Combined Vorinostat and Chloroquine Inhibit Sodium-Iodide Symporter Endocytosis and Enhance Radionuclide Uptake In Vivo.

PURPOSE: Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo. EXPERIMENTAL DESIGN: Informed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake, and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice. RESULTS: We identified an acidic dipeptide within the NIS C-terminus that mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine (CQ) modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. CONCLUSIONS: NIS internalization is specifically druggable in vivo. Our data, therefore, provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. See related commentary by Lechner and Brent, p. 1220.

Communities of practice in residential aged care: A rapid review.

BACKGROUND: Communities of practice (CoPs) have the potential to help address the residential aged care system's need for continuing education and quality improvement. CoPs have been used in healthcare to improve clinical practice; however, little is known about their application to the unique residential aged care context. OBJECTIVES: This rapid review of CoPs for residential aged care was conducted to summarise the features of CoPs, how they are developed and maintained, and assess their effectiveness. METHODS: MEDLINE and CINAHL databases were searched for studies published from January 1991 to November 2022 about CoPs in residential aged care. Data were extracted regarding the CoPs' three key features of 'domain', 'community' and 'practice' as described by Wenger and colleagues. Kirkpatrick's four levels of evaluation (members' reactions, learning, behaviour and results) was used to examine studies on the effectiveness of CoPs. The Mixed Methods Appraisal Tool was used for quality appraisal. RESULTS: Nineteen articles reported on 13 residential aged care CoPs. Most CoPs aimed to improve care quality (n = 9, 69%) while others aimed to educate members (n = 3, 23%). Membership was often multidisciplinary (n = 8, 62%), and interactions were in-person (n = 6, 46%), online (n = 3, 23%) or both (n = 4, 31%). Some CoPs were developed with the aid of a planning group (n = 4, 31%) or as part of a larger collaborative (n = 4, 31%), and were maintained using a facilitator (n = 7, 54%) or adapted to member feedback (n = 2, 15%). Thirteen (81%) studies evaluated members' reactions, and three (24%) studies assessed members' behaviour. The heterogeneity of studies and levels of reporting made it difficult to synthesise findings. CONCLUSIONS: This review revealed the variation in why, and how, CoPs have been used in residential aged care, which is consistent with previous reviews of CoPs in healthcare. While these findings can inform the development of CoPs in this context, further research is needed to understand how CoPs, including the membership makeup, delivery mode, facilitator type and frequency of meetings, impact quality of care.

Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter.

The sodium iodide symporter (NIS) functions to transport iodide and is critical for successful radioiodide ablation of cancer cells. Approaches to bolster NIS function and diminish recurrence post-radioiodide therapy are impeded by oncogenic pathways that suppress NIS, as well as the inherent complexity of NIS regulation. Here, we utilize NIS in high-throughput drug screening and undertake rigorous evaluation of lead compounds to identify and target key processes underpinning NIS function. We find that multiple proteostasis pathways, including proteasomal degradation and autophagy, are central to the cellular processing of NIS. Utilizing inhibitors targeting distinct molecular processes, we pinpoint combinatorial drug strategies giving robust >5-fold increases in radioiodide uptake. We also reveal significant dysregulation of core proteostasis genes in human tumors, identifying a 13-gene risk score classifier as an independent predictor of recurrence in radioiodide-treated patients. We thus propose and discuss a model for targetable steps of intracellular processing of NIS function.

Recurrence of Papillary Thyroid Cancer: A Systematic Appraisal of Risk Factors.

