RESUMEN
E2F3 is a transcription factor that may initiate tumorigenesis if overexpressed. Previously, we demonstrated that E2F3 mRNA is overexpressed in breast cancer and that E2F3 overexpression results in centrosome amplification and unregulated mitosis, which can promote aneuploidy and chromosome instability to initiate and sustain tumors. Further, we demonstrated that E2F3 leads to overexpression of the mitotic regulator Shugoshin-1, which until recently had unknown roles in cancer. This study aims to evaluate the roles of E2F3 and Shugoshin-1 in breast cancer metastatic potential. Here we demonstrated that E2F3 and Shugoshin-1 silencing leads to reduced cell invasion and migration in two mesenchymal triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578t). Moreover, E2F3 and Shugoshin-1 modulate the expression of epithelial-to-mesenchymal transition-associated genes such as Snail, E-Cadherin, and multiple matrix metalloproteinases. Furthermore, E2F3 depletion leads to reductions in tumor growth and metastasis in NOD-scid Gamma mice. Results from this study suggest a key role for E2F3 and a novel role for Shugoshin-1 in metastatic progression. These results can further help in the improvement of TNBC targeted therapies by interfering with pathways that intersect with the E2F3 and Shugoshin-1 signaling pathways.
Asunto(s)
Movimiento Celular/genética , Factor de Transcripción E2F3/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Masculino , Ratones , Ratones SCID , Transducción de Señal/genéticaRESUMEN
Prediction of lung cancer metastasis relies on post-resection assessment of tumor histology, which is a severe limitation since only a minority of lung cancer patients are diagnosed with resectable disease. Therefore, characterization of metastasis-predicting biomarkers in pre-resection small biopsy specimens is urgently needed. Here we report a biomarker consisting of the phosphorylation of the retinoblastoma protein (Rb) on serine 249 combined with elevated p39 expression. This biomarker correlates with epithelial-to-mesenchymal transition traits in non-small cell lung carcinoma (NSCLC) cells. Immunohistochemistry staining of NSCLC tumor microarrays showed that strong phospho-Rb S249 staining positively correlated with tumor grade specifically in the squamous cell carcinoma (SCC) subtype. Strong immunoreactivity for p39 positively correlated with tumor stage, lymph node invasion, and distant metastases, also in SCC. Linear regression analyses showed that the combined scoring for phospho-Rb S249, p39 and E-cadherin in SCC is even more accurate at predicting tumor staging, relative to each score individually. We propose that combined immunohistochemistry staining of NSCLC samples for Rb phosphorylation on S249, p39, and E-cadherin protein expression could aid in the assessment of tumor staging and metastatic potential when tested in small primary tumor biopsies. The intense staining for phospho-Rb S249 that we observed in high grade SCC could also aid in the precise sub-classification of poorly differentiated SCCs.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Proteína de Retinoblastoma/metabolismo , Biomarcadores de Tumor/genética , Cadherinas/biosíntesis , Cadherinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Adhesión Celular/genética , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Clasificación del Tumor , Metástasis de la Neoplasia , Fosforilación , Proteína de Retinoblastoma/genéticaRESUMEN
AIM: To determine serum vitamin D levels and colonic vitamin D receptor (VDR) expression in inflammatory bowel disease (IBD) and non-IBD patients and correlate these with histopathology. METHODS: Puerto Rican IBD (n = 10) and non-IBD (n = 10) patients ≥ 21 years old scheduled for colonoscopy were recruited. Each patient completed a questionnaire and provided a serum sample and a colonic biopsy of normal-appearing mucosa. For IBD patients, an additional biopsy was collected from visually diseased mucosa. