Publications by authors named "Bin T Teh"

61 Publications

Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.

Hum Cell 2020 Jul 23;33(3):695-708. Epub 2020 Mar 23.

Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand.

Three cholangiocarcinoma (CCA) cell line-formerly named, M156, M213 and M214 have been intensively used with discrepancy of their tumor origins. They were assumed to be originated from three different donors without authentication. To verify the origins of these cell lines, the short tandem repeat (STR) analysis of the currently used cell lines, the cell stocks from the establisher and the primary tumor of a CCA patient were performed. Their phenotypic and genotypic originality were compared. The currently used 3 CCA cell lines exhibited similar STR as CCA patient ID-M213 indicating the same origin of these cells. The cell stocks from the establisher, however, revealed the same STR of M213 and M214 cells, but not M156. The misidentification of M214 and M156 is probably due to the mislabeling and cross-contamination of M213 cells during culture. These currently used cell lines were renamed as KKU-213A, -213B and -213C, for the formerly M213, M214 and M156 cells, respectively. These cell lines were established from a male with an intrahepatic mass-forming CCA stage-4B. The tumor was an adenosquamous carcinoma with the liver fluke ova granuloma in evidence. All cell lines had positive CK19 with differential CA19-9 expression. They exhibited aneuploidy karyotypes, distinct cell morphology, cell growth, cytogenetic characteristic and progressive phenotypes. KKU-213C formed a adenosquamous carcinoma, whereas KKU-213A and KKU-213B formed poorly- and well-differentiated squamous cell carcinomas in xenografted mice. mRNA microarray revealed different expression profiles among these three cell lines. The three cell lines have unique characteristics and may resemble the heterogeneity of tumor origin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13577-020-00334-wDOI Listing
July 2020

Aristolochic acids and their derivatives are widely implicated in liver cancers in Taiwan and throughout Asia.

Sci Transl Med 2017 Oct;9(412)

Centre for Computational Biology, Duke-NUS Medical School, Singapore 169857, Singapore.

Many traditional pharmacopeias include and related plants, which contain nephrotoxins and mutagens in the form of aristolochic acids and similar compounds (collectively, AA). AA is implicated in multiple cancer types, sometimes with very high mutational burdens, especially in upper tract urothelial cancers (UTUCs). AA-associated kidney failure and UTUCs are prevalent in Taiwan, but AA's role in hepatocellular carcinomas (HCCs) there remains unexplored. Therefore, we sequenced the whole exomes of 98 HCCs from two hospitals in Taiwan and found that 78% showed the distinctive mutational signature of AA exposure, accounting for most of the nonsilent mutations in known cancer driver genes. We then searched for the AA signature in 1400 HCCs from diverse geographic regions. Consistent with exposure through known herbal medicines, 47% of Chinese HCCs showed the signature, albeit with lower mutation loads than in Taiwan. In addition, 29% of HCCs from Southeast Asia showed the signature. The AA signature was also detected in 13 and 2.7% of HCCs from Korea and Japan as well as in 4.8 and 1.7% of HCCs from North America and Europe, respectively, excluding one U.S. hospital where 22% of 87 "Asian" HCCs had the signature. Thus, AA exposure is geographically widespread. Asia, especially Taiwan, appears to be much more extensively affected, which is consistent with other evidence of patterns of AA exposure. We propose that additional measures aimed at primary prevention through avoidance of AA exposure and investigation of possible approaches to secondary prevention are warranted.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aan6446DOI Listing
October 2017

Clear cell sarcomas of the kidney are characterised by BCOR gene abnormalities, including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion.

Histopathology 2018 Jan 30;72(2):320-329. Epub 2017 Oct 30.

VIVA-KKH Paediatric Brain and Solid Tumour Laboratory, KK Women's and Children's Hospital, Singapore.

Aims: Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically.

Methods And Results: By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression.

Conclusions: The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/his.13366DOI Listing
January 2018

Osteoblast-specific deletion of Hrpt2/Cdc73 results in high bone mass and increased bone turnover.

Bone 2017 05 3;98:68-78. Epub 2017 Apr 3.

Program for Skeletal Disease and Tumor Microenvironment, Grand Rapids, MI, USA; Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI, USA. Electronic address:

Inactivating mutations that lead to loss of heterozygosity within the HRPT2/Cdc73 gene are directly linked to the development of primary hyperparathyroidism, parathyroid adenomas, and ossifying fibromas of the jaw (HPT-JT). The protein product of the Cdc73 gene, parafibromin, is a core member of the polymerase-associated factors (PAF) complex, which coordinates epigenetic modifiers and transcriptional machinery to control gene expression. We conditionally deleted Cdc73 within mesenchymal progenitors or within mature osteoblasts and osteocytes to determine the consequences of parafibromin loss within the mesenchymal lineage. Homozygous deletion of Cdc73 via the Dermo1-Cre driver resulted in embryos which lacked mesenchymal organ development of internal organs, including the heart and fetal liver. Immunohistochemical detection of cleaved caspase-3 revealed extensive apoptosis within the progenitor pools of developing organs. Unexpectedly, when Cdc73 was homozygously deleted within mature osteoblasts and osteocytes (via the Ocn-Cre driver), the mice had a normal life span but increased cortical and trabecular bone. OCN-Cre;Cdc73 bones displayed large cortical pores actively undergoing bone remodeling. Additionally the cortical bone of OCN-Cre;Cdc73 femurs contained osteocytes with marked amounts of cytoplasmic RNA and a high rate of apoptosis. Transcriptional analysis via RNA-seq within OCN-Cre;Cdc73 osteoblasts showed that loss of Cdc73 led to a derepression of osteoblast-specific genes, specifically those for collagen and other bone matrix proteins. These results aid in our understanding of the role parafibromin plays within transcriptional regulation, terminal differentiation, and bone homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2016.12.006DOI Listing
May 2017

Genomic profiling reveals mutational landscape in parathyroid carcinomas.

JCI Insight 2017 03 23;2(6):e92061. Epub 2017 Mar 23.

Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

Parathyroid carcinoma (PC) is an extremely rare malignancy lacking effective therapeutic intervention. We generated and analyzed whole-exome sequencing data from 17 patients to identify somatic and germline genetic alterations. A panel of selected genes was sequenced in a 7-tumor expansion cohort. We show that 47% (8 of 17) of the tumors harbor somatic mutations in the tumor suppressor, with germline inactivating variants in 4 of the 8 patients. The PI3K/AKT/mTOR pathway was altered in 21% of the 24 cases, revealing a major oncogenic pathway in PC. We observed amplification in 29% of the 17 patients, and a previously unreported recurrent mutation in putative kinase . We identified the first sporadic PCs with somatic mutations in the Wnt canonical pathway, complementing previously described epigenetic mechanisms mediating Wnt activation. This is the largest genomic sequencing study of PC, and represents major progress toward a full molecular characterization of this rare malignancy to inform improved and individualized treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.92061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358487PMC
March 2017

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines.

Head Neck 2017 05 25;39(5):840-852. Epub 2017 Feb 25.

Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor, Michigan.

Background: Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines.

Methods: E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites.

Results: All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration.

Conclusion: HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hed.24729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392184PMC
May 2017

A germline mutation in PBRM1 predisposes to renal cell carcinoma.

J Med Genet 2015 Jun 24;52(6):426-30. Epub 2015 Apr 24.

Ecole Pratique des Hautes Etudes, Paris, France Laboratoire de Génétique Oncologique EPHE, INSERM U753, Villejuif, France Faculté de Médecine Université Paris-Sud, Le Kremlin-Bicêtre, France.

Background: Many cases of familial renal cell carcinoma (RCC) remain unexplained by mutations in the known predisposing genes or shared environmental factors. There are therefore additional, still unidentified genes involved in familial RCC. PBRM1 is a tumour suppressor gene and somatic mutations are found in 30-45% of sporadic clear cell (cc) RCC.

Methods: We selected 35 unrelated patients with unexplained personal history of ccRCC and at least one affected first-degree relative, and sequenced the PBRM1 gene.

Results: A germline frameshift mutation (c.3998_4005del [p.Asp1333Glyfs]) was found in one patient. The patient's mother, his sister and one niece also had ccRCC. The mutation co-segregated with the disease as the three affected relatives were carriers, while an unaffected sister was not, according with autosomal-dominant transmission. Somatic studies supported these findings, as we observed both loss of heterozygosity for the mutation and loss of protein expression in renal tumours.

Conclusions: We show for the first time that an inherited mutation in PBRM1 predisposes to RCC. International studies are necessary to estimate the contribution of PBRM1 to RCC susceptibility, estimate penetrance and then integrate the gene into routine clinical practice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/jmedgenet-2014-102912DOI Listing
June 2015

Whole-exome sequencing studies of parathyroid carcinomas reveal novel PRUNE2 mutations, distinctive mutational spectra related to APOBEC-catalyzed DNA mutagenesis and mutational enrichment in kinases associated with cell migration and invasion.

J Clin Endocrinol Metab 2015 Feb 11;100(2):E360-4. Epub 2014 Nov 11.

Laboratory of Cancer Epigenome (W.Y., H.L.H., A.G., D.H., S.L.P., C.K.O., B.T.T.), Division of Medical Sciences, National Cancer Centre Singapore, 169610 Singapore; Division of Cancer and Stem Cell Biology (W.Y., J.R.M., H.L.H., D.H., S.L.P., C.K.O., S.G.R., P.T., B.T.T.), Duke-National University of Singapore Graduate Medical School, 169857 Singapore; National University of Singapore Graduate School for Integrative Sciences and Engineering (W.Y.), 117456 Singapore; Academic Endocrine Unit, Nuffield Department of Clinical Medicine (M.S., P.N., R.V.T.), Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, OX3 7LJ Oxford, United Kingdom; Department of Pathology (R.v.E., D.R., T.v.W., H.M.), Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; Molecular Endocrinology Group, Molecular Pathobiology Research Centre Unit (U.I.P.M.) (B.C.) of the Portuguese Institute of Oncology from Lisbon Francisco Gentil (IPOLFG) and Chronic Diseases Research Centre (CEDOC), Faculty of Medical Sciences (FCM), New University of Lisbon (UNL), 1099-023 Lisbon, Portugal; Genome Institute of Singapore (P.T.), 138672 Singapore; and Cancer Science Institute of Singapore (P.T., B.T.T.), National University of Singapore, 117599 Singapore.

Context: Cell division cycle 73 (CDC73), encoding the protein parafibromin, is the most prevalent mutated gene in familial and sporadic parathyroid carcinoma (PC).

Objective: To identify additional genetic abnormalities in PCs.

Design: Whole-exome sequencing was performed using DNA from seven pairs of matched PCs and one triplet containing double primary tumor and normal leukocyte. Somatic variants were confirmed using Sanger sequencing and recurrently mutated genes were assessed in 13 additional PCs as well as 40 parathyroid adenomas (PA).

Results: PC had an average of 51 somatic variants/tumor (range 3-176) with approximately 58% of variants occurring as nonsynonymous single nucleotide variants. The importance of CDC73 in PC is reinforced with a remarkable preferential amplification of the mutant CDC73 allele. Furthermore, recurrent germ line and somatic mutations in prune homolog 2 [Drosophila] (PRUNE2) were found in PC and computationally predicted to be deleterious; in addition, recurrent mutations in kinase genes related to cell migration and invasion were found. PRUNE2 showed recurrent mutations in 18% (4/22) of PCs with additional screening in 40 PAs revealing only one rare missense polymorphism (Asp1677Asn). For the first time, the mutational signature associated with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-catalyzed cytosine-to-uracil deamination is found in a subset of PC.

Conclusion: This study outlines the genetic landscape of PC and attempts to characterize the mutational processes shaping the PC genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/jc.2014-3238DOI Listing
February 2015

Glypican 3 overexpression in primary and metastatic Wilms tumors.

Virchows Arch 2015 Jan 4;466(1):67-76. Epub 2014 Nov 4.

Department of Pathology, University of Washington, 325 9th Ave, Seattle, WA, 98104, USA,

Glypican 3 (GPC3), a heparan sulfate proteoglycan, plays a role in cell growth and differentiation. Mutations of the GPC3 gene are responsible for Simpson-Golabi-Behmel syndrome, which is characterized by anomalies of postnatal overgrowth and an increased risk of developing pediatric malignancies, mostly Wilms tumor and liver cancer. In order to understand the possible role of GPC3 in renal development and Wilms tumor formation, we analyzed messenger RNA (mRNA) and protein levels of GPC3 in sporadic Wilms tumors and compared it to normal kidneys and other common renal epithelial tumors. By using Affymetrix HGU133 oligonucleotide gene expression microarray data from 191 renal tumors and 12 normal kidneys, we found significant overexpression of GPC3 in Wilms tumors (p < 0.01), with 3.5-fold higher expression in comparison to normal kidneys and 6.5-fold higher than any type of renal tumors. The GPC3 gene product in Wilms tumor was further evaluated by immunohistochemistry and quantified by an automated image analysis. Cytoplasmic and membranous GPC3 immunoreactivity was present in 77 % of primary Wilms tumors (23/30), 93 % of metastatic Wilms tumors (13/14), 50 % of metanephric adenomas (4/8), 33 % of congenital mesoblastic nephromas (2/6), 100 % of nephrogenic rests (11/11), and 100 % of fetal kidneys (5/5). GPC3 staining was predominantly identified in blastemal and epithelial components of Wilms tumors, similar to that of fetal non-neoplastic kidney. All adult renal tumors (n = 60) and normal kidneys (n = 15) were GPC3 negative. These findings suggest the utility of GPC3 in differential diagnosis and follow-up of Wilms tumors. Our data also indicate that GPC3 is an oncofetal protein with a potential therapeutic value.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00428-014-1669-4DOI Listing
January 2015

Next-generation sequencing of translocation renal cell carcinoma reveals novel RNA splicing partners and frequent mutations of chromatin-remodeling genes.

Clin Cancer Res 2014 Aug 4;20(15):4129-40. Epub 2014 Jun 4.

Genitourinary Medical Oncology,

Purpose: MITF/TFE translocation renal cell carcinoma (TRCC) is a rare subtype of kidney cancer. Its incidence and the genome-wide characterization of its genetic origin have not been fully elucidated.

Experimental Design: We performed RNA and exome sequencing on an exploratory set of TRCC (n = 7), and validated our findings using The Cancer Genome Atlas (TCGA) clear-cell RCC (ccRCC) dataset (n = 460).

Results: Using the TCGA dataset, we identified seven TRCC (1.5%) cases and determined their genomic profile. We discovered three novel partners of MITF/TFE (LUC7L3, KHSRP, and KHDRBS2) that are involved in RNA splicing. TRCC displayed a unique gene expression signature as compared with other RCC types, and showed activation of MITF, the transforming growth factor β1 and the PI3K complex targets. Genes differentially spliced between TRCC and other RCC types were enriched for MITF and ID2 targets. Exome sequencing of TRCC revealed a distinct mutational spectrum as compared with ccRCC, with frequent mutations in chromatin-remodeling genes (six of eight cases, three of which were from the TCGA). In two cases, we identified mutations in INO80D, an ATP-dependent chromatin-remodeling gene, previously shown to control the amplitude of the S phase. Knockdown of INO80D decreased cell proliferation in a novel cell line bearing LUC7L3-TFE3 translocation.

Conclusions: This genome-wide study defines the incidence of TRCC within a ccRCC-directed project and expands the genomic spectrum of TRCC by identifying novel MITF/TFE partners involved in RNA splicing and frequent mutations in chromatin-remodeling genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-13-3036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4167829PMC
August 2014

Gene profiling suggests a common evolution of bladder cancer subtypes.

BMC Med Genomics 2013 Oct 17;6:42. Epub 2013 Oct 17.

Department of Pathology, University of California at San Diego, 9500 Gilman Drive, MC 0612, La Jolla, CA 92093, USA.

Background: Bladder cancer exists as several distinct subtypes, including urothelial carcinoma (UCa), squamous cell carcinoma (SCCa), adenocarcinoma and small cell carcinoma. These entities, despite showing distinct morphology and clinical behavior, arise from the urothelial lining and are often accompanied by similar precursor/in situ findings. The relationship between these subtypes has not been explored in detail.

Methods: We compared gene expression analysis of the two most common subtypes of bladder cancer, UCa (n = 10) and SCCa (n = 9), with an additional comparison to normal urothelium from non-cancer patients (n = 8) using Affymetrix GeneChip Human genome arrays (Affymetrix, Santa Clara, CA). The results were stratified by supervised and unsupervised clustering analysis, as well as by overall fold change in gene expression.

Results: When compared to normal urothelium, UCa showed differential expression of 155 genes using a 5-fold cut-off. Examples of differentially regulated genes included topoisomerases, cancer-related transcription factors and cell cycle mediators. A second comparison of normal urothelium to SCCa showed differential expression of 503 genes, many of which were related to squamous-specific morphology (desmosomal complex, intermediate filaments present within squamous epithelium, squamous cornifying proteins, and molecules upregulated in squamous carcinomas from other anatomic sites). When compared, 137 genes were commonly dysregulated in both UCa and SCCa as compared to normal urothelium. All dysregulated genes in UCa were shared in common with SCCa, with the exception of only 18 genes. Supervised clustering analysis yielded correct classification of lesions in 26/27 (96%) of cases and unsupervised clustering analysis yielded correct classification in 25/27 (92.6%) of cases.

Conclusions: The results from this analysis suggest that bladder SCCa shares a significant number of gene expression changes with conventional UCa, but also demonstrates an additional set of alterations that is unique to this entity that defines the squamous phenotype. The similarity in deregulated gene products suggests that SCCa may be a much more closely related entity at the molecular level to conventional UCa than previously hypothesized.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1755-8794-6-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015777PMC
October 2013

Molecular targets on the horizon for kidney and urothelial cancer.

Nat Rev Clin Oncol 2013 Oct 27;10(10):557-70. Epub 2013 Aug 27.

Dana-Farber and Brigham and Women's Cancer Center, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215, USA and Hospital del Mar-IMIM-IMAS, Doctor Aiguader 88, 08003 Barcelona, Spain.

As whole-genome sequencing technology rapidly advances, the insights gained from deciphering cancer genomes are shifting the paradigm in the diagnosis and treatment of cancer with the promise of individualized treatment for each patient. Information gained in this way is extensive for certain cancers, but fairly limited in renal cell carcinomas and urothelial carcinoma. Mutations in multiple, potentially druggable genes have been identified in urothelial carcinomas; however, the association between molecular alterations and clinical outcome has not yet been robustly demonstrated. Data in this area are emerging in renal cell carcinoma, leading to the development of targeted agents that have improved overall survival. Unfortunately, these treatments rarely yield complete responses, are not curative, and development of resistance ensues. This Review will focus on the biology of non-hormonally driven urological cancers. We discuss how approaches using whole-genome sequencing can facilitate the discovery of biomarkers of drug sensitivity in both renal cell carcinomas and urothelial carcinomas. For renal cell carcinomas, we will describe how genomic and epigenomic mining has uncovered novel genes and pathways involved in tumorigenesis, tumour classification and mechanisms of resistance in the various subsets of this disease and the potential for exploiting these discoveries in the clinic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrclinonc.2013.155DOI Listing
October 2013

The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell-cycle progression in malignant bladder cancer cells in vitro and in vivo.

Clin Cancer Res 2013 Apr 12;19(7):1717-28. Epub 2013 Feb 12.

Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH 44195, USA.

Purpose: Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target.

Experimental Design: Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model.

Results: A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells.

Conclusions: Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-12-2383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626291PMC
April 2013

Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis.

Hum Mol Genet 2012 Dec 10;21(24):5268-79. Epub 2012 Sep 10.

Department of Medical and Molecular Genetics, Centre for Rare Diseases and Personalized Medicine, School of Clinical and Experimental Medicine, University of Birmingham College of Medical and Dental Sciences, Edgbaston, Birmingham, UK.

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/dds378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755511PMC
December 2012

Functional importance of Dicer protein in the adaptive cellular response to hypoxia.

J Biol Chem 2012 Aug 28;287(34):29003-20. Epub 2012 Jun 28.

Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5B 1W8, Canada.

The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.373365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436557PMC
August 2012

Combined gene expression profiling and RNAi screening in clear cell renal cell carcinoma identify PLK1 and other therapeutic kinase targets.

Cancer Res 2011 Aug 3;71(15):5225-34. Epub 2011 Jun 3.

Van Andel Research Institute, Grand Rapids, MI, USA.

In recent years, several molecularly targeted therapies have been approved for clear cell renal cell carcinoma (ccRCC), a highly aggressive cancer. Although these therapies significantly extend overall survival, nearly all patients with advanced ccRCC eventually succumb to the disease. To identify other molecular targets, we profiled gene expression in 90 ccRCC patient specimens for which tumor grade information was available. Gene set enrichment analysis indicated that cell-cycle-related genes, in particular, Polo-like kinase 1 (PLK1), were associated with disease aggressiveness. We also carried out RNAi screening to identify kinases and phosphatases that when inhibited could prevent cell proliferation. As expected, RNAi-mediated knockdown of PLK1 and other cell-cycle kinases was sufficient to suppress ccRCC cell proliferation. The association of PLK1 in both disease aggression and in vitro growth prompted us to examine the effects of a small-molecule inhibitor of PLK1, BI 2536, in ccRCC cell lines. BI 2536 inhibited the proliferation of ccRCC cell lines at concentrations required to inhibit PLK1 kinase activity, and sustained inhibition of PLK1 by BI 2536 led to dramatic regression of ccRCC xenograft tumors in vivo. Taken together, these findings highlight PLK1 as a rational therapeutic target for ccRCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-11-0076DOI Listing
August 2011

Deregulation of E2-EPF ubiquitin carrier protein in papillary renal cell carcinoma.

Am J Pathol 2011 Feb;178(2):853-60

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. We analyzed primary tumor specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays constructed from an additional 57 paraffin-embedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumor samples and established renal cell carcinoma cell lines, and potential associations with pathologic variables and survival in 27 patients with follow-up information were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1α expression, and is associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements within the E2-EPF promoter, and for the first time we demonstrated that E2-EPF is a hypoxia-inducible gene directly regulated via HIF1. These findings reveal deregulation of the oxygen-sensing pathway impinging on the positive feedback mechanism of HIF1-mediated regulation of E2-EPF in PRCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajpath.2010.10.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070594PMC
February 2011

Downregulation of CASR expression and global loss of parafibromin staining are strong negative determinants of prognosis in parathyroid carcinoma.

Mod Pathol 2011 May 14;24(5):688-97. Epub 2011 Jan 14.

Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands.

Parathyroid carcinoma is associated with mutations in the HRPT2/CDC73 gene and with decreased parafibromin and calcium-sensing receptor (CASR) expression, but in some cases establishing an unequivocal diagnosis remains a challenge. The aim of our study was to evaluate the prognostic value of CASR and parafibromin expression and of HRPT2/CDC73 mutations in patients with an established diagnosis of parathyroid carcinoma. Data on survival and disease-free survival were obtained from hospital records of 23 patients with an established diagnosis of parathyroid carcinoma in whom CASR and parafibromin expression and HRPT2/CDC73 mutation analyses were available from paraffin-embedded pathological specimens. Kaplan-Meier curves were used for survival analysis. Downregulation of CASR expression, global loss of parafibromin staining and a HRPT2/CDC73 mutation were, respectively, found in 7 (30%), 13 (59%) and 4 (17%) patients, and were associated with, respectively, 16-fold, 4-fold and 7-fold increased risk of developing local or distant metastasis. These findings suggest that although downregulation of CASR expression, global loss of parafibromin staining and mutations in the HRPT2/CDC73 gene are tools of proven value to assist in establishing a diagnosis of parathyroid carcinoma, their absence does not exclude it. Notwithstanding, we demonstrate a significant added value of these markers as strong determinants of increased malignant potential and thus as negative prognostic markers in this malignancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/modpathol.2010.236DOI Listing
May 2011

Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas.

Proc Natl Acad Sci U S A 2011 Jan 10;108(4):1439-44. Epub 2011 Jan 10.

Laboratory of Systems Biology, Van Andel Research Institute, Grand Rapids, MI 49503, USA.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1012500108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3029686PMC
January 2011

Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression.

BMC Med Genomics 2010 Dec 16;3:59. Epub 2010 Dec 16.

Laboratory of Computational Biology, Van Andel Research Institute, Grand Rapids, MI, USA.

Background: Germline mutations in the folliculin (FLCN) gene are associated with the development of Birt-Hogg-Dubé syndrome (BHDS), a disease characterized by papular skin lesions, a high occurrence of spontaneous pneumothorax, and the development of renal neoplasias. The majority of renal tumors that arise in BHDS-affected individuals are histologically similar to sporadic chromophobe renal cell carcinoma (RCC) and sporadic renal oncocytoma. However, most sporadic tumors lack FLCN mutations and the extent to which the BHDS-derived renal tumors share genetic defects associated with the sporadic tumors has not been well studied.

Methods: BHDS individuals were identified symptomatically and FLCN mutations were confirmed by DNA sequencing. Comparative gene expression profiling analyses were carried out on renal tumors isolated from individuals afflicted with BHDS and a panel of sporadic renal tumors of different subtypes using discriminate and clustering approaches. qRT-PCR was used to confirm selected results of the gene expression analyses. We further analyzed differentially expressed genes using gene set enrichment analysis and pathway analysis approaches. Pathway analysis results were confirmed by generation of independent pathway signatures and application to additional datasets.

Results: Renal tumors isolated from individuals with BHDS showed distinct gene expression and cytogenetic characteristics from sporadic renal oncocytoma and chromophobe RCC. The most prominent molecular feature of BHDS-derived kidney tumors was high expression of mitochondria-and oxidative phosphorylation (OXPHOS)-associated genes. This mitochondria expression phenotype was associated with deregulation of the PGC-1α-TFAM signaling axis. Loss of FLCN expression across various tumor types is also associated with increased nuclear mitochondrial gene expression.

Conclusions: Our results support a genetic distinction between BHDS-associated tumors and other renal neoplasias. In addition, deregulation of the PGC-1α-TFAM signaling axis is most pronounced in renal tumors that harbor FLCN mutations and in tumors from other organs that have relatively low expression of FLCN. These results are consistent with the recently discovered interaction between FLCN and AMPK and support a model in which FLCN is a regulator of mitochondrial function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1755-8794-3-59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012009PMC
December 2010

Comparative gene expression profiling analysis of urothelial carcinoma of the renal pelvis and bladder.

BMC Med Genomics 2010 Dec 15;3:58. Epub 2010 Dec 15.

Van Andel Research Institute, Grand Rapids, MI 49503, USA.

Background: Urothelial carcinoma (UC) can arise at any location along the urothelial tract, including the urethra, bladder, ureter, or renal pelvis. Although tumors arising in these various locations have similar morphology, it is unclear whether the gene expression profiles are similar between the upper-tract (ureter and renal pelvis) and lower-tract (bladder and urethra) carcinomas. Because differences may facilitate different screening and treatment modalities, we sought to examine the relationship between urothelial carcinoma of the renal pelvis (rUC) and urothelial carcinoma of the bladder (bUC).

Methods: Fresh tumor tissue was collected from patients with bUC (n = 10) and benign mucosa from the bladder of individuals undergoing resection for non-UC conditions (n = 7). Gene expression profiles from these samples were determined using high-throughput Affymetrix gene expression microarray chips. Bioinformatic approaches were used to compare the gene expression profiles of these samples with those of rUC samples and normal kidney samples that had been described previously.

Results: Using unsupervised analytic approaches, rUC and bUC were indistinguishable. Yet when a supervised analytic approach was used, a small number of differentially expressed genes were identified; these differences were most likely limited to a single pathway--the chloride ion binding activity pathway--which was more frequently activated in rUC than in bUC.

Conclusions: We found that the gene expression profiles of UCs from the upper and lower tract were extremely similar, suggesting that similar pathogenic mechanisms likely function in the development of these tumors. The differential expression of genes in the identified pathway may represent a new avenue for detection of upper-tract tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1755-8794-3-58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022544PMC
December 2010

Prospective clinical trial of preoperative sunitinib in patients with renal cell carcinoma.

J Urol 2010 Sep;184(3):859-64

Department of Urology, Roswell Park Cancer Institute, Buffalo, New York, USA.

Purpose: Sunitinib is an approved treatment for metastatic renal cell carcinoma. We performed a prospective clinical trial to evaluate the safety and clinical response to sunitinib administered before nephrectomy in patients with localized or metastatic clear cell renal cell carcinoma.

Materials And Methods: Patients with biopsy proven clear cell renal cell carcinoma were enrolled in the study and treated with 37.5 mg sunitinib malate daily for 3 months before nephrectomy. The primary end point was safety.

Results: In an 18-month period 20 patients were enrolled. The most common toxicities were gastrointestinal symptoms and hematological effects. Grade 3 toxicity developed in 6 patients (30%). No surgical complications were attributable to sunitinib treatment. Of the 20 patients 17 (85%) experienced reduced tumor diameter (mean change -11.8%, range -27% to 11%) and cross-sectional area (mean change -27.9%, range -43% to 23%). Enhancement on contrast enhanced computerized tomography decreased in 15 patients (mean HU change -22%, range -74% to 29%). After tumor reduction 8 patients with cT1b disease underwent laparoscopic partial nephrectomy. Surgical parameters, such as blood loss, transfusion rate, operative time and complications, were similar to those in patients who underwent surgery during the study period and were not enrolled in the trial.

Conclusions: Preoperative treatment with sunitinib is safe. Sunitinib decreased the size of primary renal cell carcinoma in 17 of 20 patients. Future trials can be considered to evaluate neoadjuvant sunitinib to maximize nephron sparing and decrease the recurrence of high risk, localized renal cell carcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.juro.2010.05.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105599PMC
September 2010

Reversible epithelial to mesenchymal transition and acquired resistance to sunitinib in patients with renal cell carcinoma: evidence from a xenograft study.

Mol Cancer Ther 2010 Jun 25;9(6):1525-35. Epub 2010 May 25.

Johns Hopkins University, Baltimore, Maryland, USA.

Tyrosine kinase inhibitors (TKI) targeting angiogenesis via inhibition of the vascular endothelial growth factor pathway have changed the medical management of metastatic renal cell carcinoma. Although treatment with TKIs has shown clinical benefit, these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. To address this question, we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response to sunitinib and eventually progressed with therapy. Tumor pieces were grafted s.c. in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density, and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor-derived cell line was established and assessed in vitro for potential direct antitumor effects of sunitinib. To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg, sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histologic examination of the original skin metastasis revealed evidence of epithelial to mesenchymal transition, whereas the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. In conclusion, the histologic examination in this xenograft study suggests that reversible epithelial to mesenchymal transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-09-1106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049816PMC
June 2010

Kinase targets in renal-cell carcinomas: reassessing the old and discovering the new.

Lancet Oncol 2010 Jun 8;11(6):571-8. Epub 2010 Apr 8.

Laboratory of Computational Biology, Van Andel Research Institute, Grand Rapids, MI, USA.

Renal-cell carcinoma is a heterogeneous group of tumours that arise in the adult kidneys. Irrespective of the type of renal tumour, traditional chemotherapeutic and radiation-based therapies have been largely ineffective at treating advanced tumours, with long-term survival being very low. Molecularly-targeted inhibitors of protein kinases are effective in delaying progression of advanced renal tumours. These therapies revolve around inhibition of the vascular endothelial growth factor receptor tyrosine kinase and the mammalian target of rapamycin serine or threonine kinase signalling pathways. The genetic complexity of renal tumours revealed by gene-expression profiling and other molecular-genetic technologies indicate that inhibition of additional kinase-associated pathways could also prevent renal tumour growth. In this review, we discuss the use of molecularly-targeted kinase inhibitors in the treatment of renal-cell carcinoma and identify the next generation of kinase inhibitors that show promise for treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S1470-2045(09)70380-8DOI Listing
June 2010

Signatures of mutation and selection in the cancer genome.

Nature 2010 Feb;463(7283):893-8

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

The cancer genome is moulded by the dual processes of somatic mutation and selection. Homozygous deletions in cancer genomes occur over recessive cancer genes, where they can confer selective growth advantage, and over fragile sites, where they are thought to reflect an increased local rate of DNA breakage. However, most homozygous deletions in cancer genomes are unexplained. Here we identified 2,428 somatic homozygous deletions in 746 cancer cell lines. These overlie 11% of protein-coding genes that, therefore, are not mandatory for survival of human cells. We derived structural signatures that distinguish between homozygous deletions over recessive cancer genes and fragile sites. Application to clusters of unexplained homozygous deletions suggests that many are in regions of inherent fragility, whereas a small subset overlies recessive cancer genes. The results illustrate how structural signatures can be used to distinguish between the influences of mutation and selection in cancer genomes. The extensive copy number, genotyping, sequence and expression data available for this large series of publicly available cancer cell lines renders them informative reagents for future studies of cancer biology and drug discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature08768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145113PMC
February 2010

Dual KS: Defining Gene Sets with Tissue Set Enrichment Analysis.

Cancer Inform 2010 Jan 21;9:1-9. Epub 2010 Jan 21.

These authors contributed equally to this work.

Background: Gene set enrichment analysis (GSEA) is an analytic approach which simultaneously reduces the dimensionality of microarray data and enables ready inference of the biological meaning of observed gene expression patterns. Here we invert the GSEA process to identify class-specific gene signatures. Because our approach uses the Kolmogorov-Smirnov approach both to define class specific signatures and to classify samples using those signatures, we have termed this methodology "Dual-KS" (DKS).

Results: The optimum gene signature identified by the DKS algorithm was smaller than other methods to which it was compared in 5 out of 10 datasets. The estimated error rate of DKS using the optimum gene signature was smaller than the estimated error rate of the random forest method in 4 out of the 10 datasets, and was equivalent in two additional datasets. DKS performance relative to other benchmarked algorithms was similar to its performance relative to random forests.

Conclusions: DKS is an efficient analytic methodology that can identify highly parsimonious gene signatures useful for classification in the context of microarray studies. The algorithm is available as the dualKS package for R as part of the bioconductor project.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816930PMC
http://dx.doi.org/10.4137/cin.s2892DOI Listing
January 2010

CDC73/HRPT2 CpG island hypermethylation and mutation of 5'-untranslated sequence are uncommon mechanisms of silencing parafibromin in parathyroid tumors.

Endocr Relat Cancer 2010 Mar 18;17(1):273-82. Epub 2010 Feb 18.

Hormones and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, E25, St Leonards, New South Wales 2065, Australia.

The tumor suppressor HRPT2/CDC73 is mutated in constitutive DNA from patients with the familial disorder hyperparathyroidism-jaw tumor syndrome and in approximately 70% of all parathyroid carcinomas. In a number of HRPT2 mutant tumors however, expression of the encoded protein parafibromin is lost in the absence of a clear second event such as HRPT2 allelic loss or the presence of a second mutation in this tumor suppressor gene. We sought to determine whether hypermethylation of a 713 bp CpG island extending 648 nucleotides upstream of the HRPT2 translational start site and 65 nucleotides into exon 1 might be a mechanism contributing to the loss of expression of parafibromin in parathyroid tumors. Furthermore, we asked whether mutations might be present in the 5'-untranslated region (5'-UTR) of HRPT2. We investigated a pool of tissue from 3 normal parathyroid glands, as well as 15 individual parathyroid tumor samples including 6 tumors with known HRPT2 mutations, for hypermethylation of the HRPT2 CpG island. Methylation was not identified in any specimens despite complete loss of parafibromin expression in two parathyroid carcinomas with a single detectable HRPT2 mutation and retention of the wild-type HRPT2 allele. Furthermore, no mutations of a likely pathogenic nature were identified in the 5'-UTR of HRPT2. These data strongly suggest that alternative mechanisms such as mutation in HRPT2 intronic regions, additional epigenetic regulation such as histone modifications, or other regulatory inactivation mechanisms such as targeting by microRNAs may play a role in the loss of parafibromin expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1677/ERC-09-0291DOI Listing
March 2010

Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis.

J Biomol Screen 2010 Jan 11;15(1):80-5. Epub 2009 Dec 11.

Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York, USA.

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen's TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers' protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan qPCR can be optimized by using the MasterPure RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057109355059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2902272PMC
January 2010

Microarray gene expression profiling using core biopsies of renal neoplasia.

Am J Transl Res 2009 Jan 1;1(1):55-61. Epub 2009 Jan 1.

We investigate the feasibility of using microarray gene expression profiling technology to analyze core biopsies of renal tumors for classification of tumor histology. Core biopsies were obtained ex-vivo from 7 renal tumors-comprised of four histological subtypes-following radical nephrectomy using 18-gauge biopsy needles. RNA was isolated from these samples and, in the case of biopsy samples, amplified by in vitro transcription. Microarray analysis was then used to quantify the mRNA expression patterns in these samples relative to non-diseased renal tissue mRNA. Genes with significant variation across all non-biopsy tumor samples were identified, and the relationship between tumor and biopsy samples in terms of expression levels of these genes was then quantified in terms of Euclidean distance, and visualized by complete linkage clustering. Final pathologic assessment of kidney tumors demonstrated clear cell renal cell carcinoma (4), oncocytoma (1), angiomyolipoma (1) and adrenalcortical carcinoma (1). Five of the seven biopsy samples were most similar in terms of gene expression to the resected tumors from which they were derived in terms of Euclidean distance. All seven biopsies were assigned to the correct histological class by hierarchical clustering. We demonstrate the feasibility of gene expression profiling of core biopsies of renal tumors to classify tumor histology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776289PMC
January 2009

The tumor suppressor parafibromin is required for posttranscriptional processing of histone mRNA.

Mol Carcinog 2010 Mar;49(3):215-23

Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.

Parafibromin, encoded by the gene HRPT2, is a tumor suppressor protein associated with the RNA polymerase II-associated complex, Paf1 complex. HRPT2 mutations were first identified in patients with the multiple endocrine neoplasia syndrome, hyperparathyroidism-jaw tumor (HPT-JT) syndrome, and have also been found in sporadic parathyroid and renal tumors. However, the mechanisms by which parafibromin suppresses tumor formation remain unknown. In this study, we identify a novel role of parafibromin in the regulation of replication-dependent histones. Both in vitro and in vivo analyses reveal a posttranscriptional role of parafibromin in histone mRNA processing. Downregulation of parafibromin through RNA interference or in vivo mutations lead to uncleaved histone mRNA with polyadenylated tails. These results indicate that parafibromin regulates the 3' processing of histone RNA, an essential component of the cell cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mc.20591DOI Listing
March 2010