Pepper: cytoscape app for protein complex expansion using protein-protein interaction networks.

UNLABELLED: We introduce Pepper (Protein complex Expansion using Protein-Protein intERactions), a Cytoscape app designed to identify protein complexes as densely connected subnetworks from seed lists of proteins derived from proteomic studies. Pepper identifies connected subgraph by using multi-objective optimization involving two functions: (i) the coverage, a solution must contain as many proteins from the seed as possible, (ii) the density, the proteins of a solution must be as connected as possible, using only interactions from a proteome-wide interaction network. Comparisons based on gold standard yeast and human datasets showed Pepper's integrative approach as superior to standard protein complex discovery methods. The visualization and interpretation of the results are facilitated by an automated post-processing pipeline based on topological analysis and data integration about the predicted complex proteins. Pepper is a user-friendly tool that can be used to analyse any list of proteins. AVAILABILITY: Pepper is available from the Cytoscape plug-in manager or online (http://apps.cytoscape.org/apps/pepper) and released under GNU General Public License version 3.

Moderate intergenerational and somatic instability of a 55-CTG repeat in transgenic mice.

Myotonic dystrophy (DM) is associated with the expansion of a (CTG)n trinucleotide repeat in the 3' untranslated region (UTR) of the DM protein kinase gene (DMPK). The (CTG)n repeat is polymorphic and varies in size between 5 and 37 repeats in unaffected individuals whereas in affected patients there are between 50 and 4,000 CTGs. The size of the (CTG)n repeat, which increases through generations, generally correlates with clinical severity and age of onset. The instability of the CTG repeat appears to depend on its size as well as on the sex of the transmitting parent. Moreover, mitotic instability analysis of different human DM tissues shows length mosaicism between different cell lineages. The molecular mechanisms of triplet instability remain elusive. To investigate the role of genomic sequences in instability, we produced transgenic mice containing a 45-kb genomic segment with a 55-CTG repeat cloned from a mildly affected patient. In contrast to other mouse models containing CAG repeats within cDNAs, these mice showed both intergenerational and somatic repeat instability.

[Update on FGFR3 mutation and multiple regional epigenetic silencing (MRES) phenotype in urothelial carcinogenesis]. / Mise au point sur la mutation FGFR3 et le phénotype épigénétique MRES dans la carcinogenèse vésicale.

FGFR3 mutation leads to a constitutive activation of the receptor 3 to Fibroblast Growth Factor. This mutation is early in urothelial carcinogenesis and is strongly associated to low grade papillary tumors. Multiple regional epigenetic silencing (MRES) phenotype corresponds to the transcriptional inactivation of chromosomal regions in muscle invasive bladder cancer, and is strongly associated to the molecular signature of carcinoma in situ. These alterations could be targeted by new specific therapies.

Assessing HER2 gene amplification as a potential target for therapy in invasive urothelial bladder cancer with a standardized methodology: results in 1005 patients.

BACKGROUND: This study assessed the human epidermal growth factor receptor-2 (HER2) protein expression and its relationship with gene amplification in invasive bladder carcinoma, using the same criteria than for breast cancer. PATIENTS AND METHODS: In 1005 patients, paraffin-embedded tissues of transurethral resection or cystectomy were evaluated by immunohistochemistry (IHC), using antibodies against HER2. All samples with a 2+ or 3+ HER2 overexpression were evaluated by FISH. RESULTS: HER2 overexpression was observed in 93 (9.2%) tumors (2+: 42 tumors and 3+: 51 tumors). Using FISH, all HER2 3+ tumors had a gene amplification, whereas no amplification was found in 2+ tumors. Intratumoral heterogeneity was observed in 35% of cases. These tumors showed the same heterogeneous pattern, with adjacent 3+ positive and negative areas by both IHC and FISH. CONCLUSIONS: This study showed that 5.1% of invasive bladder carcinomas had a HER2 gene amplification. These findings may have clinical implications for the management of patients with HER2-positive locally advanced or metastatic bladder cancer, as they could be potential candidates for targeted therapy.

[Molecular events involved in the metastatic process]. / Evénements moléculaires impliqués dans le processus métastatique.

The occurrence of metastasis is probably the least characterised process in tumour progression. It is difficult to study, due to the limited access to metastatic tissues, to the multiple steps involved and the long time required to observe metastasis growth, even in model systems. Our understanding of metastatic processes has been changed in recent years by a number of observations and the development of new concepts.

MYC activation associated with the integration of HPV DNA at the MYC locus in genital tumors.

To determine whether integration of human papillomavirus (HPV) DNA sequences could lead to the deregulation of genes implied in oncogenesis, we analysed the HPV integration sites in a series of nine cell lines derived from invasive genital carcinomas. Using in situ hybridization, HPV16 or 18 sequences were found at chromosome band 8q24, the localization of MYC, in IC1, IC2, IC3, IC6 and CAC-1 cells and at other sites in IC4, IC5, IC7 and IC8 cells. We then localized viral sequences at the molecular level and searched for alterations of MYC structure and expression in these cells. MYC genomic status and viral integration sites were also analysed in primary tumors from which IC1, IC2, IC3 and IC6 cells were derived. In IC1, IC2 and CAC-1 cells, HPV DNA was located within 58 kb of MYC, downstream, upstream, or within MYC. In IC3 and IC6 cells, HPV DNA was located 400-500 kb upstream of MYC. Amplification studies showed that, in IC1, IC2 and IC3, viral and MYC sequences were co-amplified in an amplicon between less than 50 and 800 kb in size. MYC amplification was also observed in primary tumors, indicating that this genetic alteration, together with viral insertion at the MYC locus, had already taken place in vivo. MYC was not amplified in the other cell lines. MYC mRNA and protein overexpression was observed in the five cell lines in which the HPV DNA was inserted close to the MYC locus, but in none of the lines where the insertion had occurred at other sites. MYC activation, triggered by the insertion of HPV DNA sequences, can be an important genetic event in cervical oncogenesis.

Pancreatic beta cells cultured from individual preneoplastic foci in a multistage tumorigenesis pathway: a potentially general technique for isolating physiologically representative cell lines.

Culturing and comparing the discrete stages of tumorigenesis provide a route to defining important components of the cancer phenotype and, in addition, present the opportunity to establish cell cultures more representative of normal cells than the ultimate malignant cancer cells. Herein we report that preneoplastic foci in one multistep tumorigenesis pathway can be cultured in vitro and show that they preserve distinctive characteristics of the normal cells from which they arose, pancreatic beta cells. In the RIP1-Tag2 line of transgenic mice, which express the simian virus 40 T antigen in insulin-producing beta cells, pancreatic islets develop into vascularized tumors in a multistage pathway. We established conditions for reproducible derivation of beta-cell lines from individual hyperplastic islets that have not yet developed into solid tumors. Most of these cell lines, designated beta HC, release insulin at physiological concentrations of glucose. In contrast to tumor-derived lines (beta TC), which are not properly regulated, the ability of the beta HC lines to respond correctly to glucose correlated with maintenance of normally depressed levels of low-Km hexokinases. Glutamic acid decarboxylase (GAD), an early autoantigen in type I diabetes, was detected in most of the beta HC lines. The relative levels of the two forms of this enzyme (GAD65 and GAD67) varied significantly between the different cell lines, suggesting independent regulation. Class I major histocompatibility complex antigens were detected on the beta HC cells, and the levels of surface major histocompatibility complex expression correlated with their capacity to serve as targets in a cytotoxic T-cell killing assay. The beta HC lines will be of value for studies of beta-cell physiology, autoantigenicity, and tumor development. This work suggests the possibility of culturing preneoplastic stages of other cancers, both to address the mechanisms of transformation and to provide a source of cells that maintain important qualities of their normal progenitors.

Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas.

Increased serum levels of human chorionic gonadotropin beta subunit (hCG beta) were described previously in patients with bladder cancer. To obtain insight into such production of hCG beta, the expression of hCG beta 7, 8, 5, and 3 genes in bladder carcinomas and normal urothelia was investigated by reverse transcription PCR. Surprisingly, hCG beta mRNAs were detected in both normal urothelial and carcinomatous cells. However, tumor progression was characterized by different patterns of transcription of the hCG beta genes; the beta 7 gene was the only gene transcribed in normal urothelia and Ta tumors included in this study, whereas in addition to beta 7, genes beta 5, 8, and 3 were transcribed in T1 to T4 tumors. Moreover, transcription levels of the latter three genes increased with the stage of the disease. These observations showed that dramatic modifications in the expression of hCG beta genes accompany progression of bladder carcinomas.

