Kidney functional reserve and damage biomarkers in subclinical chronic kidney disease and acute kidney injury.

Significant loss of kidney function is not easily identified by serum creatinine (sCr)-based measurements. In the presence of normal sCr, decreased kidney functional reserve (KFR) may identify a significant loss of function. We evaluated KFR in experimental subclinical chronic kidney disease (sCKD) before and after brief ischemia-reperfusion injury (IRI). Using fluorescein isothiocyanate-labeled sinistrin, glomerular filtration rate (GFR) was measured transcutaneously before and after adenine-induced sCKD, and 1 and 2 wk after brief IRI, and compared with urinary kidney damage biomarkers. sCKD reduced stimulated and unstimulated GFR by ∼20% while reducing KFR by 50%. IRI reduced unstimulated GFR for 14 days, but KFR remained relatively unchanged in sCKD and transiently increased in control kidneys at 7 days. sCr increased and creatinine clearance (CrCl) decreased only immediately after IRI; sCr and CrCl correlated poorly with measured GFR except on day 1 after IRI. Heterogeneity in sCr and CrCl resulted from variation in tubular creatinine secretion. The increase in damage biomarker concentrations persisted for up to 14 days after IRI, allowing retrospective detection of sCKD before AKI by urine clusterin/urine kidney injury molecule-1 with an area under the curve of 1.0. sCr and CrCl are unreliable unless sCr is acutely elevated. Measurement of KFR and urine damage biomarker excretion detected sCKD despite normal sCr and CrCl. After IRI, the urine clusterin-to-urine kidney injury molecule-1 ratio may identify prior sCKD.NEW & NOTEWORTHY Early kidney function loss is poorly identified by serum creatinine (sCr)-based measurements. Direct kidney functional reserve (KFR) measurement before kidney injury and elevated urinary biomarkers clusterin and kidney injury molecule-1 detect subclinical chronic kidney disease (sCKD) after kidney injury despite normal range sCr and creatinine clearance. Reliance on sCr masks underlying sCKD. Acute kidney injury risk evaluation requires direct glomerular filtration rate measurement and KFR, whereas kidney damage biomarkers facilitate identification of prior subclinical injury.

Plasma arginine metabolites in health and chronic kidney disease.

BACKGROUND: Elevated plasma asymmetric and symmetric dimethylarginine (ADMA and SDMA) are risk factors for chronic kidney disease (CKD) and cardiovascular disease. Using plasma cystatin C (pCYSC)-based estimated glomerular filtration rate (eGFR) trajectories, we identified a cohort at high risk of poor kidney-related health outcomes amongst members of the Dunedin Multidisciplinary Health and Development Study (DMHDS). We therefore examined associations between methylarginine metabolites and kidney function in this cohort. METHODS: ADMA, SDMA, L-arginine and L-citrulline were measured in plasma samples from 45-year-olds in the DMHDS cohort by liquid chromatography-tandem mass spectrometry. RESULTS: In a healthy DMHDS subset (n = 376), mean concentrations were: ADMA (0.40 ± 0.06 µmol/L), SDMA (0.42 ± 0.06 µmol/L), L-arginine (93.5 ± 23.1 µmol/L) and L-citrulline (24.0 ± 5.4 µmol/L). In the total cohort (n = 857), SDMA correlated positively with serum creatinine (Pearson's r = 0.55) and pCYSC (r = 0.55), and negatively with eGFR (r = 0.52). A separate cohort of 38 patients with stage 3-4 CKD (eGFR 15-60 mL/min/1.73 m2) confirmed significantly higher mean ADMA (0.61 ± 0.11 µmol/L), SDMA (0.65 ± 0.25 µmol/L) and L-citrulline (42.7 ± 11.8 µmol/L) concentrations. DMHDS members classified as high-risk of poor kidney health outcomes had significantly higher mean concentrations of all four metabolites compared with individuals not at risk. ADMA and SDMA individually predicted high-risk of poor kidney health outcomes with areas under the ROC curves (AUCs) of 0.83 and 0.84, and together with an AUC of 0.90. CONCLUSIONS: Plasma methylarginine concentrations facilitate stratification for risk of CKD progression.

Dynamic intron retention modulates gene expression in the monocytic differentiation pathway.

