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

Steroid sulphatase (STS) liberates sulphated oestrogens into their active forms. In the colon, evidence suggests that although initially pro-apoptotic in healthy mucosa, once malignancy occurs, oestrogens may stimulate colorectal cancer (CRC) proliferation. Moreover, greater intratumoural oestrogen synthesis is negatively associated with survival outcomes in CRC patients. However, little is known about oestrogen metabolism pathways in CRC

The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.

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

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

Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

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

Abstract Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake. Cancer Res; 71(19); 6153–64. ©2011 AACR.