CONTEXT: Thyroid cancer recurrence is associated with increased mortality and adverse outcomes. Recurrence risk is currently predicted using clinical tools, often restaging patients after treatment. Detailed understanding of recurrence risk at disease onset could lead to personalized and improved patient care. OBJECTIVE: We aimed to perform a comprehensive bioinformatic and experimental analysis of 3 levels of genetic change (mRNA, microRNA, and somatic mutation) apparent in recurrent tumors and construct a new combinatorial prognostic risk model. METHODS: We analyzed The Cancer Genome Atlas data (TCGA) to identify differentially expressed genes (mRNA/microRNA) in 46 recurrent vs 455 nonrecurrent thyroid tumors. Two exonic mutational pipelines were used to identify somatic mutations. Functional gene analysis was performed in cell-based assays in multiple thyroid cell lines. The prognostic value of genes was evaluated with TCGA datasets. RESULTS: We identified 128 new potential biomarkers associated with recurrence, including 40 mRNAs, 39 miRNAs, and 59 genetic variants. Among differentially expressed genes, modulation of FN1, ITGα3, and MET had a significant impact on thyroid cancer cell migration. Similarly, ablation of miR-486 and miR-1179 significantly increased migration of TPC-1 and SW1736 cells. We further utilized genes with a validated functional role and identified a 5-gene risk score classifier as an independent predictor of thyroid cancer recurrence. CONCLUSION: Our newly proposed risk model based on combinatorial mRNA and microRNA expression has potential clinical utility as a prognostic indicator of recurrence. These findings should facilitate earlier prediction of recurrence with implications for improving patient outcome by tailoring treatment to disease risk and increasing posttreatment surveillance.

The potential interaction between medical treatment and radioiodine treatment success: A systematic review.

Introduction: Radioactive iodine (RAI) therapy is a critical component in the post-surgical management of thyroid cancer patients, as well as being a central therapeutic option in the treatment of hyperthyroidism. Previous work suggests that antithyroid drugs hinder the efficacy of RAI therapy in patients. However, the effects of other background medications on RAI treatment efficacy have not been evaluated. Therefore, we performed a systematic review and meta-analysis investigating the potential off-target effects of medication on RAI therapy in patients with thyroid cancer and hyperthyroidism. Methods: Systematic review and meta-analysis according to the 2020 PRISMA guidelines. Databases searched: MEDLINE, EMBASE and Cochrane Library for studies published between 2001 and 2021. Results: Sixty-nine unique studies were identified. After screening, 17 studies with 3313 participants were included. One study investigated thyroid cancer, with the rest targeted to hyperthyroidism. The majority of studies evaluated the effects of antithyroid drugs; the other drugs studied included lithium, prednisone and glycididazole sodium. Antithyroid drugs were associated with negative impacts on post-RAI outcomes (n = 5 studies, RR = 0.81, p = 0.02). However, meta-analysis found moderate heterogeneity between studies (I2 = 51%, τ2 = 0.0199, p = 0.08). Interestingly, lithium (n = 3 studies), prednisone (n = 1 study) and glycididazole (n = 1 study) appeared to have positive impacts on post-RAI outcomes upon qualitative analysis. Conclusion: Our systematic review strengthens previous work on antithyroid medication effects on RAI, and highlights that this field remains under researched especially for background medications unrelated to thyroid disease, with very few papers on non-thyroid medications published. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php, identifier CRD42021274026.

Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake.

The sodium iodide symporter (NIS) is required for iodide uptake, which facilitates thyroid hormone biosynthesis. NIS has been exploited for over 75 years in ablative radioiodine (RAI) treatment of thyroid cancer, where its ability to transport radioisotopes depends on its localization to the plasma membrane. The advent of NIS-based in vivo imaging and theranostic strategies in other malignancies and disease modalities has recently increased the clinical importance of NIS. However, NIS trafficking remains ill-defined. Here, we used tandem mass spectrometry followed by coimmunoprecipitation and proximity ligation assays to identify and validate two key nodes-ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP)-controlling NIS trafficking. Using cell-surface biotinylation assays and highly inclined and laminated optical sheet microscopy, we demonstrated that ARF4 enhanced NIS vesicular trafficking from the Golgi to the plasma membrane, whereas VCP-a principal component of endoplasmic reticulum (ER)-associated degradation-governed NIS proteolysis. Gene expression analysis indicated VCP expression was particularly induced in aggressive thyroid cancers and in patients who had poorer outcomes following RAI treatment. Two repurposed FDA-approved VCP inhibitors abrogated VCP-mediated repression of NIS function, resulting in significantly increased NIS at the cell-surface and markedly increased RAI uptake in mouse and human thyroid models. Collectively, these discoveries delineate NIS trafficking and highlight the new possibility of systemically enhancing RAI therapy in patients using FDA-approved drugs. SIGNIFICANCE: These findings show that ARF4 and VCP are involved in NIS trafficking to the plasma membrane and highlight the possible therapeutic role of VCP inhibitors in enhancing radioiodine effectiveness in radioiodine-refractory thyroid cancer.