Monocytes play a crucial role in maintaining homeostasis and mediating a successful innate immune response. They also act as central players in diverse pathological conditions, thus making them an attractive therapeutic target. Within the bone marrow, monocytes arise from a committed precursor termed Common Monocyte Progenitor (cMoP). However, molecular mechanisms that regulate the differentiation of cMoP to various monocytic subsets remain unclear. Herein, we purified murine myeloid precursors for deep poly-A-enriched RNA sequencing to understand the role of alternative splicing in the development and differentiation of monocytes under homeostasis. Our analyses revealed intron retention to be the major alternative splicing mechanism involved in the monocyte differentiation cascade, especially in the differentiation of Ly6Chi monocytes to Ly6Clo monocytes. Furthermore, we found that the intron retention of key genes involved in the differentiation of murine Ly6Chi to Ly6Clo monocytes was also conserved in humans. Our data highlight the unique role of intron retention in the regulation of the monocytic differentiation pathway.

Towards resolution of the intron retention paradox in breast cancer.

BACKGROUND: After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in cancer. Many solid and liquid tumours are associated with drastic increases in IR, and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue. METHODS: In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR. RESULTS: Strikingly, we found that aberrantly decreasing IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA-binding protein expression and changes in tissue composition are among the causes of aberrantly decreasing IR in BrCa. CONCLUSIONS: Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.

p53 status determines the role of autophagy in pancreatic tumour development.

Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy's role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)--the most common mutational event in PDAC--develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.

Intron retention in mRNA: No longer nonsense: Known and putative roles of intron retention in normal and disease biology.

Until recently, retention of introns in mature mRNAs has been regarded as a consequence of mis-splicing. Intron-retaining transcripts are thought to be non-functional because they are readily degraded by nonsense-mediated decay. However, recent advances in next-generation sequencing technologies have enabled the detection of numerous transcripts that retain introns. As we review herein, intron-retaining mRNAs play an essential conserved role in normal physiology and an emergent role in diverse diseases. Intron retention should no longer be overlooked as a key mechanism that independently reduces gene expression in normal biology. Exploring its contribution to the development and/or maintenance of diseases is of increasing importance.

RBM3 regulates temperature sensitive miR-142-5p and miR-143 (thermomiRs), which target immune genes and control fever.

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

Detection of alternative lengthening of telomeres by telomere quantitative PCR.

Alternative lengthening of telomeres (ALT) is one of the two known telomere length maintenance mechanisms that are essential for the unlimited proliferation potential of cancer cells. Existing methods for detecting ALT in tumors require substantial amounts of tumor material and are labor intensive, making it difficult to study prevalence and prognostic significance of ALT in large tumor cohorts. Here, we present a novel strategy utilizing telomere quantitative PCR to diagnose ALT. The protocol is more rapid than conventional methods and scrutinizes two distinct characteristics of ALT cells concurrently: long telomeres and the presence of C-circles (partially double-stranded circles of telomeric C-strand DNA). Requiring only 30 ng of genomic DNA, this protocol will facilitate large-scale studies of ALT in tumors and can be readily adopted by clinical laboratories.

Targeting mTOR dependency in pancreatic cancer.

OBJECTIVE: Pancreatic cancer is a leading cause of cancer-related death in the Western world. Current chemotherapy regimens have modest survival benefit. Thus, novel, effective therapies are required for treatment of this disease. DESIGN: Activating KRAS mutation almost always drives pancreatic tumour initiation, however, deregulation of other potentially druggable pathways promotes tumour progression. PTEN loss leads to acceleration of Kras(G12D)-driven pancreatic ductal adenocarcinoma (PDAC) in mice and these tumours have high levels of mammalian target of rapamycin (mTOR) signalling. To test whether these KRAS PTEN pancreatic tumours show mTOR dependence, we compared response to mTOR inhibition in this model, to the response in another established model of pancreatic cancer, KRAS P53. We also assessed whether there was a subset of pancreatic cancer patients who may respond to mTOR inhibition. RESULTS: We found that tumours in KRAS PTEN mice exhibit a remarkable dependence on mTOR signalling. In these tumours, mTOR inhibition leads to proliferative arrest and even tumour regression. Further, we could measure response using clinically applicable positron emission tomography imaging. Importantly, pancreatic tumours driven by activated KRAS and mutant p53 did not respond to treatment. In human tumours, approximately 20% of cases demonstrated low PTEN expression and a gene expression signature that overlaps with murine KRAS PTEN tumours. CONCLUSIONS: KRAS PTEN tumours are uniquely responsive to mTOR inhibition. Targeted anti-mTOR therapies may offer clinical benefit in subsets of human PDAC selected based on genotype, that are dependent on mTOR signalling. Thus, the genetic signatures of human tumours could be used to direct pancreatic cancer treatment in the future.

Normal mammalian cells negatively regulate telomere length by telomere trimming.

