Fuzzy methods for analysis of microarrays and networks

Bioinformatics involves analyses of biological data such as DNA sequences, microarrays and protein-protein interaction (PPI) networks. Its two main objectives are the identification of genes or proteins and the prediction of their functions. Biological data often contain uncertain and imprecise information. Fuzzy theory provides useful tools to deal with this type of information, hence has played an important role in analyses of biological data. In this thesis, we aim to develop some new fuzzy techniques and apply them on DNA microarrays and PPI networks. We will focus on three problems: (1) clustering of microarrays; (2) identification of disease-associated genes in microarrays; and (3) identification of protein complexes in PPI networks. The first part of the thesis aims to detect, by the fuzzy C-means (FCM) method, clustering structures in DNA microarrays corrupted by noise. Because of the presence of noise, some clustering structures found in random data may not have any biological significance. In this part, we propose to combine the FCM with the empirical mode decomposition (EMD) for clustering microarray data. The purpose of EMD is to reduce, preferably to remove, the effect of noise, resulting in what is known as denoised data. We call this method the fuzzy C-means method with empirical mode decomposition (FCM-EMD). We applied this method on yeast and serum microarrays, and the silhouette values are used for assessment of the quality of clustering. The results indicate that the clustering structures of denoised data are more reasonable, implying that genes have tighter association with their clusters. Furthermore we found that the estimation of the fuzzy parameter m, which is a difficult step, can be avoided to some extent by analysing denoised microarray data. The second part aims to identify disease-associated genes from DNA microarray data which are generated under different conditions, e.g., patients and normal people. We developed a type-2 fuzzy membership (FM) function for identification of diseaseassociated genes. This approach is applied to diabetes and lung cancer data, and a comparison with the original FM test was carried out. Among the ten best-ranked genes of diabetes identified by the type-2 FM test, seven genes have been confirmed as diabetes-associated genes according to gene description information in Gene Bank and the published literature. An additional gene is further identified. Among the ten best-ranked genes identified in lung cancer data, seven are confirmed that they are associated with lung cancer or its treatment. The type-2 FM-d values are significantly different, which makes the identifications more convincing than the original FM test. The third part of the thesis aims to identify protein complexes in large interaction networks. Identification of protein complexes is crucial to understand the principles of cellular organisation and to predict protein functions. In this part, we proposed a novel method which combines the fuzzy clustering method and interaction probability to identify the overlapping and non-overlapping community structures in PPI networks, then to detect protein complexes in these sub-networks. Our method is based on both the fuzzy relation model and the graph model. We applied the method on several PPI networks and compared with a popular protein complex identification method, the clique percolation method. For the same data, we detected more protein complexes. We also applied our method on two social networks. The results showed our method works well for detecting sub-networks and give a reasonable understanding of these communities.

Beyond barcoding : a mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.

A biological staging model for operable non-small cell lung cancer

Background Currently the best prognostic index for operable non-small cell lung cancer (NSCLC) is the TNM staging system. Molecular biology holds the promise of predicting outcome for the individual patient and identifying novel therapeutic targets. Angiogenesis, matrix metalloproteinases (MMP)-2 and -9, and the erb/HER type I tyrosine kinase receptors are all implicated in the pathogenesis of NSCLC. Methods A retrospective analysis of 167 patients with resected stage I-IIIa NSCLC and >60 days postoperative survival with a minimum follow up of 2 years was undertaken. Immunohistochemical analysis was performed on paraffin embedded sections for the microvessel marker CD34, MMP-2 and MMP-9, EGFR, and c-erbB-2 to evaluate the relationships between and impact on survival of these molecular markers. Results Tumour cell MMP-9 (HR 1.91 (1.23-2.97)), a high microvessel count (HR 1.97 (1.28-3.03)), and stage (stage II HR 1.44 (0.87-2.40), stage IIIa HR 2.21 (1.31-3.74)) were independent prognostic factors. Patients with a high microvessel count and tumour cell MMP-9 expression had a worse outcome than cases with only one (HR 1.68 (1.04-2.73)) or neither (HR 4.43 (2.29-8.57)) of these markers. EGFR expression correlated with tumour cell MMP-9 expression (p<0.001). Immunoreactivity for both of these factors within the same tumour was associated with a poor prognosis (HR 2.22 (1.45-3.41)). Conclusion Angiogenesis, EGFR, and MMP-9 expression provide prognostic information independent of TNM stage, allowing a more accurate outcome prediction for the individual patient. The development of novel anti-angiogenic agents, EGFR targeted therapies, and MMP inhibitors suggests that target specific adjuvant treatments may become a therapeutic option in patients with resected NSCLC.

Herceptest: HER2 expression and gene amplification in non-small cell lung cancer

HER2 is an erbB/HER type I tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3 + cases demonstrated gene amplification. One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting. © 2001 Wiley-Liss, Inc.

Safety and efficacy of the combination of trastuzumab with docetaxel for HER2-positive women with advanced breast cancer : a review of the existing clinical trials and results of the expanded access programme in the UK

Trastuzumab is a humanised monoclonal antibody against the extracellular domain of HER2 (human epidermal growth factor receptor-2) that is overexpressed in about 25% of human breast cancers. It has shown clinical benefit in HER2-positive breast cancer cases when used alone or in combination with chemotherapy. Trastuzumab increases the response rate to chemotherapy and prolongs survival when used in combination with taxanes. In this article, we review the clinical trials where trastuzumab has been administered together with docetaxel, and we present the results of the trastuzumab expanded access programme (EAP) in the UK. Combination of trastuzumab with docetaxel results in similar response rates and time-to-progression with the trastuzumab/paclitaxel combinations. The toxicity of the combination and the risk of heart failure are low. The clinical data for the docetaxel/trastuzumab combination indicate a favourable profile from both the efficacy and the safety point of view and confirm the feasibility and safety of trastuzumab administration both as monotherapy and in combination with docetaxel. © 2004 Blackwell Publishing Ltd.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer : the EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p<0.0001). TP53 mutation status was not associated with prognosis. G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second primary lung cancer or recurrence/metastasis (progressive disease). EGFR mutations were almost exclusively found in never-smoking females (p=0.0067). KRAS mutations were detected in 18.5% of cases, mainly ADC (p<0.0001), and showed a tendency toward association with progressive disease status. These results suggest that mutations are good markers of different aetiologies and histopathological forms of lung cancers but have little prognostic value, with the exception of KRAS mutation, which may have a prognostic value in ADC. Copyright©ERS 2012.

Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.

Modulation of breast cancer progression and differentiation by the gp30/neregulin

In the last decade we have come to understand that the growth of cancer cells in general and of breast cancer in particular depends, in many cases, upon growth factors that will bind to and activate their receptors. One of these growth factor receptors is the erbB-2 protein which plays an important role in the prognosis of breast cancer and is overexpressed in nearly 30% of human breast cancer patients. While evidence accumulates to support the relationship between erbB-2 overexpression and poor overall survival in breast cancer, understanding of the biological consequence(s) of erbB-2 overexpression remains elusive. Our recent discovery of the gp30 has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB-2 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in breast cancer. We have shown that gp30 induces a biphasic growth effect on cells with erbB-2 over-expression. We have recently determined the protein sequence of gp30 and cloned its full length cDNA sequence. We have also cloned two additional forms to the ligand, that are believed to be different isoforms. We are currently expressing the different forms in order to determine their biological effects. To elucidate the cellular mechanisms underlying cell growth inhibition by gp30, we tested the effect of this ligand on cell growth and differentiation of the human breast cancer cells which overexpress erbB-2 and cells which express low levels of this protooncogene. High concentrations of ligand induced differentiation of cells overexpressing erbB-2, as measured by inhibition of cell growth, and increased synthesis of milk components, and modulation of E-cadherin and up- regulation of c-jun and c-fos. These findings indicate that ligand-induced growth inhibition in cells overexpressing erbB-2 is associated with an apparent induction of differentiation. The availability of gp30 derived synthetic peptides and its full cDNAs provides tools necessary to acquire a better understanding of the mechanism of action of the this ligands and the erbB-2 receptor in breast cancer.

The complete mitochondrial genome of the tarnished plant bug, Lygus lineolaris (Heteroptera: Miridae)

The complete mitochondrial genome of the tarnished plant bug, Lygus lineolaris, comprised 17,027 bp. The genome contained 13 protein coding regions, 22 tRNA genes and 2 ribosomal RNA genes. The gene arrangement corresponded to the common order found among insect mtDNAs which was considered to be the ancestral arrangement. The protein coding genes started with ATN and stopped with TAA or TAG. The nucleotide distribution was 76.0% A + T. The control region contained two repeat regions, one was 24 bp and the other was 161 bp. The Genbank accession for the complete L. lineolaris mt genome is EU401991.

HER2: a 'predictive factor' ready to use in the daily management of breast cancer patients?

The past few years have witnessed an exponential increase in studies trying to identify molecular markers in patients with breast tumours that might predict for the success or failure of hormonal therapy or chemotherapy. HER2, a tyrosine kinase membrane receptor of the epidermal growth factor receptor family, has been the most widely studied marker in this respect. This paper attempts to critically review to what extent HER2 may improve 'treatment individualisation' for the breast cancer patient. Copyright (C) 2000.

Estimating the magnitude of trastuzumab effects within patient subgroups in the HERA trial.

BACKGROUND: Trastuzumab (Herceptin(R)) improves disease-free survival (DFS) and overall survival for patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We aimed to assess the magnitude of its clinical benefit for subpopulations defined by nodal and steroid hormone receptor status using data from the Herceptin Adjuvant (HERA) study. PATIENTS AND METHODS: HERA is an international multicenter randomized trial comparing 1 or 2 years of trastuzumab treatment with observation after standard chemotherapy in women with HER2-positive breast cancer. In total, 1703 women randomized to 1-year trastuzumab and 1698 women randomized to observation were included in these analyses. Median follow-up was 23.5 months. The primary endpoint was DFS. RESULTS: The overall hazard ratio (HR) for trastuzumab versus observation was 0.64 [95% confidence interval (CI) 0.54-0.76; P < 0.0001], ranging from 0.46 to 0.82 for subgroups. Estimated improvement in 3-year DFS in subgroups ranged from +11.3% to +0.6%. Patients with the best prognosis (those with node-negative disease and tumors 1.1-2.0 cm) had benefit similar to the overall cohort (HR 0.53, 95% CI 0.26-1.07; 3-year DFS improvement +4.6%, 95% CI -4.0% to 13.2%). CONCLUSIONS: Adjuvant trastuzumab therapy reduces the risk of relapse similarly across subgroups defined by nodal status and steroid hormone receptor status, even those at relatively low risk for relapse.

A dual role for ErbB2 signaling in cardiac trabeculation.

Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.

Neuroprotection resulting from insufficiency of RANBP2 is associated with the modulation of protein and lipid homeostasis of functionally diverse but linked pathways in response to oxidative stress.

Oxidative stress is a deleterious stressor associated with a plethora of disease and aging manifestations, including neurodegenerative disorders, yet very few factors and mechanisms promoting the neuroprotection of photoreceptor and other neurons against oxidative stress are known. Insufficiency of RAN-binding protein-2 (RANBP2), a large, mosaic protein with pleiotropic functions, suppresses apoptosis of photoreceptor neurons upon aging and light-elicited oxidative stress, and promotes age-dependent tumorigenesis by mechanisms that are not well understood. Here we show that, by downregulating selective partners of RANBP2, such as RAN GTPase, UBC9 and ErbB-2 (HER2; Neu), and blunting the upregulation of a set of orphan nuclear receptors and the light-dependent accumulation of ubiquitylated substrates, light-elicited oxidative stress and Ranbp2 haploinsufficiency have a selective effect on protein homeostasis in the retina. Among the nuclear orphan receptors affected by insufficiency of RANBP2, we identified an isoform of COUP-TFI (Nr2f1) as the only receptor stably co-associating in vivo with RANBP2 and distinct isoforms of UBC9. Strikingly, most changes in proteostasis caused by insufficiency of RANBP2 in the retina are not observed in the supporting tissue, the retinal pigment epithelium (RPE). Instead, insufficiency of RANBP2 in the RPE prominently suppresses the light-dependent accumulation of lipophilic deposits, and it has divergent effects on the accumulation of free cholesterol and free fatty acids despite the genotype-independent increase of light-elicited oxidative stress in this tissue. Thus, the data indicate that insufficiency of RANBP2 results in the cell-type-dependent downregulation of protein and lipid homeostasis, acting on functionally interconnected pathways in response to oxidative stress. These results provide a rationale for the neuroprotection from light damage of photosensory neurons by RANBP2 insufficiency and for the identification of novel therapeutic targets and approaches promoting neuroprotection.

A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.