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1.
Mol Cancer Res ; 15(12): 1678-1691, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28851816

RESUMEN

The 5' (α)-promoter of the human doublecortin-like kinase 1 (DCLK1) gene becomes epigenetically silenced during colon carcinogenesis, resulting in loss of expression of the canonical long(L)-isoform1 (DCLK1-L) in human colon adenocarcinomas (hCRCs). Instead, hCRCs express a short(S)-isoform2 (DCLK1-S) from an alternate (ß)-promoter of DCLK1. The current study, examined if the transcriptional activity of the (ß)-promoter is suppressed in normal versus cancerous cells. On the basis of in silico and molecular approaches, it was discovered that FOXD3 potently inhibits the transcriptional activity of the (ß)-promoter. FOXD3 becomes methylated in human colon cancer cells (hCCC), with loss of FOXD3 expression, allowing expression of the DCLK1(S) variant in hCCCs/hCRCs. Relative levels of FOXD3/DCLK1(S/L) were measured in a cohort of CRC patient specimens (n = 92), in relation to overall survival (OS). Patients expressing high DCLK1(S), with or without low FOXD3, had significantly worse OS compared with patients expressing low DCLK1(S). The relative levels of DCLK1-L did not correlate with OS. In a pilot retrospective study, colon adenomas from high-risk patients (who developed CRCs in <15 years) demonstrated significantly higher staining for DCLK1(S) + significantly lower staining for FOXD3, compared with adenomas from low-risk patients (who remained free of CRCs). Latter results strongly suggest a prognostic value of measuring DCLK1(S)/FOXD3 in adenomas. Overexpression of DCLK1(S), but not DCLK1(L), caused a significant increase in the invasive potential of hCCCs, which may explain worse outcomes for patients with high DCLK1-S-expressing tumors. On the basis of these data, FOXD3 is a potent repressor of DCLK1-S expression in normal cells; loss of FOXD3 in hCCCs/hCRCs allows upregulation of DCLK1-S, imparting a potent invasive potential to the cells. Mol Cancer Res; 15(12); 1678-91. ©2017 AACR.


Asunto(s)
Neoplasias del Colon/genética , Metilación de ADN/genética , Factores de Transcripción Forkhead/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Anciano , Carcinogénesis/genética , Neoplasias del Colon/patología , Quinasas Similares a Doblecortina , Epigénesis Genética/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Células HCT116 , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/genética
2.
Lab Invest ; 97(10): 1245-1261, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28414327

RESUMEN

DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). Human colorectal tumors (CRCs) and hCCCs express a novel short isoform of DCLK1 (DCLK1-S; isoform 2) from ß-promoter of hDCLK1 gene, while normal colons express long isoform (DCLK1-L; isoform 1) from 5'(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer stem cells (CSCs). Even though DCLK1-S differs from DCLK1-L by only six amino acids, we succeeded in generating a monospecific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects CSCs. Subcellular localization of S/L-isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria. Sporadic CRCs develop from adenomas. Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients will never develop cancer. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (high-risk), or did not develop (low-risk) adenocarcinomas within 10-15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (three to fivefold) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC risk. CRC patients, with high DCLK1-S-expressing tumors (by qRT-PCR), were reported to have worse overall survival than low expressers. We now report that DCLK1-S-specific Ab may help to identify high-risk patients at the time of index/screening colonoscopy.


Asunto(s)
Anticuerpos/metabolismo , Biomarcadores de Tumor/análisis , Neoplasias del Colon/diagnóstico , Detección Precoz del Cáncer/métodos , Péptidos y Proteínas de Señalización Intracelular/análisis , Proteínas Serina-Treonina Quinasas/análisis , Anticuerpos/análisis , Biomarcadores de Tumor/metabolismo , Colon/química , Colon/patología , Colon/cirugía , Neoplasias del Colon/cirugía , Colonoscopía , Quinasas Similares a Doblecortina , Células HCT116 , Células HEK293 , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estudios Retrospectivos
3.
Int J Cancer ; 138(8): 1971-81, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26595254

RESUMEN

IL-6 is a pleiotropic cytokine increased in CRC and known to directly promote tumor growth. Colonic myofibroblasts/fibroblasts (CMFs or stromal cells) are CD90(+) innate immune cells representing up to 30% of normal colonic mucosal lamina propria cells. They are expanded in CRC tumor stroma, where they also known as a cancer associated fibroblasts (CAFs). Cells of mesenchymal origin, such as normal myofibroblasts/fibroblasts, are known to secrete IL-6; however, their contribution to the increase in IL-6 in CRC and to tumor-promoting inflammation is not well defined. Using in situ, ex vivo and coculture analyses we have demonstrated that the number of IL-6 producing CMFs is increased in CRC (C-CMFs) and they represent the major source of IL-6 in T2-T3 CRC tumors. Activity/expression of stem cell markers-aldehyde dehydrogenase and LGR5- was significantly up-regulated in colon cancer cells (SW480, Caco-2 or HT29) cultured in the presence of conditioned medium from tumor isolated C-CMFs in an IL-6 dependent manner. C-CMF and its derived condition medium, but not normal CMF isolated from syngeneic normal colons, induced differentiation of tumor promoting inflammatory T helper 17 cells (Th17) cell responses in an IL-6 dependent manner. Our study suggests that CD90(+) fibroblasts/myofibroblasts may be the major source of IL-6 in T2-T3 CRC tumors, which supports the stemness of tumor cells and induces an immune adaptive inflammatory response (a.k.a. Th17) favoring tumor growth. Taken together our data supports the notion that IL-6 producing CAFs (a.k.a. C-CMFs) may provide a useful target for treating or preventing CRCs.


Asunto(s)
Neoplasias Colorrectales/patología , Fibroblastos/inmunología , Interleucina-6/biosíntesis , Células Madre Neoplásicas/patología , Western Blotting , Técnicas de Cocultivo , Neoplasias Colorrectales/inmunología , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Inflamación/patología , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Células del Estroma/inmunología , Células del Estroma/metabolismo , Linfocitos T/inmunología , Antígenos Thy-1/inmunología , Antígenos Thy-1/metabolismo , Microambiente Tumoral/inmunología
4.
Sci Rep ; 5: 14983, 2015 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-26447334

RESUMEN

DCLK1 specifically marks colon/pancreatic cancers in mice, and is expressed by human colon adenocarcinomas (hCRCs). Down-regulation of DCLK1 results in loss of cancer-stem-cells (CSCs), and inhibits spheroidal/xenograft growths from hCRC-cells. The 5'-promoter of DCLK1-gene is reportedly hypermethylated in hCRCs, resulting in loss of expression of DCLK1-transcripts, originating from 5'(α)-promoter (termed DCLK1-L, in here). However, in mouse colon-tumors, 5'-promoter of DCLK1-gene remains unchanged, and DCLK1-L, originating from 5'(α)-promoter, is expressed. We hypothesized that elevated levels of DCLK1-protein in hCRC-cells, may be transcribed/translated from an alternate-promoter. Several in silico and molecular biology approaches were used to test our hypothesis. We report for the first time that majority of hCRCs express short-transcripts of DCLK1 (termed DCLK1-S, in here) from an alternate ß-promoter in IntronV of the gene, while normal-colons mainly express DCLK1-L from 5'(α)-promoter. We additionally report an important role of ß-catenin and TCF4/LEF binding-sites for activating (α)-promoter, while activated NF-κBp65 (bound to NF-κB-cis-element), activates (ß)-promoter in cancer-cells. DCLK1-S expression was examined in a cohort of 92 CRC patients; high-expressors had significantly worse overall-survival compared to low-expressors. Our novel findings' regarding usage of alternate (ß)-promoter by hCRCs, suggests that DCLK1-S may represent an important target for preventing/inhibiting colon-cancers, and for eliminating colon-CSCs.


Asunto(s)
Neoplasias del Colon/genética , Epigénesis Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/genética , Anciano , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Metilación de ADN , Quinasas Similares a Doblecortina , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido
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