In human cancer cells with telomeres that have been over-lengthened by exogenous telomerase activity, telomere shortening can occur by a process that generates circles of double-stranded telomeric DNA (t-circles). Here, we demonstrate that this telomeretrimming process occurs in cells of the male germline and in normal lymphocytes following mitogen-stimulated upregulation of telomerase activity. Mouse tissues also contain abundant t-circles, suggesting that telomere trimming also contributes to telomere length regulation in mice. In cancer cells and stimulated lymphocytes, the mechanism involves the XRCC3 homologous recombination (HR) protein and generates single-stranded C-rich telomeric DNA. This suggests that, in addition to the well-documented gradual telomere attrition that accompanies cellular replication, there is also a more rapid form of negative telomere length control in normal mammalian cells, which most likely involves HR-mediated removal of telomere loops in the form of t-circles. We therefore propose that this telomere trimming mechanism is an additional factor in the balance between telomere lengthening and telomere shortening in normal human germline and somatic cells that may prevent excessive lengthening by processes such as telomerase activity.

A gene therapy approach to eliminate HIV-1-infected cells.

The ideal therapy for HIV infection requires a method to eliminate all HIV-harboring cells in the infected individual. The authors are developing an HIV-specific promoter to drive the expression of suicide genes that would induce cell death specifically in HIV-infected cells. The authors constructed a promoter that is 100-fold more responsive to the HIV transcriptional activator, Tat, than cellular transcription factors, using a plasmid expressing luciferase under the control of the mutated LTR promoter.

Contribution of a common variant in the promoter of the 1-α-hydroxylase gene (CYP27B1) to fracture risk in the elderly.

CYP27B1 encodes mitochondrial 1α-hydroxylase, which converts 25-hydroxyvitamin D to its active 1,25-dihydroxylated metabolite. We tested the hypothesis that common variants in the CYP27B1 promoter are associated with fracture risk. The study was designed as a population-based genetic association study, which involved 153 men and 596 women aged 65-101 years, who had been followed for 2.2 years (range 0.1-5.5) between 1999 and 2006. During the follow-up period, the incidence of fragility fractures was ascertained. Bone ultrasound attenuation (BUA) was measured in all individuals, as were serum 25-hydroxyvitamin D and PTH concentrations; 86% subjects had vitamin D insufficiency. Genotypes were determined for the -1260C>A (rs10877012) and +2838T>C (rs4646536) CYP27B1 polymorphisms. A reporter gene assay was used to assess functional expression of the -1260C>A CYP27B1 variants. The association between genotypes and fracture risk was analyzed by Cox's proportional hazards model. We found that genotypic distribution of CYP27B1 -1260 and CYP27B1 +2838 polymorphisms was consistent with the Hardy-Weinberg equilibrium law. The two polymorphisms were in high linkage disequilibrium, with D' = 0.96 and r² = 0.94. Each C allele of the CYP27B1 -1260 polymorphism was associated with increased risk of fracture (hazard ratio = 1.34, 95% CI 1.03-1.73), after adjustment for age, sex, number of falls, and BUA. In transient transfection studies, a reporter gene downstream of the -1260(A)-containing promoter was more highly expressed than that containing the C allele. These data suggest that a common but functional variation within the CYP27B1 promoter gene is associated with fracture risk in the elderly.

Selection and validation of reference genes for normalisation of gene expression in ischaemic and toxicological studies in kidney disease.

Normalisation to standard reference gene(s) is essential for quantitative real-time polymerase chain reaction (RT-qPCR) to obtain reproducible and comparable results of a gene of interest (GOI) between subjects and under varying experimental conditions. There is limited evidence to support selection of the commonly used reference genes in rat ischaemic and toxicological kidney models. Employing these models, we determined the most stable reference genes by comparing 4 standard methods (NormFinder, qBase+, BestKeeper and comparative ΔCq) and developed a new 3-way linear mixed-effects model for evaluation of reference gene stability. This new technique utilises the intra-class correlation coefficient as the stability measure for multiple continuous and categorical covariates when determining the optimum normalisation factor. The model also determines confidence intervals for each candidate normalisation gene to facilitate selection and allow sample size calculation for designing experiments to identify reference genes. Of the 10 candidate reference genes tested, the geometric mean of polyadenylate-binding nuclear protein 1 (PABPN1) and beta-actin (ACTB) was the most stable reference combination. In contrast, commonly used ribosomal 18S and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were the most unstable. We compared the use of PABPN1×ACTB and 2 commonly used genes 18S and GAPDH on the expression of 4 genes of interest know to vary after renal injury and expressed by different kidney cell types (KIM-1, HIF1α, TGFß1 and PECAM1). The less stable reference genes gave varying patterns of GOI expression in contrast to the use of the least unstable reference PABPN1×ACTB combination; this improved detection of differences in gene expression between experimental groups. Reduced within-group variation of the now more accurately normalised GOI may allow for reduced experimental group size particularly for comparison between various models. This objective selection of stable reference genes increased the reliability of comparisons within and between experimental groups.

Advances in Detection of Kidney Transplant Injury.

Early detection of graft injury after kidney transplantation is key to maintaining long-term good graft function. Graft injury could be due to a multitude of factors including ischaemia reperfusion injury, cell or antibody-mediated rejection, progressive interstitial fibrosis and tubular atrophy, infections and toxicity from the immunosuppressive drugs themselves. The current gold standard for assessing renal graft dysfunction is renal biopsy. However, biopsy is usually late when triggered by a change in serum creatinine and of limited utility in diagnosis of early injury when histological changes are equivocal. Therefore, there is a need for timely, objective and non-invasive diagnostic techniques with good early predictive value to determine graft injury and provide precision in titrating immunosuppression. We review potential novel plasma and urine biomarkers that offer sensitive new strategies for early detection and provide major insights into mechanisms of graft injury. This is a rapidly expanding field, but it is likely that a combination of biomarkers will be required to provide adequate sensitivity and specificity for detecting graft injury.

Array-CGH identifies cyclin D1 and UBCH10 amplicons in anaplastic thyroid carcinoma.

Anaplastic thyroid cancer (ATC) is a rare but highly aggressive disease with largely unexplained etiology and molecular pathogenesis. In this study, we analyzed genome-wide copy number changes, BRAF (V-raf sarcoma viral oncogene homolog B1) mutations, and p16 and cyclin D1 expressions in a panel of ATC primary tumors. Three ATCs harbored the common BRAF mutation V600E. Using array-comparative genomic hybridisation (array-CGH), several distinct recurrent copy number alterations were revealed including gains in 16p11.2, 20q11.2, and 20q13.12. Subsequent fluorescence in situ hybridization revealed recurrent locus gain of UBCH10 in 20q13.12 and Cyclin D1 (CCND1) in 11q13. The detection of a homozygous loss encompassing the CDKN2A locus in 9p21.3 motivated the examination of p16 protein expression, which was undetectable in 24/27 ATCs (89%). Based on the frequent gain in 11q13 (41%; n=11), the role of CCND1 was further investigated. Expression of cyclin D1 protein was observed at varying levels in 18/27 ATCs (67%). The effect of CCND1 on thyroid cell proliferation was assessed in vitro in ATC cells by means of siRNA and in thyroid cells after CCND1 transfection. In summary, the recurrent chromosomal copy number changes and molecular alterations identified in this study may provide an insight into the pathogenesis and development of ATC.

A PCR-based expression signature of malignancy in follicular thyroid tumors.

The diagnosis of follicular thyroid carcinoma (FTC) in the absence of metastasis can only be established postoperatively. Moreover, high-risk FTCs are often not identifiable at the time of diagnosis. In this study, we aimed to identify transcriptional markers of malignancy and high-risk disease in follicular thyroid tumors. The expression levels of 26 potential markers of malignancy were determined in a panel of 75 follicular thyroid tumors by a TaqMan quantitative RT-PCR approach. Logistic regression analysis (LRA) was used for gene selection and generation of diagnostic and prognostic algorithms. An algorithm based on the expression levels of five genes (TERT, TFF3, PPARgamma, CITED1, and EGR2) could effectively predict high-risk disease with a specificity of 98.5%. The metastatic potential could be predicted in all four cases with apparently benign or minimally invasive (MI) disease at the time of diagnosis, but poor long-term outcome. In addition, a second model was produced by implementing two genes (TERT and TFF3), which was able to distinguish adenomas from de facto carcinomas. When this model was tested in an independent series of atypical adenomas (AFTA) and MI-FTCs, 16 out of 17 AFTAs were classified as 'benign', while MI-FTCs with vascular invasion (sometimes referred to as 'moderately invasive') and/or large tumor size tended to classify in the 'malignant' group. The reported models can be the foundation for the development of reliable preoperative diagnostic and prognostic tests that can guide the therapeutic approach of follicular thyroid neoplasms with indeterminate cytology.

Delineation, functional validation, and bioinformatic evaluation of gene expression in thyroid follicular carcinomas with the PAX8-PPARG translocation.