Dimerization of the Sodium/Iodide Symporter.

Background: The ability of thyroid follicular epithelial cells to accumulate iodide via the sodium/iodide symporter (NIS) is exploited to successfully treat most thyroid cancers, although a subset of patients lose functional NIS activity and become unresponsive to radioiodide therapy, with poor clinical outcome. Our knowledge of NIS regulation remains limited, however. While numerous membrane proteins are functionally regulated via dimerization, there is little definitive evidence of NIS dimerization, and whether this might impact upon radioiodide uptake and treatment success is entirely unknown. We hypothesized that NIS dimerizes and that dimerization is a prerequisite for iodide uptake. Methods: Coimmunoprecipitation, proximity ligation, and Förster resonance energy transfer (FRET) assays were used to assess NIS:NIS interaction. To identify residues involved in dimerization, a homology model of NIS structure was built based on the crystal structure of the dimeric bacterial protein vSGLT. Results: Abundant cellular NIS dimerization was confirmed in vitro via three discrete methodologies. FRET and proximity ligation assays demonstrated that while NIS can exist as a dimer at the plasma membrane (PM), it is also apparent in other cellular compartments. Homology modeling revealed one key potential site of dimeric interaction, with six residues <3Å apart. In particular, NIS residues Y242, T243, and Q471 were identified as critical to dimerization. Individual mutation of residues Y242 and T243 rendered NIS nonfunctional, while abrogation of Q471 did not impact radioiodide uptake. FRET data show that the putative dimerization interface can tolerate the loss of one, but not two, of these three clustered residues. Conclusions: We show for the first time that NIS dimerizes in vitro, and we identify the key residues via which this happens. We hypothesize that dimerization of NIS is critical to its trafficking to the PM and may therefore represent a new mechanism that would need to be considered in overcoming therapeutic failure in patients with thyroid cancer.

Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication.

Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication.IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.

The putative tumour suppressor protein Latexin is secreted by prostate luminal cells and is downregulated in malignancy.

Loss of latexin (LXN) expression negatively correlates with the prognosis of several human cancers. Despite association with numerous processes including haematopoietic stem cell (HSC) fate, inflammation and tumour suppression, a clearly defined biological role for LXN is still lacking. Therefore, we sought to understand LXN expression and function in the normal and malignant prostate to assess its potential as a therapeutic target. Our data demonstrate that LXN is highly expressed in normal prostate luminal cells but downregulated in high Gleason grade cancers. LXN protein is both cytosolic and secreted by prostate cells and expression is directly and potently upregulated by all-trans retinoic acid (atRA). Whilst overexpression of LXN in prostate epithelial basal cells did not affect cell fate, LXN overexpression in the luminal cancer line LNCaP reduced plating efficiency. Transcriptome analysis revealed that LXN overexpression had no direct effects on gene expression but had significant indirect effects on important genes involved in both retinoid metabolism and IFN-associated inflammatory responses. These data highlight a potential role for LXN in retinoid signaling and inflammatory pathways. Investigating the effects of LXN on immune cell function in the tumour microenvironment (TME) may reveal how observed intratumoural loss of LXN affects the prognosis of many adenocarcinomas.

