Chromosome instability drives phenotypic switching to metastasis.

Chromosome instability (CIN) is the most striking feature of human cancers. However, how CIN drives tumor progression to metastasis remains elusive. Here we studied the role of chromosome content changes in generating the phenotypic dynamics that are required for metastasis. We isolated epithelial and mesenchymal clones from human carcinoma cell lines and showed that the epithelial clones were able to generate mesenchymal variants, which had the potential to further produce epithelial revertants autonomously. The successive acquisition of invasive mesenchymal and then epithelial phenotypes recapitulated the steps in tumor progression to metastasis. Importantly, the generation of mesenchymal variants from clonal epithelial populations was associated with subtle changes in chromosome content, which altered the chromosome transcriptome and influenced the expression of genes encoding intercellular junction (IJ) proteins, whereas the loss of chromosome 10p, which harbors the ZEB1 gene, was frequently detected in epithelial variants generated from mesenchymal clones. Knocking down these IJ genes in epithelial cells induced a mesenchymal phenotype, whereas knocking down the ZEB1 gene in mesenchymal cells induced an epithelial phenotype, demonstrating a causal role of chromosome content changes in phenotypic determination. Thus, our studies suggest a paradigm of tumor metastasis: primary epithelial carcinoma cells that lose chromosomes harboring IJ genes acquire an invasive mesenchymal phenotype, and subsequent chromosome content changes such as loss of 10p in disseminated mesenchymal cells generate epithelial variants, which can be selected for to generate epithelial tumors during metastatic colonization.

Disruption of tubular Flcn expression as a mouse model for renal tumor induction.

The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression of tumorigenesis. The mTOR and TGF-ß signalings were upregulated in Flcn-deficient tumors, and these two activated pathways may synergetically cause renal tumorigenesis. Treatment of knockout mice with the mTOR inhibitor rapamycin for 10 months led to the suppression of tumor growth. Thus, our model recapitulates human Birt-Hogg-Dubé kidney tumorigenesis, provides a valuable tool for further study of Flcn-deficient renal tumorigenesis, and tests new drugs/approaches to their treatment.

Hyperplasia and fibrosis in mice with conditional loss of the TSC2 tumor suppressor in Müllerian duct mesenchyme-derived myometria.

Uterine leiomyomata are the most common tumors found in the female reproductive tract. Despite the high prevalence and associated morbidities of these benign tumors, little is known about the molecular basis of uterine leiomyoma development and progression. Loss of the Tuberous Sclerosis 2 (TSC2) tumor suppressor has been proposed as a mechanism important for the etiology of uterine leiomyomata based on the Eker rat model. However, conflicting evidence showing increased TSC2 expression has been reported in human uterine leiomyomata, suggesting that TSC2 might not be involved in the pathogenesis of this disorder. We have produced mice with conditional deletion of the Tsc2 gene in the myometria to determine whether loss of TSC2 leads to leiomyoma development in murine uteri. Myometrial hyperplasia and increased collagen deposition was observed in Tsc2(cKO) mice compared with control mice, but no leiomyomata were detected by post-natal week 24. Increased signaling activity of mammalian target of rapamycin complex 1, which is normally repressed by TSC2, was also detected in the myometria of Tsc2(cKO) mice. Treatment of the mutant mice with rapamycin significantly inhibited myometrial expansion, but treatment with the progesterone receptor modulator, mifepristone, did not. The ovaries of the Tsc2(cKO) mice appeared normal, but half the mice were infertile and most of the other half became infertile after a single litter, which was likely due to oviductal blockage. Our study shows that although TSC2 loss alone does not lead to leiomyoma development, it does lead to myometrial hyperplasia and fibrosis.

Gene profiling suggests a common evolution of bladder cancer subtypes.

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.

Overexpression of asparagine synthetase and matrix metalloproteinase 19 confers cisplatin sensitivity in nasopharyngeal carcinoma cells.

Platinum-based concurrent chemoradiotherapy is considered a standard treatment approach for locoregionally advanced nasopharyngeal carcinoma. However, only a minority of patients benefit from this treatment regimen compared with radiotherapy alone. Identification of a set of molecular markers predicting sensitivity of platinum-based chemotherapy may contribute to personalized treatment of patients with nasopharyngeal carcinoma for better clinical outcome with less toxicity. Previously, we generated a cisplatin-sensitive nasopharyngeal carcinoma cell line, S16, by clonal selection from CNE-2 cells and found that eIF3a is upregulated and contributes to cisplatin sensitivity by downregulating the synthesis of nucleotide excision repair proteins. In this study, we conducted a gene expression profiling analysis and found three other genes, asparagine synthetase (ASNS), choriogonadotropin α subunit (CGA), and matrix metalloproteinase 19 (MMP19), that are upregulated in the cisplatin-sensitive S16 cells compared with the CNE-2 cells. However, only ASNS and MMP19, but not CGA, contributes to cisplatin sensitivity by potentiating cisplatin-induced DNA damage and apoptosis. Thus, ASNS and MMP19, along with eIF3a, are the sensitivity factors for cisplatin treatment and may serve as potential candidate molecular markers for predicting cisplatin sensitivity of advanced nasopharyngeal carcinoma.

