RESUMEN
BACKGROUND: Epithelial cell adhesion molecule (epcam) is a multifunctional transmembrane glycoprotein expressed on both normal epithelium and epithelial neoplasms such as gastric, breast, and renal carcinomas. Recent studies have proposed that the proteolytic cleavage of the intracellular domain of epcam (epcam-icd) can trigger signalling cascades leading to aggressive tumour behavior. The expression profile of epcam-icd has not been elucidated for primary colorectal carcinoma. In the present study, we examined epcam-icd immunohistochemical staining in a large cohort of patients with primary colorectal adenocarcinoma and assessed its performance as a potential prognostic marker. METHODS: Immunohistochemical staining for epcam-icd was assessed on tissue microarrays consisting of 137 primary colorectal adenocarcinoma samples. Intensity of staining for each core was scored by 3 independent pathologists. The membranous epcam-icd staining score was calculated as a weighted average from 3 core samples per tumour. Univariate analysis of the average scores and clinical outcome measures was performed. RESULTS: The level of membranous epcam-icd staining was positively associated with well-differentiated tumours (p = 0.01); low preoperative carcinoembryonic antigen (p = 0.001); and several measures of survival, including 2-year (p = 0.02) and 5-year survival (p = 0.05), and length of time post-diagnosis (p = 0.03). A number of other variables-including stage, grade, and lymph node status-showed correlations with epcam staining and markers of poor outcome, but did not reach statistical significance. CONCLUSIONS: Low membranous epcam-icd staining might be a useful marker to identify tumours with aggressive clinical behavior and potential poor prognosis and might help to select candidates who could potentially benefit from treatment targeting epcam.
RESUMEN
In previous studies, we demonstrated that miR-193b expression is reduced in melanoma relative to benign nevi, and also that miR-193b represses cyclin D1 and Mcl-1 expression. We suggested that stathmin 1 (STMN1) might be a target of miR-193b. STMN1 normally regulates microtubule dynamics either by sequestering free tubulin heterodimers or by promoting microtubule catastrophe. Increased expression of STMN1 has been observed in a variety of human malignancies, but its association with melanoma is unknown. We now report that STMN1 is upregulated during the progression of melanoma relative to benign nevi, and that STMN1 is directly regulated by miR-193b. Using an experimental cell culture approach, overexpression of miR-193b using synthetic microRNAs repressed STMN1 expression, whereas inhibition of miR-193b with anti-miR oligos increased STMN1 expression in melanoma cells. The use of a luciferase reporter assay confirmed that miR-193b directly regulates STMN1 by targeting the 3'-untranslated region of STMN1 mRNA. We further demonstrated that STMN1 is overexpressed in malignant melanoma compared with nevi in two independent melanoma cohorts, and that its level is inversely correlated with miR-193b expression. However, STMN1 expression was not significantly associated with patient survival, Breslow depth, mitotic count or patient age. STMN1 knockdown by small-interfering RNA in melanoma cells drastically repressed cell proliferation and migration potential, whereas ectopic expression of STMN1 using lentivirus increased cell proliferation and migration rates. Subsequent gene expression analysis indicated that interconnected cytoskeletal networks are directly affected following STMN1 knockdown. In addition, we identified deregulated genes associated with proliferation and migration, and revealed that p21(Cip1/Waf1) and p27(Kip) could be downstream effectors of STMN1 signaling. Taken together, our study suggests that downregulation of miR-193b may contribute to increased STMN1 expression in melanoma, which consequently promotes migration and proliferation of tumor cells.
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Melanoma/genética , Estatmina/genética , Regiones no Traducidas 3' , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patología , MicroARNs/genética , MicroARNs/metabolismo , Oncogenes , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estatmina/biosíntesis , Estatmina/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
A universal response to elevated temperature and other forms of physiological stress is the induction of heat shock proteins (HSPs). Hsp16 in Schizosaccharomyces pombe encodes a polypeptide of predicted molecular weight 16 kDa that belongs to the HSP20/alpha-crystallin family whose members range in size from 12 to 43 kDa. Heat shock treatment increases expression of the hsp16 gene by 64-fold in wild-type cells and 141-fold in cdc22-M45 (ribonucleotide reductase) mutant cells. Hsp16 expression is mediated by the spc1 MAPK signaling pathway through the transcription factor atf1 and in addition through the HSF pathway. Nucleotide depletion or DNA damage as occurs in cdc22-M45 mutant cells, or during hydroxyurea or camptothecin treatment, is sufficient to activate hsp16 expression through atf1. Our findings suggest a novel role for small HSPs in the stress response following nucleotide depletion and DNA damage. This extends the types of damage that are sensed by the spc1 MAPK pathway via atf1.
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Chaperonina 60/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nucleótidos/farmacología , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Chaperonina 60/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , Nitrógeno/farmacología , ARN de Hongos/efectos de los fármacos , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transducción de Señal/efectos de los fármacos , Técnicas del Sistema de Dos HíbridosRESUMEN
We examined a panel of sporadic breast carcinomas for loss of heterozygosity (LOH) in a 10-cM interval on chromosome 10 known to encompass the PTEN gene. We detected allele loss in 27 of 70 breast tumour DNAs. Fifteen of these showed loss limited to a subregion of the area studied. The most commonly deleted region was flanked by D10S215 and D10S541 and encompasses the PTEN locus. We used a combination of denaturing gradient gel electrophoresis and single-strand conformation polymorphism analyses to investigate the presence of PTEN mutations in tumours with LOH in this region. We did not detect mutations of PTEN in any of these tumours. Our data show that, in sporadic breast carcinoma, loss of heterozygosity of the PTEN locus is frequent, but mutation of PTEN is not. These results are consistent with loss of another unidentified tumour suppressor in this region in sporadic breast carcinoma.
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Neoplasias de la Mama/genética , Cromosomas Humanos Par 10 , Genes Supresores de Tumor , Pérdida de Heterocigocidad , Monoéster Fosfórico Hidrolasas/genética , Polimorfismo Conformacional Retorcido-Simple , Proteínas Supresoras de Tumor , Centrómero/genética , Mapeo Cromosómico , ADN de Neoplasias/genética , Femenino , Humanos , Repeticiones de Microsatélite , Fosfohidrolasa PTEN , Reacción en Cadena de la PolimerasaRESUMEN
Deletions involving chromosome 10q23 occur frequently in prostatic carcinomas. Recently, a novel tumour suppressor gene, PTEN, mapping to this interval, has been identified. Mutation or deletion of PTEN has been observed in a proportion of prostate cancer cell lines; however, primary prostate carcinomas have not been studied. We have investigated the involvement of PTEN in primary prostatic adenocarcinomas using a panel of 51 matched normal and prostate tumour DNAs. We first determined the proportion of tumours with allele loss at loci in 10q23 which span the region containing the PTEN gene. Our results show that LOH involving 10q23 is common in primary prostate carcinomas. Twenty-five of 51 (49%) tumours showed loss of heterozygosity (LOH) over the region spanning the PTEN locus. We next directly analysed the PTEN gene for mutations of the coding region using single strand conformation polymorphism (SSCP) and sequence analyses. Of those tumours with LOH, only a single tumour was found to carry a missense mutation in PTEN. No mutations in PTEN were identified in tumours without LOH. Our results suggest either that mutation of PTEN is a late event in prostate tumorigenesis, or that another tumour suppressor gene important in prostate cancer may lie close to PTEN in 10q23.