The fibroblast growth factor receptor 3 (FGFR3) mutation is a strong indicator of superficial bladder cancer with low recurrence rate.

We analyzed the possible prognostic value of the recently discovered fibroblast growth factor receptor 3 (FGFR3) mutations in bladder cancer. A FGFR3 mutation was found in 34 of 53 pTaG1-2 bladder cancers, whereas none of the 19 higher-staged tumors had a mutation (P < 0.0001). In 57 patients with superficial disease followed prospectively by cystoscopy for 12 months, 14 of 23 patients in the wild-type FGFR3 group developed recurrent bladder cancer compared with only 7 of 34 patients in the mutant group (P = 0.004). The recurrence rate per year was 0.24 for the FGFR3 mutant tumors and 1.12 for tumors with a wild-type FGFR3 gene. In addition, FGFR3 mutation status was the strongest predictor of recurrence when compared with stage and grade (P = 0.008). This is the first mutation in bladder cancer that selectively identifies patients with favorable disease characteristics. Our results suggest that the frequency of cystoscopic examinations can be reduced considerably in patients with FGFR3-positive tumors.

Frequent loss of heterozygosity on chromosome 10q in muscle-invasive transitional cell carcinomas of the bladder.

Loss of heterozygosity (LOH) on chromosome 10 has been observed in several human cancers including glioblastomas, meningiomas, melanomas and endometrial and prostate carcinomas. We have investigated the incidence of LOH on chromosome 10 in 36 human transitional cell carcinomas (TCCs) of the bladder, three upper urinary tract TCCs and one lymph node metastasis, using a panel of 27 highly polymorphic markers spanning 10p (short arm) and 10q (long arm). Fourteen bladder tumours (39%), the three upper urinary tract tumours and the lymph node metastasis showed LOH for at least one locus on chromosome 10. Remarkably, LOH on chromosome 10 was observed mainly in muscle-invasive (P = 0.01) and high grade tumours (P = 0.03). For five tumours and the lymph node metastasis, LOH was found at all informative loci, indicating monosomy or isodisomy of chromosome 10. The deletion mapping of the tumours with partial loss delineated two minimal regions of loss on chromosome 10q. One region, the most telomeric, was bounded by markers D10S214 and D10S169 and the other, the most proximal, was bounded by markers D10S222 and D10S531. Our results demonstrate that chromosome 10q LOH is common in muscle-invasive bladder cancers and that two potential tumour suppressor loci, at 10q24.1-q24.3 and 10q26.1-q26.2, may contribute to the malignant progression of these tumours. Localization of the smallest common regions of loss in bladder tumours provides a starting point for the identification of the genes involved.

Androgen suppressed apoptosis is modified in p53 deficient mice.

Several in vitro studies have provided evidence that the tumor suppressor protein, p53, is involved in the cell death process referred to as apoptosis. The recent development of p53 knock-out mice has enabled further investigation into the function of p53 for apoptosis, in vivo. Radiation-induced apoptosis is suppressed in such mice, yet other forms of apoptosis do not seem to be significantly affected. In this report, we present evidence that such male p53 nullizygous mice have less apoptosis in the prostate glands associated with the first 4 days following castration. Ventral prostate glands were obtained from normal, heterozygous p53-null and p53 nullizygous mice at daily intervals after castration. These tissues were stained for apoptosis with the use of the in situ and labeling method and apoptotic bodies were quantified by microscopy. Although labeled apoptotic bodies were observed in post-castrated tissues from all of these genetic variant mice, the onset of apoptosis was delayed and the occurrence of apoptosis was significantly reduced in the p53 nullizygous mice when compared to normal controls. Heterozygous p53-null mice were intermediate for these criteria. Examination of the internucleosomal DNA fragmentation pattern at 2 days of castration supports a significant diminution of prostate cell apoptosis in nullizygous p53 mice. Additionally, large nucleated and multinucleated cells were detected in the prostate epithelium of noncastrated p53 nullizygous mice and these abnormal cells were increased after castration. Flow cytometric analysis of these tissues confirmed a high number of 4C and 8C DNA content cells in the p53 nullizygous prostates and their frequency was increased by castration. In concordance with an earlier study, we conclude that functional p53 protein is not essential for prostate epithelial cells to undergo castration-induced apoptosis. However, wild-type p53 does appear to enhance this process, especially in the early period following castration, and this protein may regulate an aberrant prostate cell cycling activity that follows castration.

Decreased expression of keratinocyte growth factor receptor in a subset of human transitional cell bladder carcinomas.

Growth factors and growth factor receptors are involved in tumor progression. The fibroblast growth factor receptor 2 gene encodes distinct isoforms. The isoforms which bind KGF (keratinocyte growth factor or FGF-7) are called KGF-R or FGFR2b. KGF-R is expressed in different epithelia and is involved in the control of epithelial-mesenchymal interactions. Expression of KGF-R mRNA was examined in normal human bladder and transitional cell carcinoma of the bladder (TCC) by semi-quantitative RT-PCR using TFIID and GAPDH as internal standards. In normal bladder, the KGF-R mRNA was detected in the urothelium but not in the underlying stroma. In TCCs, the level of KGF-R mRNA was generally either normal or low. Eighteen out of 54 TCCs had a KGF-R mRNA level below 30% of that found in normal urothelium. This decrease in KGF-R mRNA was not accompanied by an increase in BEK (FGFR2c) mRNA, the other major splice variant of the fibroblast growth factor receptor 2 gene. Expression of the KGF-R was also monitored by immunohistochemistry using a functional KGF-immunoglobulin chimera. The receptor was uniformly expressed throughout the normal urothelium except for the umbrella cells. Immunoreactivity for KGF-R was found to be negative in tumors with low levels of KGF-R mRNA, while the peritumoral normal urothelium was positive. Among patients with muscle invasive tumors, those exhibiting a low level of KGF-R mRNA had a significantly higher proportion of cancer deaths. Our results suggest that decreased expression of KGF-R can be considered as a marker of tumor progression in muscle invasive TCCs.

Relationship between E-cadherin and fibroblast growth factor receptor 2b expression in bladder carcinomas.

E-cadherin is a cell-cell adhesion molecule expressed predominantly by epithelial cells. Reduction or loss of E-cadherin immunoreactivity has been associated with tumour progression in many epithelial cancers, including bladder carcinomas. The fibroblast growth factor receptor 2b (FGFR2b) recognized specifically by FGF7 is expressed only by epithelial cells. Recently, decreased expression of FGFR2b protein and mRNA was found to be associated with tumour progression in bladder carcinomas. The purpose of this investigation was to look for a possible relationship between E-cadherin and FGFR2b expression in bladder carcinomas. As decreased E-cadherin immunoreactivity was found to correlate directly with decreased expression at the mRNA level, the possible relationship between E-cadherin and FGFR2b was investigated at the mRNA level using semi-quantitative RT - PCR in 92 transitional cell carcinomas (TCCs) and four lymph node metastases. All tumours with low E-cadherin expression had low expression of FGFR2b, whereas tumours with low FGFR2b mRNA could express any level of E-cadherin mRNA. The same observation was equally valid for bladder and colon cancer cell lines suggesting that, besides bladder tumours, this relationship could apply to other carcinomas types. These results suggest that a relationship exists between the transcription of the E-cadherin and FGFR2b genes preventing high expression of FGFR2b where expression of E-cadherin is low. We suggest that reduced expression of FGFR2b in conjunction with decreased expression of E-cadherin may contribute to the aggressive behaviour attributable to high grade TCCs.

Tumour suppressive properties of fibroblast growth factor receptor 2-IIIb in human bladder cancer.

FGFRs (fibroblast growth factor receptors) are encoded by four genes (FGFR1-4). Alternative splicing results in various receptor isoforms. The FGFR2-IIIb variant is present in a wide variety of epithelia, including the bladder epithelium. Recently, we have shown that FGFR2-IIIb is downregulated in a subset of transitional cell carcinomas of the bladder, and that this downregulation is associated with a poor prognosis. We investigated possible tumour suppressive properties of FGFR2-IIIb by transfecting two human bladder tumour cell lines, J82 and T24, which have no endogenous FGFR2-IIIb expression, with FGFR2-IIIb cDNA. No stable clones expressing FGFR2-IIIb were isolated with the J82 cell line. For the T24 cell line, stable transfectants expressing FGFR2-IIIb had reduced growth in vitro and formed fewer tumours in nude mice which, in addition, grew more slowly. The potential mechanisms leading to decreased FGFR2-IIIb mRNA levels were also investigated. The 5' region of the human FGFR2 gene was isolated and found to contain a CpG island which was partially methylated in more than half the cell lines and tumours which do not express FGFR2-IIIb. No homozygous deletion was identified in any of the tumours or cell lines with reduced levels of FGFR2-IIIb. Mutational analysis of the entire coding region of FGFR2-IIIb at the transcript level was performed in 33 bladder tumours. In addition to normal FGFR2-IIIb mRNA, abnormal transcripts were detected in two tumour samples. These abnormal mRNAs resulted from exon skipping which affected the region encoding the kinase domain. Altogether, these results show that FGFR2-IIIb has tumour growth suppressive properties in bladder carcinomas and suggest possible mechanisms of FGFR2 gene inactivation.

No evidence of somatic FGFR3 mutation in various types of carcinoma.

Germline specific point mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3) are associated with autosomal dominant human skeletal dysplasia and craniosynostosis syndromes. Mutations identical to the germinal activating mutations found in severe skeletal dysplasias have been identified in certain types of cancer: at low frequency in multiple myeloma and cervix carcinoma and at high frequency in bladder carcinoma. We analysed, by SSCP and sequencing, the prevalence of FGFR3 mutations in 116 primary tumours of various types (upper aerodigestive tract, oesophagus, stomach, lung and skin). The regions analysed encompassed all FGFR3 point mutations previously described in severe skeletal dysplasia and cancers. No mutations were detected in the tumour types examined, suggesting that FGFR3 mutations are restricted to a few tumour types, the evidence to date suggesting that they are very specific to bladder carcinomas.

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids.

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.

Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells.

After differential screening of a cDNA library constructed from quail neuroretina cells infected with the v-myc containing avian retrovirus MC29, we have isolated a cDNA clone Pax-QNR, homologous to the murine Pax6 which is mutated in the autosomal dominant mutation small eye (Sey) of the mouse and aniridia in man. Here we report the characterization of Pax-QNR/Pax-6 expression in the chicken, quail, and mouse pancreas. In situ hybridization performed with E3 chick embryos demonstrated that, in addition to the documented expression of Pax-QNR/Pax-6 in the neural tube, this gene is also expressed in the pancreatic bud. This expression is later restricted to discrete parts of the organ. From bacterially expressed Pax-QNR peptides we obtained rabbit antisera (paired domain, serum 11; domain between paired and homeo, serum 12; homeodomain, serum 13; and carboxyl-terminal part, serum 14) capable of specifically recognizing Pax-QNR/Pax-6 proteins (48, 46 kilodaltons) in cell lines derived from alpha- and beta-pancreatic cells, but not from exocrine derived cell lines. We conclude that Pax-QNR/Pax-6 represents another gene expressed both in the endocrine pancreas and neuro-ectodermic tissues.

PRC2-independent chromatin compaction and transcriptional repression in cancer.

The silencing of large chromosomal regions by epigenetic mechanisms has been reported to occur frequently in cancer. Epigenetic marks, such as histone methylation and acetylation, are altered at these loci. However, the mechanisms of formation of such aberrant gene clusters remain largely unknown. Here, we show that, in cancer cells, the epigenetic remodeling of chromatin into hypoacetylated domains covered with histone H3K27 trimethylation is paralleled by changes in higher-order chromatin structures. Using fluorescence in situ hybridization, we demonstrate that regional epigenetic silencing corresponds to the establishment of compact chromatin domains. We show that gene repression is tightly correlated to the state of chromatin compaction and not to the levels of H3K27me3-its removal through the knockdown of EZH2 does not induce significant gene expression nor chromatin decompaction. Moreover, transcription can occur with intact high-H3K27me3 levels; treatment with histone deacetylase inhibitors can relieve chromatin compaction and gene repression, without altering H3K27me3 levels. Our findings imply that compaction and subsequent repression of large chromatin domains are not direct consequences of PRC2 deregulation in cancer cells. By challenging the role of EZH2 in aberrant gene silencing in cancer, these findings have therapeutical implications, notably for the choice of epigenetic drugs for tumors with multiple regional epigenetic alterations.