RESUMO
Heat shock protein 70-2 (Hsp70-2) is a chaperone protein essential for the growth of spermatocytes and cancer cells. Here, we show that Hsp70-2 depletion triggers lysosomal membrane permeabilization and cathepsin-dependent cell death and identify lens epithelium-derived growth factor (LEDGF) as an Hsp70-2-regulated guardian of lysosomal stability in human cancer. Knockdown of LEDGF in cancer cells induces destabilization of lysosomal membranes followed by caspase-independent and Bcl-2-resistant cell death. Accordingly, ectopic LEDGF stabilizes lysosomes and protects cancer cells against cytotoxicity induced by anticancer agents that trigger the lysosomal cell death pathway. Remarkably, ectopic LEDGF also increases the tumorigenic potential of human cancer cells in immunodeficient mice, and LEDGF expression is increased in human breast and bladder carcinomas correlating with that of Hsp70-2 in invasive bladder cancer. Taken together, these data reveal LEDGF as an oncogenic protein that controls a caspase-independent lysosomal cell death pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Neoplasias da Mama/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Lisossomos/metabolismo , Fatores de Transcrição/fisiologia , Neoplasias da Bexiga Urinária/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Morte Celular/fisiologia , Linhagem da Célula , Permeabilidade da Membrana Celular , Regulação para Baixo , Feminino , Proteínas de Choque Térmico HSP70/deficiência , Células HeLa , Humanos , Lisossomos/patologia , Camundongos , Camundongos SCID , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Transfecção , Transplante Heterólogo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
The human transcription factor SOX4 was 5-fold up-regulated in bladder tumors compared with normal tissue based on whole-genome expression profiling of 166 clinical bladder tumor samples and 27 normal urothelium samples. Using a SOX4-specific antibody, we found that the cancer cells expressed the SOX4 protein and, thus, did an evaluation of SOX4 protein expression in 2,360 bladder tumors using a tissue microarray with clinical annotation. We found a correlation (P < 0.05) between strong SOX4 expression and increased patient survival. When overexpressed in the bladder cell line HU609, SOX4 strongly impaired cell viability and promoted apoptosis. To characterize downstream target genes and SOX4-induced pathways, we used a time-course global expression study of the overexpressed SOX4. Analysis of the microarray data showed 130 novel SOX4-related genes, some involved in signal transduction (MAP2K5), angiogenesis (NRP2), and cell cycle arrest (PIK3R3) and others with unknown functions (CGI-62). Among the genes regulated by SOX4, 25 contained at least one SOX4-binding motif in the promoter sequence, suggesting a direct binding of SOX4. The gene set identified in vitro was analyzed in the clinical bladder material and a small subset of the genes showed a high correlation to SOX4 expression. The present data suggest a role of SOX4 in the bladder cancer disease.
Assuntos
Proteínas de Grupo de Alta Mobilidade/biossíntese , Transativadores/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Amplificação de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição SOXC , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Transfecção , Regulação para Cima , Neoplasias da Bexiga Urinária/genéticaRESUMO
We have analysed the occurrence of the extracellular glycoprotein vitronectin in carcinomas and normal tissue of human breast. Immunohistochemical analysis of carcinomas revealed a strong vitronectin accumulation in extracellular matrix (ECM) around some cancer cell clusters and in the subendothelial area of some blood vessels. In normal tissue, vitronectin had a homogeneous periductal occurrence, with local accumulation much lower than that in the carcinomas. Using a new solid phase radioligand assay, the vitronectin concentrations of extracts of carcinomas and normal breast tissue were determined and found to be indistinguishable. Comparison of the vitronectin and the hemoglobin concentrations of the extracts showed that their vitronectin content was not derived from blood contamination. Vitronectin mRNA was undetectable in both carcinomas and normal tissue. We conclude that vitronectin is not synthesised locally in breast tissue but derived by leakage from vessels, followed by extracellular accumulation in patterns distinctly different in carcinomas and normal tissue. The observation of a high vitronectin content in the carcinomas and its localisation in the tissue contributes to the clarification of the role of vitronectin in tumour biology in interaction with the plasminogen activation system and integrins.
Assuntos
Neoplasias da Mama/metabolismo , Vitronectina/metabolismo , Sequência de Bases , Mama/irrigação sanguínea , Mama/metabolismo , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/irrigação sanguínea , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Estudos de Casos e Controles , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaio Radioligante , Células Tumorais Cultivadas , Vitronectina/genéticaRESUMO
The aims were to evaluate the common reference design approach in microarray experiments and to evaluate the technical performance and the normalisation of cDNA microarrays with a limited number of spots. Total RNA from 3 normal and 3 tumour sample biopsies were used for synthesis of amino-allyl labelled cRNA. Equal amounts of cRNA from all samples were mixed as reference. After conjugation of cRNA with fluorophores (Cy3/Cy5), each individual tumour cRNA was hybridised to a cDNA microarray together with reference cRNA, scanned and analysed. We show that our procedures for producing cDNA microarrays are reproducible. The concordance between duplicated spots and replicate hybridisation was found to be high. We have demonstrated that our cDNA microarrays are of a high technical quality. The majority of the cDNA microarrays had low local spot background levels. Despite the high background levels for some local spots, variation could be minimized by locally weighted scatter plot smooth normalisation (LOWESS), which we showed was also suitable for normalisation of cDNA microarrays with a limited number of probes.