CUL3 and NRF2 mutations confer an NRF2 activation phenotype in a sporadic form of papillary renal cell carcinoma.

Sustained activation of the stress-regulated transcription factor NRF2 (NFE2L2) is a prominent feature of many types of cancer, implying that mutations driving NRF2 may be important to tumor progression. In hereditary type 2 papillary renal cell carcinoma (PRCC2, also known as hereditary leiomyomatosis and renal cell cancer), NRF2 activation is a direct consequence of the accumulation of intracellular fumarate, a result of fumarate hydratase (FH) inactivation, but it is not clear how NRF2 may be activated in sporadic forms of PRCC2. Here we show that somatic mutations in NRF2, CUL3, and SIRT1 are responsible for driving the NRF2 activation phenotype in sporadic PRCC2. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens. Our results show a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2.

Expression of the PTTG1 oncogene is associated with aggressive clear cell renal cell carcinoma.

The pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene implicated in malignant progression of both endocrine and nonendocrine malignancies. Clear cell renal cell carcinoma (ccRCC) is cytogenetically characterized by chromosome 3p deletions that harbor the ccRCC-related von Hippel-Lindau, PBRM1, BAP1, and SETD2 tumor suppressor genes, along with chromosome 5q amplifications where the significance has been unclear. PTTG1 localizes to the chromosome 5q region where amplifications occur in ccRCC. In this study, we report a functional role for PTTG1 in ccRCC tumorigenesis. PTTG1 was amplified in ccRCC, overexpressed in tumor tissue, and associated with high-grade tumor cells and poor patient prognosis. In preclinical models, PTTG1 ablation reduced tumorigenesis and invasion. An analysis of gene expression affected by PTTG1 indicated an association with invasive and metastatic disease. PTTG1-dependent expression of the RhoGEF proto-oncogene ECT2 was observed in a number of ccRCC cell lines. Moreover, ECT2 expression correlated with PTTG1 expression and poor clinical features. Together, our findings reveal features of PTTG1 that are consistent with its identification of an oncogene amplified on chromsome 5q in ccRCC, where it may offer a novel therapeutic target of pathologic significance in this disease.

Plasma-based circulating MicroRNA biomarkers for Parkinson's disease.

BACKGROUND: The current "gold-standard" for Parkinson's disease (PD) diagnosis is based primarily on subjective clinical rating scales related with motor features. Molecular biomarkers that are objective and quantifiable remain attractive as clinical tools to detect PD prior to its motor onsets. OBJECTIVE: Here, we aimed to identify, develop, and validate plasma-based circulating microRNA (miRNAs) as biomarkers for PD. METHODS: Global miRNA expressions were acquired from a discovery set of 32 PD/32 controls using microarrays. k-Top Scoring Pairs (k-TSP) algorithm and significance analysis of microarrays (SAM) were applied to obtain comprehensive panels of PD-predictive biomarkers. TaqMan miRNA-specific real-time PCR assays were performed to validate the microarray data and to evaluate the biomarker performance using a new replication set of 42 PD/30 controls. Data was analyzed in a paired PD-control fashion. The validation set was composed of 30 PD, 5 progressive supranuclear palsy, and 4 multiple system atrophy samples from a new clinical site. RESULTS: We identified 9 pairs of PD-predictive classifiers using k-TSP analysis and 13 most differentially-expressed miRNAs by SAM. A combination of both data sets produced a panel of PD-predictive biomarkers: k-TSP1 (miR-1826/miR-450b-3p), miR-626, and miR-505, and achieved the highest predictive power of 91% sensitivity, 100% specificity, 100% positive predicted value, and 88% negative predicted value in the replication set. However, low predictive values were shown in the validation set. CONCLUSIONS: This proof-of-concept study demonstrates the feasibility of using plasma-based circulating miRNAs as biomarkers for neurodegenerative disorders such as PD and shows the challenges of molecular biomarker research using samples from multiple clinical sites.

An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma.

Fumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2). The main effect of FH mutation is fumarate accumulation. The current paradigm posits that the main consequence of fumarate accumulation is HIF-α stabilization. Paradoxically, FH mutation differs from other HIF-α stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We identified that fumarate can directly up-regulate antioxidant response element (ARE)-controlled genes. We demonstrated that aldo-keto reductase family 1 member B10 (AKR1B10) is an ARE-controlled gene and is up-regulated upon FH knockdown as well as in FH null cell lines. AKR1B10 overexpression is also a prominent feature in both hereditary and sporadic PRCC2. This phenotype better explains the similarities between hereditary and sporadic PRCC2.

Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette) transporters gene enrichment in typhoid fever-infected Nigerian children.

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) is a human-specific pathogen that causes typhoid fever, and remains a global health problem especially in developing countries. Its pathogenesis is complex and host response is poorly understood. In Africa, typhoid fever can be a major cause of morbidity in young infected children. The onset of the illness is insidious and clinical diagnosis is often unreliable. Gold standard blood culture diagnostic services are limited, thus rapid, sensitive, and affordable diagnostic test is essential in poor-resourced clinical settings. Routine typhoid fever vaccination is highly recommended but currently licensed vaccines provide only 55-75% protection. Recent epidemiological studies also show the rapid emergence of multi-drug resistant S. Typhi strains. High-throughput molecular technologies, such as microarrays, can dissect the molecular mechanisms of host responses which are S. Typhi-specific to provide a comprehensive genomic component of immunological responses and suggest new insights for diagnosis and treatment. METHODS: Global transcriptional profiles of S. Typhi-infected young Nigerian children were obtained from their peripheral blood and compared with that of other bacteremic infections using Agilent gene expression microarrays. The host-response profiles of the same patients in acute vs. convalescent phases were also determined. The top 96-100 differentially-expressed genes were identified and four genes were validated by quantitative real-time PCR. Gene clusters were obtained and functional pathways were predicted by DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: Transcriptional profiles from S. Typhi-infected children could be distinguished from those of other bacteremic infections. Enriched gene clusters included genes associated with extracellular peptides/components such as lipocalin (LCN2) and systemic immune response which is atypical in bacterial invasion. Distinct gene expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We found novel down-regulation of ABC (ATP-binding cassette) transporters genes such as ABCA7, ABCC5, and ABCD4 and ATPase activity as the highest enriched pathway. CONCLUSIONS: We identified unique extracellular components and ABC transporters gene enrichments in typhoid fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for typhoid fever in Africa.

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

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.

Serglycin is a theranostic target in nasopharyngeal carcinoma that promotes metastasis.

Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial-mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients.

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

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.

Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes.

Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer, characterized by the presence of inactivating mutations in the VHL gene in most cases, and by infrequent somatic mutations in known cancer genes. To determine further the genetics of ccRCC, we have sequenced 101 cases through 3,544 protein-coding genes. Here we report the identification of inactivating mutations in two genes encoding enzymes involved in histone modification-SETD2, a histone H3 lysine 36 methyltransferase, and JARID1C (also known as KDM5C), a histone H3 lysine 4 demethylase-as well as mutations in the histone H3 lysine 27 demethylase, UTX (KMD6A), that we recently reported. The results highlight the role of mutations in components of the chromatin modification machinery in human cancer. Furthermore, NF2 mutations were found in non-VHL mutated ccRCC, and several other probable cancer genes were identified. These results indicate that substantial genetic heterogeneity exists in a cancer type dominated by mutations in a single gene, and that systematic screens will be key to fully determining the somatic genetic architecture of cancer.

Interleukin-8 mediates resistance to antiangiogenic agent sunitinib in renal cell carcinoma.

The broad spectrum kinase inhibitor sunitinib is a first-line therapy for advanced clear cell renal cell carcinoma (ccRCC), a deadly form of kidney cancer. Unfortunately, most patients develop sunitinib resistance and progressive disease after about 1 year of treatment. In this study, we evaluated the mechanisms of resistance to sunitinib to identify the potential tactics to overcome it. Xenograft models were generated that mimicked clinical resistance to sunitinib. Higher microvessel density was found in sunitinib-resistant tumors, indicating that an escape from antiangiogenesis occurred. Notably, escape coincided with increased secretion of interleukin-8 (IL-8) from tumors into the plasma, and coadministration of an IL-8 neutralizing antibody resensitized tumors to sunitinib treatment. In patients who were refractory to sunitinib treatment, IL-8 expression was elevated in ccRCC tumors, supporting the concept that IL-8 levels might predict clinical response to sunitinib. Our results reveal IL-8 as an important contributor to sunitinib resistance in ccRCC and a candidate therapeutic target to reverse acquired or intrinsic resistance to sunitinib in this malignancy.

p38delta/MAPK13 as a diagnostic marker for cholangiocarcinoma and its involvement in cell motility and invasion.