A subset of follicular thyroid carcinomas contains a balanced translocation, t(2;3)(q13;p25), that results in fusion of the paired box gene 8 (PAX8) and peroxisome proliferator-activated receptor gamma (PPARG) genes with concomitant expression of a PAX8-PPARgamma fusion protein, PPFP. PPFP is thought to contribute to neoplasia through a mechanism in which it acts as a dominant-negative inhibitor of wild-type PPARgamma. To better understand this type of follicular carcinoma, we generated global gene expression profiles using DNA microarrays of a cohort of follicular carcinomas along with other common thyroid tumors and used the data to derive a gene expression profile characteristic of PPFP-positive tumors. Transient transfection assays using promoters of four genes whose expression was highly associated with the translocation showed that each can be activated by PPFP. PPFP had unique transcriptional activities when compared with PAX8 or PPARgamma, although it had the potential to function in ways qualitatively similar to PAX8 or PPARgamma depending on the promoter and cellular environment. Bioinformatics analyses revealed that genes with increased expression in PPFP-positive follicular carcinomas include known PPAR target genes; genes involved in fatty acid, amino acid, and carbohydrate metabolism; micro-RNA target genes; and genes on chromosome 3p. These results have implications for the neoplastic mechanism of these follicular carcinomas.

IRFinder: assessing the impact of intron retention on mammalian gene expression.

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.

PAX8-peroxisome proliferator-activated receptor gamma (PPARgamma) disrupts normal PAX8 or PPARgamma transcriptional function and stimulates follicular thyroid cell growth.

Follicular thyroid carcinomas are associated with a chromosomal translocation that fuses the thyroid-specific transcription factor paired box gene 8 (PAX8) with the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). This study investigated the transcriptional mechanisms by which PAX8-PPARgamma regulates follicular thyroid cells. In HeLa cells, rat follicular thyroid (FRTL-5) cells, or immortalized human thyroid cells, PAX8-PPARgamma stimulated transcription from PAX8-responsive thyroperoxidase and sodium-iodide symporter promoters in a manner at least comparable with wild-type PAX8. In contrast, PAX8-PPARgamma failed to stimulate transcription from the thyroglobulin promoter and blocked the synergistic stimulation of this promoter by wild-type PAX8 and thyroid transcription factor-1. Unexpectedly, PAX8-PPARgamma transcriptional function on a PPARgamma-responsive promoter was cell-type dependent; in HeLa cells, PAX8-PPARgamma dominantly inhibited expression of the PPARgamma-responsive promoter, whereas in FRTL-5 and immortalized human thyroid cells PAX8-PPARgamma stimulated this promoter. In gel shift analyses, PAX8-PPARgamma bound a PPARgamma-response element suggesting that its transcriptional function is mediated via direct DNA contact. A biological model of PAX8-PPARgamma function in follicular thyroid cells was generated via constitutive expression of the fusion protein in FRTL-5 cells. In this model, PAX8-PPARgamma expression was associated with enhanced growth as assessed by soft agar assays and thymidine uptake. Therefore, PAX8-PPARgamma disrupts normal transcriptional regulation by stimulating some genes and inhibiting others, the net effect of which may mediate follicular thyroid cell growth and loss of differentiation that ultimately leads to carcinogenesis.

The Ras effector NORE1A is suppressed in follicular thyroid carcinomas with a PAX8-PPARgamma fusion.

CONTEXT: The Ras effector NORE1A (RASSF5A) is a putative tumor suppressor and is inactivated in several human cancers. NORE1A has not been studied in thyroid cancer. OBJECTIVE: The objective of this study was to investigate whether NORE1A is involved in follicular thyroid cancer (FTC) development. DESIGN: We analyzed NORE1A expression in 25 FTCs, eight follicular thyroid adenomas, and seven normal thyroid tissues by TaqMan quantitative RT-PCR. The results were evaluated in relation to RASSF1A expression, RAS mutations, and PAX8-PPARgamma fusions assessed in the same material. NORE1A promoter methylation was assessed by the combined bisulfite restriction endonuclease assay. RESULTS: Although the NORE1A mRNA levels of the majority of the tumors were similar to those in the normal controls, the cases harboring a PAX8-PPARgamma translocation (n = 6) exhibited dramatically reduced NORE1A expression (P < 0.001). In contrast, RAS mutations (n = 5) and NORE1A down-regulation were mutually exclusive. A significant reduction in the expression of the NORE1A homolog and the bona fide tumor suppressor gene RASSF1A was observed, but with weak correlation to the respective NORE1A values. No NORE1A promoter methylation was detected in the 32 thyroid tumors analyzed. CONCLUSIONS: Our experiments demonstrate the suppression of NORE1A, a known Ras effector, in PAX8-PPARgamma carrying FTCs.