretion via PTTG binding factor (PBF). The aim of this study was to ascertain whether PBF expression is associated with thyroid cancer outcome. PBF expression was measured at the mRNA and protein level. Tissue was collected during surgery, with normal samples being taken from the contralateral lobe. In vitro studies ascertained the ability of PBF to transform cells and form tumors in nude mice and its subcellular localization. The study was conducted at a primary care/referral center. Thyroid tumors were collected from a series of 27 patients undergoing surgical excision of papillary and follicular thyroid tumors. No intervention was conducted. The expression of PBF in thyroid cancers compared with normal thyroid, hypothesized before the investigation to be raised in tumors, was the main outcome measure. PBF mRNA expression was higher in differentiated thyroid carcinomas than in normal thyroid (P < 0.001; n = 27) and was independently associated with tumor recurrence (P = 0.002; R(2) = 0.49). PTTG was able to up-regulate PBF mRNA expression in vitro (P < 0.001; n = 12), and stable overexpression of PBF in NIH3T3 cells resulted in significant colony formation (P < 0.001; n = 12). In vivo, stable sc overexpression of PBF induced tumor formation in athymic nude mice. PBF is an additional prognostic indicator in differentiated thyroid cancer that is transforming in vitro and tumorigenic in vivo.