PTTG and PBF Functionally Interact with p53 and Predict Overall Survival in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and poses a significant health burden due to its rising incidence. Although the proto-oncogene pituitary tumor-transforming gene 1 (PTTG) predicts poor patient outcome, its mechanisms of action are incompletely understood. We show here that the protein PBF modulates PTTG function, is overexpressed in HNSCC tumors, and correlates with significantly reduced survival. Lentiviral shRNA attenuation of PTTG or PBF expression in HNSCC cells with either wild-type or mutant p53, and with and without HPV infection, led to dysregulated expression of p53 target genes involved in DNA repair and apoptosis. Mechanistically, PTTG and PBF affected each other's interaction with p53 and cooperated to reduce p53 protein stability in HNSCC cells independently of HPV. Depletion of either PTTG or PBF significantly repressed cellular migration and invasion and impaired colony formation in HNSCC cells, implicating both proto-oncogenes in basic mechanisms of tumorigenesis. Patients with HNSCC with high tumoral PBF and PTTG had the poorest overall survival, which reflects a marked impairment of p53-dependent signaling.Significance: These findings reveal a complex and novel interrelationship between the expression and function of PTTG, PBF, and p53 in human HNSCC that significantly influences patient outcome. Cancer Res; 78(20); 5863-76. ©2018 AACR.

Estrogen Activation by Steroid Sulfatase Increases Colorectal Cancer Proliferation via GPER.

Context: Estrogens affect the incidence and progression of colorectal cancer (CRC), although the precise molecular mechanisms remain ill-defined. Objective: The present study investigated prereceptor estrogen metabolism through steroid sulphatase (STS) and 17ß-hydroxysteroid dehydrogenase activity and subsequent nongenomic estrogen signaling in human CRC tissue, in The Cancer Genome Atlas colon adenocarcinoma data set, and in in vitro and in vivo CRC models. We aimed to define and therapeutically target pathways through which estrogens alter CRC proliferation and progression. Design, Setting, Patients, and Interventions: Human CRC samples with normal tissue-matched controls were collected from postmenopausal female and age-matched male patients. Estrogen metabolism enzymes and nongenomic downstream signaling pathways were determined. CRC cell lines were transfected with STS and cultured for in vitro and in vivo analysis. Estrogen metabolism was determined using an ultra-performance liquid chromatography-tandem mass spectrometry method. Primary Outcome Measure: The proliferative effects of estrogen metabolism were evaluated using 5-bromo-2'-deoxyuridine assays and CRC mouse xenograft studies. Results: Human CRC exhibits dysregulated estrogen metabolism, favoring estradiol synthesis. The activity of STS, the fundamental enzyme that activates conjugated estrogens, is significantly (P < 0.001) elevated in human CRC compared with matched controls. STS overexpression accelerates CRC proliferation in in vitro and in vivo models, with STS inhibition an effective treatment. We defined a G-protein-coupled estrogen receptor (GPER) proproliferative pathway potentially through increased expression of connective tissue growth factor in CRC. Conclusion: Human CRC favors estradiol synthesis to augment proliferation via GPER stimulation. Further research is required regarding whether estrogen replacement therapy should be used with caution in patients at high risk of developing CRC.

Pro-invasive Effect of Proto-oncogene PBF Is Modulated by an Interaction with Cortactin.

CONTEXT: Metastatic disease is responsible for the majority of endocrine cancer deaths. New therapeutic targets are urgently needed to improve patient survival rates. OBJECTIVE: The proto-oncogene PTTG1-binding factor (PBF/PTTG1IP) is overexpressed in multiple endocrine cancers and circumstantially associated with tumor aggressiveness. This study aimed to understand the role of PBF in tumor cell invasion and identify possible routes to inhibit its action. Design, Setting, Patients, and Interventions: Thyroid, breast, and colorectal cells were transfected with PBF and cultured for in vitro analysis. PBF and cortactin (CTTN) expression was determined in differentiated thyroid cancer and The Cancer Genome Atlas RNA-seq data. PRIMARY OUTCOME MEASURE: Pro-invasive effects of PBF were evaluated by 2D Boyden chamber, 3D organotypic, and proximity ligation assays. RESULTS: Our study identified that PBF and CTTN physically interact and co-localize, and that this occurs at the cell periphery, particularly at the leading edge of migrating cancer cells. Critically, PBF induces potent cellular invasion and migration in thyroid and breast cancer cells, which is entirely abrogated in the absence of CTTN. Importantly, we found that CTTN is over-expressed in differentiated thyroid cancer, particularly in patients with regional lymph node metastasis, which significantly correlates with elevated PBF expression. Mutation of PBF (Y174A) or pharmacological intervention modulates the PBF: CTTN interaction and attenuates the invasive properties of cancer cells. CONCLUSION: Our results demonstrate a unique role for PBF in regulating CTTN function to promote endocrine cell invasion and migration, as well as identify a new targetable interaction to block tumor cell movement.

Germline ESR2 mutation predisposes to medullary thyroid carcinoma and causes up-regulation of RET expression.

Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERß expression in the MTC tumour. ERα and ERß form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERß represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis.

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.

Picomolar Inhibition of Plasmepsin V, an Essential Malaria Protease, Achieved Exploiting the Prime Region.

Malaria is an infectious disease caused by Plasmodium parasites. It results in an annual death-toll of ~ 600,000. Resistance to all medications currently in use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV) is an essential Plasmodium protease and a highly promising antimalarial target, which still lacks molecular characterization and drug-like inhibitors. PmV, cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an indispensable parasitic process for virulence and infection. Here, we describe the accessibility of PmV catalytic pockets to inhibitors and propose a novel strategy for PmV inhibition. We also provide molecular and structural data suitable for future drug development. Using high-throughput platforms, we identified a novel scaffold that interferes with PmV in-vitro at picomolar ranges (~ 1,000-fold more active than available compounds). Via systematic replacement of P and P' regions, we assayed the physico-chemical requirements for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic groups upstream to P3, the arginine and other possible substitutions in position P3 proved to be critically important elements in achieving potent inhibition. In-silico analyses provided essential QSAR information and model validation. Our inhibitors act 'on-target', confirmed by cellular interference of PmV function and biochemical interaction with inhibitors. Our inhibitors are poorly performing against parasite growth, possibly due to poor stability of their peptidic component and trans-membrane permeability. The lowest IC50 for parasite growth inhibition was ~ 15 µM. Analysis of inhibitor internalization revealed important pharmacokinetic features for PExEl-based molecules. Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for picomolar activity against PmV. All the presented data are discussed in respect to human aspartic proteases and previously reported inhibitors, highlighting differences and proposing new strategies for drug development.

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.

Drug repositioning as a route to anti-malarial drug discovery: preliminary investigation of the in vitro anti-malarial efficacy of emetine dihydrochloride hydrate.

BACKGROUND: Drug repurposing or repositioning refers to the usage of existing drugs in diseases other than those it was originally used for. For diseases like malaria, where there is an urgent need for active drug candidates, the strategy offers a route to significantly shorten the traditional drug development pipelines. Preliminary high-throughput screens on patent expired drug libraries have recently been carried out for Plasmodium falciparum. This study reports the systematic and objective further interrogation of selected compounds reported in these studies, to enable their repositioning as novel stand-alone anti-malarials or as combinatorial partners. METHODS: SYBR Green flow cytometry and micro-titre plate assays optimized in the laboratory were used to monitor drug susceptibility of in vitro cultures of P. falciparum K1 parasite strains. Previously described fixed-ratio methods were adopted to investigate drug interactions. RESULTS: Emetine dihydrochloride hydrate, an anti-protozoal drug previously used for intestinal and tissue amoebiasis was shown to have potent inhibitory properties (IC50 doses of ~ 47 nM) in the multidrug resistant K1 strain of P. falciparum. The sum 50% fractional inhibitory concentration (∑FIC50, 90) of the interaction of emetine dihydrochloride hydrate and dihydroartemisinin against the K1 strains of P. falciparum ranged from 0.88-1.48. CONCLUSION: The results warrant further investigation of emetine dihydrochloride hydrate as a potential stand-alone anti-malarial option. The interaction between the drug and the current front line dihydroartemisinin ranged from additive to mildly antagonistic in the fixed drug ratios tested.

Regulation of pituitary tumor transforming gene (PTTG) expression and phosphorylation in thyroid cells.

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.

Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment.

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.