Cholangiocarcinoma (CC) and hepatocellularcarcinoma (HCC) are two main forms of liver malignancies, which exhibit differences in drug response and prognosis. Immunohistotochemical staining for cytokeratin markers has been used to some success in the differential diagnosis of CC from HCC. However, there remains a need for additional markers for increased sensitivity and specificity of diagnosis. In this study, we have identified a p38 MAP kinase, p38delta (also known as MAPK13 or SAPK4) as a protein that is upregulated in CC relative to HCC and to normal biliary tract tissues. We performed microarray gene expression profiling on 17 cases of CC, 12 cases of adjacent normal liver tissue, and three case of normal bile duct tissue. p38delta was upregulated in 16 out of 17 cases of CC relative to normal tissue. We subsequently performed immunohistochemical staining of p38delta in 54 cases of CC and 54 cases of HCC. p38delta staining distinguished CC from HCC with a sensitivity of 92.6% and a specificity of 90.7%. To explore the possible functional significance of p38delta expression in CC, we examined the effects of overexpression and knockdown of p38delta expression in human CC cell lines. Our results indicate that p38delta is important for motility and invasion of CC cells, suggesting that p38delta may play an important role in CC metastasis. In summary, p38delta may serve as a novel diagnostic marker for CC and may also serve as a new target for molecular based therapy of this disease.

MicroRNA profiling of human kidney cancer subtypes.

Although the functions of most of the identified microRNAs (miRNAs) have yet to be determined, their use as potential biomarkers has been considered in several human diseases and cancers. In order to understand their role in renal tumorigenesis, we screened the expression levels of miRNAs in four subtypes of human renal neoplasms: clear cell, papillary, and chromophobe renal cell carcinomas (RCC) as well as benign renal oncocytomas. We found a unique miRNA signature for each subtype of renal tumor. Furthermore, we identified unique patterns of miRNA expression distinguishing clear cell RCC cases with favorable vs. unfavorable outcome. Specifically, we documented the overexpression of miRs 424 and 203 in clear cell RCC relative to papillary RCC, as well as the inversion of expression of miR-203 in the benign oncocytomas (where it is underexpressed relative to normal kidney) as compared to the malignant chromophobe RCC (where it is overexpressed relative to normal kidney). Our results further suggest that overexpression of S-has-miR-32 is associated with poor outcome. While previous studies have identified unique miRNA expression pattern distinguishing tumors from different anatomical locations, here we extend this principle to demonstrate the utility of miRNA expression profiling to identify a signature unique to various tumor subtypes at a single anatomic locus.

Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia.

The Birt-Hogg-Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHD(flox/flox)/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHD(flox/flox)/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD (flox/+)/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome-related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.

A comparison study reveals important features of agreement and disagreement between summarized DNA and RNA data obtained from renal cell carcinoma.

Cytogenetic abnormalities, such as DNA amplifications and deletions, often lead to significant changes in gene expression levels within a chromosomal region. Instead of generating additional DNA copy number data, one method to identify DNA copy number abnormalities has been to search existing gene expression data for regional perturbations in gene expression. However, it is not clear how well this surrogate method performs in the examination of individual tumors and how we can use both DNA and RNA data to identify candidate genes that may be mutated. Here we report a comparison study using summarized DNA and RNA data to identify chromosomal abnormalities in human samples. Forty-four tissue samples from patients diagnosed as having renal cell carcinoma (RCC) were collected, together with 15 normal kidney samples as controls, and for each sample the genome-wide DNA and RNA data were obtained for comparison using Affymetrix 100K SNP and HGU133plus2 gene expression chips, respectively. The DNA and RNA data was summarized by both chromosome arm and cytogenetic banding patterns and compared. The result of this analysis revealed that the two summarized data sets used to identify cytogenetic changes agreed well. However, some differences between the two were also identified. These differences of large-scale gene expression deregulation without evidence of the comparable DNA copy number alterations may be the result of known mechanisms, such as large-scale methylation or chromosome inactivation, or may be the result of some new mechanism of DNA-RNA translation. The usefulness of the combined data set for identifying regions of mutated genes is also discussed.

Somatic pairing of chromosome 19 in renal oncocytoma is associated with deregulated EGLN2-mediated [corrected] oxygen-sensing response.

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase EGLN2 [corrected] a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of EGLN2 [corrected] in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma.