Waleska K. Martins

Molecular classifiers for gastric cancer and nonmalignant diseases of the gastric mucosa

High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher’s linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.

Characterization of a cancer/testis (CT) antigen gene family capable of eliciting humoral response in cancer patients

Cancer/testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CT antigens are promising candidates for cancer immunotherapy, and the identification of novel CT antigens is a prerequisite for the development of cancer vaccines. We have identified a CT antigen, named CTSP-1, with partial similarity to the breast differentiation antigen NY-BR-1. CTSP-1 presents several splicing and polyadenylation variants and has a very restricted expression pattern among normal tissues. CTSP-1 is exclusively expressed in normal testis and is aberrantly expressed in 47.6% (10 of 21) of tumor cell lines and in 44.4% (75 of 169) of tumors from different histological types. The highest percentages of positive expression were observed in melanomas (59.0%) followed by prostate (58.0%) and lung (57.0%) tumors. CTSP-1 is part of a highly conserved gene family, and members of this family also have a restricted expression pattern and similar protein structure. Antibodies against members of this gene family were detected in 10% (14 of 141) of plasma samples from patients with a wide spectrum of tumors. The highest percentages of antibody response were observed in patients with prostate (20.8%), thyroid (20.0%), and breast (16.6%) tumors. Because of its very restricted expression pattern in normal tissues and immunogenicity in different types of tumors, CTSP-1 should be considered a promising candidate for cancer immunotherapy.

Differentially expressed genes in gastric tumors identified by cDNA array

Using cDNA fragments from the FAPESP/lICR Cancer Genome Project, we constructed a cDNA array having 4512 elements and determined gene expression in six normal and six tumor gastric tissues. Using t-statistics, we identified 80 cDNAs whose expression in normal and tumor samples differed more than 3.5 sample standard deviations. Using Self-Organizing Map, the expression profile of these cDNAs allowed perfect separation of malignant and non-malignant samples. Using the supervised learning procedure Support Vector Machine, we identified trios of cDNAs that could be used to classify samples as normal or tumor, based on single-array analysis. Finally, we identified genes with altered linear correlation when their expression in normal and tumor samples were compared. Further investigation concerning the function of these genes could contribute to the understanding of gastric carcinogenesis and may prove useful in molecular diagnostics.

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

BackgroundThe ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers.ResultsOf the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes.ConclusionsThe versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

Differential expression of IGFBP-5 and two human ESTs in thyroid glands with goiter, adenoma and papillary or follicular carcinomas.

Here, we describe the identification of three human genes with altered expression in thyroid diseases. One of them corresponds to insulin-like growth factor binding protein 5 (IGFBP5), which has already been described as over expressed in other cancers and, for the first time, is identified as overexpressed in thyroid tumors. The other genes, named 44 and 199, are ESTs with yet unknown function and were mapped on human chromosomes seven and four, respectively. We determined by RT-PCR the expression level of these genes in ten samples of disease-free thyroid, ten of goiter, nine of papillary carcinoma, ten of adenoma and seven of follicular carcinoma and the significance of observed differences was statistically determined. IGFBP-5 and gene 44 were significantly overexpressed in papillary carcinoma when compared to normal and goiter. Genes 44 and 199 were differentially expressed in follicular carcinoma and adenoma when compared to normal thyroid tissue.

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

BackgroundPersistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours.MethodsIn this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts.ResultsWe observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells.ConclusionThis is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

Parallel damage in mitochondrial and lysosomal compartments promotes efficient cell death with autophagy: The case of the pentacyclic triterpenoids

The role of autophagy in cell death is still controversial and a lot of debate has concerned the transition from its pro-survival to its pro-death roles. The similar structure of the triterpenoids Betulinic (BA) and Oleanolic (OA) acids allowed us to prove that this transition involves parallel damage in mitochondria and lysosome. After treating immortalized human skin keratinocytes (HaCaT) with either BA or OA, we evaluated cell viability, proliferation and mechanism of cell death, function and morphology of mitochondria and lysosomes, and the status of the autophagy flux. We also quantified the interactions of BA and OA with membrane mimics, both in-vitro and in-silico. Essentially, OA caused mitochondrial damage that relied on autophagy to rescue cellular homeostasis, which failed upon lysosomal inhibition by Chloroquine or Bafilomycin-A1. BA caused parallel damage on mitochondria and lysosome, turning autophagy into a destructive process. The higher cytotoxicity of BA correlated with its stronger efficiency in damaging membrane mimics. Based on these findings, we underlined the concept that autophagy will turn into a destructive outcome when there is parallel damage in mitochondrial and lysosomal membranes. We trust that this concept will help the development of new drugs against aggressive cancers.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Autophagy Modulation for Organelle-Targeting Therapy

Autophagy is a crucial metabolic pathway that sustains cellular homeostasis in health and that can also play either a protective or a destructive role in disease. During the last decade, progress made in understanding of the molecular basis of autophagy has uncovered an exciting opportunity to target it for the treatment of several human illnesses. In fact, there is emerging interest in autophagy-modulating and autophagytargeted therapy with a variety of pharmacologic agents. However, to develop effective autophagy-targeted therapy, it is essential to identify the pharmacologic key targets in the autophagy pathway. In this chapter, we reviewed the cases of success and pitfalls of activating or inhibiting autophagy attempting therapeutic intervention of diseases, including cancer, neurologic disorders, and infectious diseases. In all these histopathologic states, autophagy is considered as the principal cellular mechanisms of defense and immunochemical homeostasis. In the last section of this chapter, we discuss main directions that may be of particular use in the future investigations, including a promissory avenue for autophagy modulation for organelle-targeting therapy through a promotion of parallel damage in lysosomal and mitochondrial membranes.

Photosensitized Membrane Permeabilization Requires Contact-Dependent Reactions between Photosensitizer and Lipids

Although the general mechanisms of lipid oxidation are known, the chemical steps through which photosensitizers and light permeabilize lipid membranes are still poorly understood. Herein we characterized the products of lipid photooxidation and their effects on lipid bilayers, also giving insight into their formation pathways. Our experimental system was designed to allow two phenothiazinium-based photosensitizers (methylene blue, MB, and DO15) to deliver the same amount of singlet oxygen molecules per second to 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine liposome membranes, but with a substantial difference in terms of the extent of direct physical contact with lipid double bonds; that is, DO15 has a 27-times higher colocalization with ω-9 lipid double bonds than MB. Under this condition, DO15 permeabilizes membranes at least 1 order of magnitude more efficiently than MB, a result that was also valid for liposomes made of polyunsaturated lipids. Quantification of reaction products uncovered a mixture of phospholipid hydroperoxides, alcohols, ketones, and aldehydes. Although both photosensitizers allowed the formation of hydroperoxides, the oxidized products that require direct reactions between photosensitizer and lipids were more prevalent in liposomes oxidized by DO15. Membrane permeabilization was always connected with the presence of lipid aldehydes, which cause a substantial decrease in the Gibbs free energy barrier for water permeation. Processes depending on direct contact between photosensitizers and lipids were revealed to be essential for the progress of lipid oxidation and consequently for aldehyde formation, providing a molecular-level explanation of why membrane binding correlates so well with the cell-killing efficiency of photosensitizers.