RESUMO
The gene encoding CRASH, a human asparaginase-like protein, has been cloned and its transcriptional activation has been detected in gynecologic cancers. To define the expression of CRASH in human tumors and its possible functional role, monoclonal antibodies against the CRASH protein have been generated. In non-transformed tissues CRASH was only detected in testis, brain, esophagus, prostate and proliferating endometrium. On the other hand, 36/50 ovarian carcinomas, 16/78 mammary carcinomas, 6/6 uroepithelial bladder carcinomas and 5/33 colon carcinomas scored positive for CRASH, with the absence of reactivity in the corresponding normal tissues. Strikingly, 11 out of the 16 breast cancers that expressed CRASH were metastatic, nominating CRASH to be functionally relevant in tumor progression. Twenty-eight out of 42 endometrium tumors expressed CRASH at high levels as did 5/41 prostate carcinomas, as well as ovary and breast cancers, indicating a regulation of CRASH expression by sex hormones. A bona fide estrogen responsive element was detected at bases -201/-183. This proved to be highly preserved across species, supporting an actual functional role. Asparaginase-like proteins play a role in growth regulation and signaling by p70 S6 kinase. The somatic knock-out of CRASH resulted in significant inhibition of growth of KM12L4A colon carcinoma cells, which abundantly express CRASH, whereas the proliferation of the syngeneic, weakly-expressing, slowly-growing KL12SM was not affected. These results are consistent with a selective growth advantage for aggressive cancers expressing CRASH, and nominate CRASH as a novel diagnostic and therapeutic tumor target.
Assuntos
Asparaginase/metabolismo , Autoantígenos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias Ovarianas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Asparaginase/antagonistas & inibidores , Asparaginase/genética , Autoantígenos/genética , Western Blotting , Neoplasias da Mama/patologia , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/secundário , Linhagem Celular Tumoral , Cistadenocarcinoma/metabolismo , Cistadenocarcinoma/secundário , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neoplasias Ovarianas/patologia , Prognóstico , RNA Interferente Pequeno/farmacologia , Homologia de Sequência de AminoácidosRESUMO
We have identified CRASH, a human asparaginase-like protein which is composed of 308 amino acids and exhibits 32% homology to human aspartylglucosaminadase at the amino acid level. Database analysis revealed that the gene corresponding to CRASH is composed of 7 exons and 6 introns. Steady-state level of CRASH mRNA was found to be increased in 5 cell lines derived from metastatic lesions compared with 2 cell lines derived from primary mammary carcinoma and HMEC (human mammary epithelial cells). We found that the mRNA level of CRASH correlates with the metastatic propensity of several isogenic human colon cancer and pancreatic carcinoma cell lines. CRASH corresponds to a recently identified sperm autoantigen and furthermore we have demonstrated inducibility of CRASH mRNA by androgen and progesterone. Investigation of several types of human cancers and their corresponding normal tissues revealed high levels of CRASH mRNA in uterine, mammary and ovarian tumors compared with the corresponding normal tissues. CRASH mRNA expression was analysed in breast cancer samples with disclosed clinico-pathological features and corresponding normal tissues. The levels of CRASH mRNA were significantly up-regulated in tumors compared with normal breast tissues and correlate with lack of estrogen receptor expression of the tumors.
Assuntos
Asparaginase/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias dos Genitais Femininos/genética , Sequência de Aminoácidos , Asparaginase/metabolismo , Autoantígenos , Sequência de Bases , Northern Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Éxons , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes/genética , Neoplasias dos Genitais Femininos/metabolismo , Neoplasias dos Genitais Femininos/patologia , Células HL-60 , Células HeLa , Humanos , Imuno-Histoquímica , Íntrons , Células K562 , Dados de Sequência Molecular , Norgestrel/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Cell lines 4A4 and 2C5 are the respective metastatic and non-metastatic variants (nude mouse system) derived from the human mammary carcinoma cell line MDA-MB-435. In order to identify genes associated with or functionally involved in metastasis, we have extended our previous transcriptional profile from 5000 to 12,000 genes using the Affymetrix Gene Chip array technology. Based on a threshold level of a change factor of > or = 2.5, we found that the steady-state level of 40 genes (0.3%) was up-regulated and conversely 89 genes (0.7%) were down-regulated in the metastatic cell line 4A4. The de-regulated genes were classified into categories such as tumor antigens/transmembrane receptors, enzymes, mediators of signaling, cell migration and angiogenesis, cell-cycle-, apoptosis-, differentiation- and growth-factor related genes, tumor suppression, transcription factors and genes encoding components of the extracellular matrix and the cytoskeleton. As possible mediators of invasion we identified DGCR6, osteopontin, autotaxin and the 65 kD phosphoprotein p65. In addition, three sugar-modifying enzymes were up-regulated in cell line 4A4. Profound differences in G-protein-mediated signaling and down-regulation of the tumor-suppressor genes DPC4, BARDI and DLC-1 were noted in the metastatic cell line 4A4.
Assuntos
Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Animais , Movimento Celular/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Nus , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células Tumorais CultivadasRESUMO
We derived the transcriptional profiles of four invasive and four noninvasive mammary carcinoma cell lines by Affymetrix GeneChip((R)) Technology with the profile of human mammary epithelial cells as a reference. We focused on the identification of genes which are upregulated in the invasive cell lines based on the following threshold levels: -fold change of 2 or higher in at least three or more cell lines. According to the scoring criteria as described above, we identified 18 transmembrane receptors, 18 secreted proteins and 5 kinases. Several of the genes described have already been put into context with respect to invasion of mammary carcinoma. We therefore focused on deregulated genes for which such an association has not been described before: transmembrane receptor tyrosine kinase DDR2, transmembrane receptors PMP22 and EMP3 and cell adhesion molecule N-cadherin. Making use of real-time PCR, consistently increased steady-state levels of mRNAs for these genes were found in an extended panel of invasive and noninvasive mammary cancer cell lines.