Avaliação da ação antitumoral in vitro da pterocarpanoquinona LQB 118 e estudo de alguns mecanismos de ação

Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata.

Aberrant expression of X-linked genes RbAp46, Rsk4, and Cldn2 in breast cancer

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes.

Esquemas terapêuticos no carcinoma da bexiga

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

A study of the regulation of Trib family members expression in normal and malignant haematopoiesis

The Tribbles family of genes consist of three members; TRIB1, TRIB2 and TRIB3. Trib1 and Trib2 have been identified as oncogenes that can induce AML in mice. However little is known about how the expressions of the Tribbles family genes are controlled in the cell during haematopoiesis or leukaemogenesis. To investigate the Tribbles genes in leukaemia a bioinformatics approach was used. TRIB2 expression was found to be elevated in T-ALL and ALL with t(1;19). TRIB1 was found not to be significantly elevated in any leukaemic subtypes. Analyses of the TRIB1 and TRIB2 gene signatures in both leukaemic and normal haematopoietic cells identified pathways and transcription factors associated with these signatures. Pathways enriched for the TRIB1 signature included TLR signalling pathways and NF-κB pathways. Transcription factors enriched for this signature include C/EBP and SRF. Enriched for the TRIB2 signature includes T cell signalling pathways and Notch signalling pathways. Transcription factors enriched for this signature include E2F and ETS. Further investigation in vitro confirmed the finding that E2F1 was as a potential regulator of TRIB2 expression. E2F1 is able to directly bind to the TRIB2 promoter region and induce TRIB2 expression. C/EBPα p42 was found to inhibit E2F1 and the p30 isoform was found to cooperate with E2F1 induced activation of the TRIB2 promoter. Indicating the potential presence of a regulatory loop involved in the regulation of the TRIB2 gene. In conclusion we have investigated the Tribbles gene signatures in both normal haematopoietic and leukaemic cells. This has led to the identification of a number of pathways and transcription factors associated with these genes. We have also identified a family of transcription factors directly responsible for the regulation of TRIB2 expression. This regulatory pathway has the potential to be targeted in the treatment of leukaemia with a high TRIB2 signature.

Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement.

We studied the pattern of BCR involvement in 52 patients with chronic myeloid leukemia by Southern blotting. Of 33 Philadelphia (Ph)-positive patients, 30 had evidence of M-BCR rearrangement, two cases were difficult to interpret, and one clearly lacked evidence of M-BCR rearrangement. Of 19 Ph-negative patients, nine showed M-BCR rearrangement, nine showed no rearrangement, and one result was uncertain. We selected for more detailed study eight patients (three Ph-positive and five Ph-negative). Two of the Ph-positive patients, whose Southern blots were difficult to interpret, had rearranged bands when the BCR gene was studied by pulsed field gel electrophoresis (PFGE). Results of PFGE studies and in situ hybridization to metaphase chromosomes in the third Ph-positive patient, whose DNA clearly lacked M-BCR rearrangement on Southern analysis, were consistent with a breakpoint on chromosome 22 located 3' of all known exons of the BCR gene. However, mRNA studied with the polymerase chain reaction showed evidence of a classical b2-a2 linkage. The findings in this patient may be explained by an unusual genomic breakpoint downstream of the BCR gene associated with long range splicing that excluded all of the 3' BCR exons. Of the five patients with Ph-negative M-BCR non-rearranged CML studied by PFGE for BCR gene rearrangement, none had evidence of rearranged bands. We conclude that PFGE is a valuable adjunct to standard molecular techniques for the study of atypical cases of CML. Occasional patients with Ph-positive CML have breakpoints outside M-BCR. The BCR gene is probably not involved in patients with Ph-negative, M-BCR non-rearranged CML.

Animal models of soft-tissue sarcoma.

Soft-tissue sarcomas (STSs) are rare mesenchymal tumors that arise from muscle, fat and connective tissue. Currently, over 75 subtypes of STS are recognized. The rarity and heterogeneity of patient samples complicate clinical investigations into sarcoma biology. Model organisms might provide traction to our understanding and treatment of the disease. Over the past 10 years, many successful animal models of STS have been developed, primarily genetically engineered mice and zebrafish. These models are useful for studying the relevant oncogenes, signaling pathways and other cell changes involved in generating STSs. Recently, these model systems have become preclinical platforms in which to evaluate new drugs and treatment regimens. Thus, animal models are useful surrogates for understanding STS disease susceptibility and pathogenesis as well as for testing potential therapeutic strategies.

Age-specific differences in oncogenic pathway deregulation seen in human breast tumors.

PURPOSE: To define the biology driving the aggressive nature of breast cancer arising in young women. EXPERIMENTAL DESIGN: Among 784 patients with early stage breast cancer, using prospectively-defined, age-specific cohorts (young or=65 years), 411 eligible patients (n = 200or=65 years) with clinically-annotated Affymetrix microarray data were identified. GSEA, signatures of oncogenic pathway deregulation and predictors of chemotherapy sensitivity were evaluated within the two age-defined cohorts. RESULTS: In comparing deregulation of oncogenic pathways between age groups, a higher probability of PI3K (p = 0.006) and Myc (p = 0.03) pathway deregulation was observed in breast tumors arising in younger women. When evaluating unique patterns of pathway deregulation, a low probability of Src and E2F deregulation in tumors of younger women, concurrent with a higher probability of PI3K, Myc, and beta-catenin, conferred a worse prognosis (HR = 4.15). In contrast, a higher probability of Src and E2F pathway activation in tumors of older women, with concurrent low probability of PI3K, Myc and beta-catenin deregulation, was associated with poorer outcome (HR = 2.7). In multivariate analyses, genomic clusters of pathway deregulation illustrate prognostic value. CONCLUSION: Results demonstrate that breast cancer arising in young women represents a distinct biologic entity characterized by unique patterns of deregulated signaling pathways that are prognostic, independent of currently available clinico-pathologic variables. These results should enable refinement of targeted treatment strategies in this clinically challenging situation.

Evidence that SOX2 overexpression is oncogenic in the lung.

BACKGROUND: SOX2 (Sry-box 2) is required to maintain a variety of stem cells, is overexpressed in some solid tumors, and is expressed in epithelial cells of the lung. METHODOLOGY/PRINCIPAL FINDINGS: We show that SOX2 is overexpressed in human squamous cell lung tumors and some adenocarcinomas. We have generated mouse models in which Sox2 is upregulated in epithelial cells of the lung during development and in the adult. In both cases, overexpression leads to extensive hyperplasia. In the terminal bronchioles, a trachea-like pseudostratified epithelium develops with p63-positive cells underlying columnar cells. Over 12-34 weeks, about half of the mice expressing the highest levels of Sox2 develop carcinoma. These tumors resemble adenocarcinoma but express the squamous marker, Trp63 (p63). CONCLUSIONS: These findings demonstrate that Sox2 overexpression both induces a proximal phenotype in the distal airways/alveoli and leads to cancer.

G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity.

The alpha 1B-adrenergic receptor (alpha 1B-ADR) is a member of the G-protein-coupled family of transmembrane receptors. When transfected into Rat-1 and NIH 3T3 fibroblasts, this receptor induces focus formation in an agonist-dependent manner. Focus-derived, transformed fibroblasts exhibit high levels of functional alpha 1B-ADR expression, demonstrate a catecholamine-induced enhancement in the rate of cellular proliferation, and are tumorigenic when injected into nude mice. Induction of neoplastic transformation by the alpha 1B-ADR, therefore, identifies this normal cellular gene as a protooncogene. Mutational alteration of this receptor can lead to activation of this protooncogene, resulting in an enhanced ability of agonist to induce focus formation with a decreased latency and quantitative increase in transformed foci. In contrast to cells expressing the wild-type alpha 1B-ADR, focus formation in "oncomutant"-expressing cell lines appears constitutively activated with the generation of foci in unstimulated cells. Further, these cell lines exhibit near-maximal rates of proliferation even in the absence of catecholamine supplementation. They also demonstrate an enhanced ability for tumor generation in nude mice with a decreased period of latency compared with cells expressing the wild-type receptor. Thus, the alpha 1B-ADR gene can, when overexpressed and activated, function as an oncogene inducing neoplastic transformation. Mutational alteration of this receptor gene can result in the activation of this protooncogene, enhancing its oncogenic potential. These findings suggest that analogous spontaneously occurring mutations in this class of receptor proteins could play a key role in the induction or progression of neoplastic transformation and atherosclerosis.

Evaluation of Altered Kras Codon Bias and NOS Inhibition During Lung Tumorigenesis

The small GTPases HRAS, NRAS and KRAS are mutated in approximately one-third of all human cancers, rendering the proteins constitutively active and oncogenic. Lung cancer is the leading cause of cancer deaths worldwide, and more than 20% of human lung cancers harbor mutations in RAS, with 98% of those occurring in the KRAS isoform. While there have been many advances in the understanding of KRAS–driven lung tumorigenesis, it remains a therapeutic challenge. To further this understanding and assess novel approaches for treatment, I have investigated two aspects of Kras–driven tumorigenesis in the lung:

(I) Despite nearly identical protein sequences, the three RAS proto-oncogenes exhibit divergent codon usage. Of the three isoforms, KRAS contains the most rare codons resulting in lower levels of KRAS protein expression relative to HRAS and NRAS. To determine the consequences of rare codon bias during de novo tumorigenesis, we created a knock-in Krasex3op mouse in which synonymous mutations in exon 3 converted codons from rare to common. These mice had reduced tumor burden and fewer oncogenic mutations in the Krasex3op allele following carcinogen exposure. The reduction in tumorigenesis appeared to be a product of rare codons affecting both the oncogenic and non–oncogenic alleles. Converting rare codons to common codons yielded a more potent oncogenic allele that promoted growth arrest and enhanced tumor suppression by the non-oncogenic allele. Thus, rare codons play an integral role in Kras tumorigenesis.

(II) Lung cancer patients exhale higher levels of NO and iNOS-/- mice are resistant to chemically induced lung tumorigenesis. I hypothesize that NO promotes Kras–driven lung adenocarcinoma, and NOS inhibition may decrease Kras–driven lung tumorigenesis. To test this hypothesis, I assessed efficacy of the NOS inhibitor L–NAME in a genetically engineered mouse model of Kras-driven lung adenocarcinoma. Adenoviral Cre recombinase was delivered into the lungs intranasally, resulting in expression of oncogenic KrasG12D and dominant-negative Trp53R172H in lung epithelial cells. L–NAME treatment was provided in the water and continued until survival endpoints. In this model, L–NAME treatment decreased tumor growth and prolonged survival. These data establish a potential clinical role for NOS inhibition in lung cancer treatment.

Chromatin accessibility mapping identifies mediators of basal transcription and retinoid-induced repression of OTX2 in medulloblastoma.

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi.

Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.

Molecular biology of cancer: mechanisms, targets, and therapeutics

1: Introduction 2: DNA structure and stability: mutations vs. repair 3: Regulation of gene expression 4: Growth factor signaling and oncogenes 5: The cell cycle 6: Growth inhibition and tumor suppressor genes 7: Apoptosis 8: Stem cells and differentiation 9: Metastasis 10: Infections and inflammation 11: Nutrients, hormones, and gene interactions 12: The Cancer Industry: drug development and clinical trial design 13: Cancer in the future: focus on diagnostics and immunotherapy

High incidence of proviral integrations in the Hoxa locus in a new model of E2a-PBX1-induced B-cell leukemia

Relevant mouse models of E2a-PBX1-induced pre-B cell leukemia are still elusive. We now report the generation of a pre-B leukemia model using E2a-PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3epsilon expression (CD3epsilon(-/-)). Using insertional mutagenesis and inverse-PCR, we show that B-cell leukemia development in the E2a-PBX1 x CD3epsilon(-/-) compound transgenic animals is significantly accelerated when compared to control littermates, and document several known and novel integrations in these tumors. Of all common integration sites, a small region of 19 kb in the Hoxa gene locus, mostly between Hoxa6 and Hoxa10, represented 18% of all integrations in the E2a-PBX1 B-cell leukemia and was targeted in 86% of these leukemias compared to 17% in control tumors. Q-PCR assessment of expression levels for most Hoxa cluster genes in these tumors revealed an unprecedented impact of the proviral integrations on Hoxa gene expression, with tumors having one to seven different Hoxa genes overexpressed at levels up to 6600-fold above control values. Together our studies set the stage for modeling E2a-PBX1-induced B-cell leukemia and shed new light on the complexity pertaining to Hox gene regulation. In addition, our results show that the Hoxa gene cluster is preferentially targeted in E2a-PBX1-induced tumors, thus suggesting functional collaboration between these oncogenes in pre-B-cell tumors.

Identification of PP2A as a novel interactor and regulator of TRIP-Br1

TRIP-Br proteins area novel family of transcriptional coregulators involved in E2F-mediated cell cycle progression. Three of the four mammalian members of TRIP-Br family, including TRIP-Br1, are known oncogenes. We now report the identification of the Bot regulatory subunit of serine/threonine protein phosphatase 2A (MA) as a novel TRIP-Br1 interactor, based on an affinity binding assay coupled with mass spectrometry. A GST-TRIP-Br1 fusion protein associates with catalytically active PP2A-AB alpha C holoenzyme in vitro. Coimmunoprecipitation confirms this association in vivo. Immunofluorescence staining with a monoclonal antibody against TRIP-Br1 reveals that endogenous TRIP-Br1 and PP2A-B alpha colocalize mainly in the cytoplasm. Consistently, immunoprecipitation followed by immunodetection with anti-phosphoserine antibody suggest that TRIP-Br1 exists in a serine-phosphorylated form. Inhibition of PP2A activity by okadaic acid or transcriptional silencing of the PP2A catalytic subunit by small interfering RNA results in downregulation of total TRIP-Br1 protein levels but upregulation of serine-phosphorylated TRIP-Br1. Overexpression of PP2A catalytic subunit increases TRIP-Br1 protein levels and TRIP-Br1 co-activated E2F1/DP1 transcription. Our data support a model in which association between PP2A-AB alpha C holoenzyme and TRIP-Br1 in vivo in mammalian cells represents a novel mechanism for regulating the level of TRIP-Br1 protooncoprotein. (C) 2008 Elsevier Inc. All rights reserved.