Bhavika Modasia

The PTTG1-Binding Factor (PBF/PTTG1IP) Regulates p53 Activity in Thyroid Cells

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.

The proto‐oncogene PBF binds p53 and is associated with prognostic features in colorectal cancer

The PTTG1‐binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione–S–transferase (GST) pull‐down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126–132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53‐responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild‐type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.

Meeting abstracts from the 64th British Thyroid Association Annual Meeting

• Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain.Graves’ orbitopathy (GO) is the main extrathyroidal manifestation of Graves’ disease. When fully expressed, it is characterized by inflammatory soft tissue changes, exophthalmos, ocular dysmotility causing diplopia, and, rarely, sight-threatening dysthyroid optic neuropathy (DON). The prevalence of GO among Graves’ patients seems lately declining, probably due to early diagnosis, early intervention on risk factors associated with its occurrence or progression (smoking, uncontrolled thyroid dysfunction), early correction of hyper and hypothyroidism. Only about 25–30% of newly diagnosed Graves’ hyperthyroids are affected with GO, which is usually mild and rarely progressive. Assessment of activity and severity of GO according to standardized criteria is fundamental to plan management. The European Thyroid Association and the European Group on Graves’ Orbitopathy (EUGOGO) have recently published the first guideline on management of GO. Mild GO usually requires only a watchful strategy, in addition to local measures (eye drops, ointments) and removal of risk factors. Intravenous glucocorticoids (ivGCs) are the first-line treatment for moderate-to-severe and active GO, as demonstrated by randomized clinical trials. When ivGCs fail or GO recurs after treatment withdrawal, options include a second course of ivGCs, oral GCs combined with orbital radiotherapy or cyclosporine, rituximab. Evidence that the any of the above treatment be effective in the context of a poor response to a first course of ivGCs is limited and should be investigated in larger studies. In addition to rituximab, ongoing investigations are exploring the role of other biologics targeting, e.g., the IGF-1 receptor or the IL-6 receptor, and results will probably available in 1–2 years. When GO has been treated medically and is inactive, rehabilitative surgery (orbital decompression, squint surgery, eyelid surgery) is often needed.

PO-124 PTTG and PBF functionally interact with P53 and predict overall survival in head and neck cancer

Introduction Worldwide,~6 87 000 new cases of head and neck cancer are diagnosed annually with more than 3 80 000 deaths. Dysregulation of the tumour suppressor p53 is common in head and neck squamous cell carcinoma (HNSCC) especially in aggressive tumours. TP53 is mutated in 50%–80% of HNSCC but relatively little is understood about the biological and clinical impact of proto-oncogenes that regulate p53. Here, we show that proto-oncogenes pituitary tumour transforming gene 1 (PTTG) and its interacting partner PBF cooperate to regulate p53 activity in HNSCC. In addition, we probe TCGA data and reveal that PTTG and PBF represent new and useful prognostic indicators. Material and methods PTTG/PBF expression was determined in matched (tumour/normal) HNSCC tissue (n=24). p53 expression and function was assessed in 92-VU-040T [wild-type TP53 (wtTP53)] and 93-VU-147T [mutant TP53 (mTP53)] cells with lentiviral shRNA ablation of PTTG and PBF. Expression of PTTG, PBF and p53-target genes, as well as patient overall survival (OS), was analysed using HNSCC TCGA data. Results and discussions HNSCC tissue revealed significant upregulation of PTTG (1.9-fold, p<0.05) and PBF mRNA (1.6-fold, p<0.01), which was consistent with expression in a larger TCGA dataset (n=520). We found a striking correlation between PTTG and a panel of 129 p53-target genes in HNSCC TCGA data (wtTP53: 82%; mTP53: 53%, p<0.05). In contrast, PBF expression correlated with fewer p53-target genes (wtTP53: 18%; mTP53: 31%, p<0.05). In agreement, there were significant mRNA changes in PTTG- and PBF-depleted HNSCC cells for key p53-responsive genes such as BCL2. Co-immunoprecipitation studies confirmed that PTTG and PBF are specific interactors of p53 in HNSCC. PTTG retained the ability to bind p53 in the absence of PBF, but the degree of interaction was significantly attenuated (PBF-depleted 92-VU-040T cells: 0.25±0.06 fold, p<0.001) suggesting that PBF facilitates binding of PTTG to p53. Half-life studies showed that PTTG and PBF inhibit p53 stability, with joint over-expression giving the most pronounced decrease in p53 protein stability (~13 fold, p<0.001). Subsequent analysis of TCGA HNSCC patients with mTP53 and high tumoural PBF/PTTG showed poorer overall survival (p<0.05, median OS=28.98 months) than those with low PBF/PTTG (median OS=71.16 months). Conclusion These findings provide compelling evidence that PTTG and PBF cooperate to dysregulate p53 in HNSCC which results in greater impairment of p53-dependent signalling and contributes towards worse clinical outcomes.

Pharmacological enhancement of radioiodine uptake using Src kinase inhibitors

• Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain.Graves’ orbitopathy (GO) is the main extrathyroidal manifestation of Graves’ disease. When fully expressed, it is characterized by inflammatory soft tissue changes, exophthalmos, ocular dysmotility causing diplopia, and, rarely, sight-threatening dysthyroid optic neuropathy (DON). The prevalence of GO among Graves’ patients seems lately declining, probably due to early diagnosis, early intervention on risk factors associated with its occurrence or progression (smoking, uncontrolled thyroid dysfunction), early correction of hyper and hypothyroidism. Only about 25–30% of newly diagnosed Graves’ hyperthyroids are affected with GO, which is usually mild and rarely progressive. Assessment of activity and severity of GO according to standardized criteria is fundamental to plan management. The European Thyroid Association and the European Group on Graves’ Orbitopathy (EUGOGO) have recently published the first guideline on management of GO. Mild GO usually requires only a watchful strategy, in addition to local measures (eye drops, ointments) and removal of risk factors. Intravenous glucocorticoids (ivGCs) are the first-line treatment for moderate-to-severe and active GO, as demonstrated by randomized clinical trials. When ivGCs fail or GO recurs after treatment withdrawal, options include a second course of ivGCs, oral GCs combined with orbital radiotherapy or cyclosporine, rituximab. Evidence that the any of the above treatment be effective in the context of a poor response to a first course of ivGCs is limited and should be investigated in larger studies. In addition to rituximab, ongoing investigations are exploring the role of other biologics targeting, e.g., the IGF-1 receptor or the IL-6 receptor, and results will probably available in 1–2 years. When GO has been treated medically and is inactive, rehabilitative surgery (orbital decompression, squint surgery, eyelid surgery) is often needed.

Meeting abstracts from the 64th British Thyroid Association Annual Meeting: Newcastle, UK. 13/05/2016

• Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain.Graves’ orbitopathy (GO) is the main extrathyroidal manifestation of Graves’ disease. When fully expressed, it is characterized by inflammatory soft tissue changes, exophthalmos, ocular dysmotility causing diplopia, and, rarely, sight-threatening dysthyroid optic neuropathy (DON). The prevalence of GO among Graves’ patients seems lately declining, probably due to early diagnosis, early intervention on risk factors associated with its occurrence or progression (smoking, uncontrolled thyroid dysfunction), early correction of hyper and hypothyroidism. Only about 25–30% of newly diagnosed Graves’ hyperthyroids are affected with GO, which is usually mild and rarely progressive. Assessment of activity and severity of GO according to standardized criteria is fundamental to plan management. The European Thyroid Association and the European Group on Graves’ Orbitopathy (EUGOGO) have recently published the first guideline on management of GO. Mild GO usually requires only a watchful strategy, in addition to local measures (eye drops, ointments) and removal of risk factors. Intravenous glucocorticoids (ivGCs) are the first-line treatment for moderate-to-severe and active GO, as demonstrated by randomized clinical trials. When ivGCs fail or GO recurs after treatment withdrawal, options include a second course of ivGCs, oral GCs combined with orbital radiotherapy or cyclosporine, rituximab. Evidence that the any of the above treatment be effective in the context of a poor response to a first course of ivGCs is limited and should be investigated in larger studies. In addition to rituximab, ongoing investigations are exploring the role of other biologics targeting, e.g., the IGF-1 receptor or the IL-6 receptor, and results will probably available in 1–2 years. When GO has been treated medically and is inactive, rehabilitative surgery (orbital decompression, squint surgery, eyelid surgery) is often needed.

Comparative analysis of human and mouse expression data identifies proto-oncogene PTTG- and PBF-associated genes in thyroid cancer

• Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain.Graves’ orbitopathy (GO) is the main extrathyroidal manifestation of Graves’ disease. When fully expressed, it is characterized by inflammatory soft tissue changes, exophthalmos, ocular dysmotility causing diplopia, and, rarely, sight-threatening dysthyroid optic neuropathy (DON). The prevalence of GO among Graves’ patients seems lately declining, probably due to early diagnosis, early intervention on risk factors associated with its occurrence or progression (smoking, uncontrolled thyroid dysfunction), early correction of hyper and hypothyroidism. Only about 25–30% of newly diagnosed Graves’ hyperthyroids are affected with GO, which is usually mild and rarely progressive. Assessment of activity and severity of GO according to standardized criteria is fundamental to plan management. The European Thyroid Association and the European Group on Graves’ Orbitopathy (EUGOGO) have recently published the first guideline on management of GO. Mild GO usually requires only a watchful strategy, in addition to local measures (eye drops, ointments) and removal of risk factors. Intravenous glucocorticoids (ivGCs) are the first-line treatment for moderate-to-severe and active GO, as demonstrated by randomized clinical trials. When ivGCs fail or GO recurs after treatment withdrawal, options include a second course of ivGCs, oral GCs combined with orbital radiotherapy or cyclosporine, rituximab. Evidence that the any of the above treatment be effective in the context of a poor response to a first course of ivGCs is limited and should be investigated in larger studies. In addition to rituximab, ongoing investigations are exploring the role of other biologics targeting, e.g., the IGF-1 receptor or the IL-6 receptor, and results will probably available in 1–2 years. When GO has been treated medically and is inactive, rehabilitative surgery (orbital decompression, squint surgery, eyelid surgery) is often needed.

Functional consequences of the first reported mutations of the proto-oncogene PTTG1IP/PBF

Pituitary tumor-transforming gene 1-binding factor (PTTG1IP; PBF) is a multifunctional glycoprotein, which is overexpressed in a wide range of tumours, and significantly associated with poorer oncological outcomes, such as early tumour recurrence, distant metastasis, extramural vascular invasion and decreased disease-specific survival. PBF transforms NIH 3T3 fibroblasts and induces tumours in nude mice, while mice harbouring transgenic thyroidal PBF expression show hyperplasia and macrofollicular lesions. Our assumption that PBF becomes an oncogene purely through increased expression has been challenged by the recent report of mutations in PBF within the Catalogue of Somatic Mutations in Cancer (COSMIC) database. We therefore sought to determine whether the first 10 PBF missense substitutions in human cancer might be oncogenic. Anisomycin half-life studies revealed that most mutations were associated with reduced protein stability compared to wild-type (WT) PBF. Proliferation assays narrowed our interest to two mutational events which significantly altered cell turnover: C51R and R140W. C51R was mainly confined to the endoplasmic reticulum while R140W was apparent in the Golgi apparatus. Both C51R and R140W lost the capacity to induce cellular migration and significantly reduced cell invasion. Colony formation and soft agar assays demonstrated that, in contrast to WT PBF, both mutants were unable to elicit significant colony formation or anchorage-independent growth. However, C51R and R140W retained the ability to repress radioiodide uptake, a functional hallmark of PBF. Our data reveal new insight into PBF function and confirm that, rather than being oncogenic, mutations in PBF are likely to be passenger effects, with overexpression of PBF the more important aetiological event in human cancer.

Identification of novel sodium iodide symporter (NIS) interactors which modulate iodide uptake

Patients termed to have radioiodine-refractory differentiated thyroid cancer (RR-DTC) cannot accumulate sufficient radioiodine for a therapeutic response due to sodium iodide symporter (NIS) dysregulation via diminished expression and/or altered plasma membrane localisation. Previous studies identified novel therapeutics to increase NIS expression, but these are associated with poor tolerance and resistance. Meanwhile, regulation of NIS plasma membrane localisation remains poorly defined and, despite protein-protein interactions being well-described to modulate trafficking events, the NIS interactome is limited. Here, two novel functional NIS interactors – ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP) – were identified by mass spectrometry, which positively and negatively modulated radioiodine uptake respectively. In papillary thyroid cancer (PTC), ARF4 is downregulated, and VCP overexpressed, which may provide a putative explanation for repressed NIS function. Subsequent investigations identified co-localisation between ARF4 and NIS at the Golgi as well as co-incident trafficking at the plasma membrane, which led to the hypothesis that ARF4 promotes NIS trafficking to the plasma membrane. In contrast, VCP decreased NIS protein expression, which was suggestive of a role for VCP in NIS processing and degradation. Pharmacological inhibitors Eeyarestatin-1 and NMS-873 overcame VCP inhibition of NIS function, which may highlight a novel therapeutic strategy for RR-DTC.

Distinct p53 response profiles in transgenic mouse models of thyroid-specific PBF and PTTG expression

• Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain.