Molecular Biology and Genomics

Group Leader: Anamaria A. Camargo

Our group has a strong background in Genomics, and has participated over the past years in several genome projects carried out in Brazil, including the Xylella fastidiosa (Simpson et al. 2000) and the Human Cancer Genome Projects (Camargo et al. 2001 and Brentani et al. 2003). We have initially applied our knowledge to identify novel human genes located on chromosomes 22 (De Souza et al. 2000) and 21 (Camargo et al. 2002) and at the Hereditary Prostate Cancer Locus 1 (HPC1) at 1q25 (Silva et al. 2003). We have also coordinated a large collaborative project, known as the Transcript Finishing Initiative, aimed to identify and validate the structure of thousands of new human genes (TFI consortium, 2004). We are now concentrating our efforts on the identification and characterization of human genes related to cancer and new this is being achieved using both computational and experimental approaches. Three research projects are currently under way at LMBG:

Identification and Characterization of Differentially Methylated Genes in Breast Cancer
Genome stability and normal gene expression are maintained by a fixed and predetermined DNA methylation pattern, which becomes abnormal in malignant cells. The characterization of differentially methylated genes in tumors offers great potential for the identification of biomarkers, which can be used to detect and diagnose cancer in its earliest stages and to accurately assess individual risk. In order to identify differentially methylated sequences in breast tumors, we have initially used the Methylation-Sensitive Arbitrarily Primed PCR (MS-APPCR). Recently, two other techniques that allow genome-wide methylation analysis were implemented in the laboratory: i) Differential Methylation Hybridization (DMH) and ii) treatment of tumor cell lines with demethylating agents followed by genome-wide gene expression analysis. Using these methodologies two differentially methylated fragments were identified: a satellite sequence (SATR-1) mapped to chromosome 5 (Costa et al. 2006) and the promoter region of the ADAM23 gene (Costa et al. 2004).

The ADAMs (A Desintegrin And Metalloprotease domain) comprise a family of multifunctional membrane-anchored cell surface glycoproteins with a common structural organization including metalloprotease, disintegrin, cystein-rich, epidermal growth factor-like, transmembrane and cytoplasmatic domains. As a further contribution to understand the role of ADAM23 in human cancer, we evaluated the prognostic significance of ADAM23 hypermethylation in 106 breast tumors. Patients with ADAM23 hypermethylation had a significantly shorter metastasis-free and overall survival than did those without it. ADAM23 hypermethylation was also shown to be an independent prognostic factor for metastasis-free and overall survival. Functional studies using RNAi methodology are being undertaken in order to investigate the role of the ADAM23 in the progression of breast tumors.

Identification and Characterization of Novel c-erbB2 Regulated Genes Using MPSS.
Overexpression of the proto-oncogene ERBB2 is observed in 25-30% of breast cancers. A model of ERBB2 overexpression in conditionally immortalized human mammary luminal epithelial cells was established (Harris et al. 1999) in order to elucidate the role of ERBB2 in breast cancer. MPSS (Massively Parallel Signature Sequencing) was used to address transcriptional differences between an immortalized normal breast epithelial cell (HB4a) and a variant cell line expressing high levels of c-erbB2 (HB4a-C5.2). A significant fraction (9%) of the MPSS tags could not be associated with a known transcript and were designated orphan tags. Among the orphan tags, there were many (1%) that showed differential expression in the c-erbB2 transfected cell line and that probably represent new human transcripts regulated by this oncogene. In order to further characterize these novel human transcripts, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3' cDNA fragments (Silva et al. 2004). GLGI-MPSS allowed the identification of 13 human transcripts putatively regulated by c-erbB2. Functional characterization of these novel ERBB2 regulated genes is underway.

Identification and Characterization of Novel Cancer-Testis (CT) Antigens by EST Data Mining.
Cancer/testis (CT) antigens are a subgroup of tumor antigens with a restricted expression in testis and in different types of tumors and are considered promising candidates for cancer immunotherapy. We have used a computational approach, based on the alignment between ESTs and the human genomic sequence, to identify novel CT candidates. Among these candidates, we identified a novel CT antigen, named CTSP-1. CTSP-1 is exclusively expressed in normal testis and is aberrantly expressed in tumor cell lines and in tumors from different histological types. Antibodies against CTSP-1 were detected in plasma samples from patients with a wide spectrum of tumors. We have also analyzed the presence of antibodies against this antigen in serum samples from 147 prostate cancer patients. Patients that did not present antibodies against CTSP-1 had a shorter biochemical recurrence interval than did those with anti-CTSP-1 antibodies. Interestingly, none of the patients with CTSP-1 antibodies developed clinical symptoms of the disease after surgery. In the multivariate analysis, antibodies against CTSP-1 showed a protective effect being the second most powerful prognostic factor among Gleason score and levels of PSA after surgery.

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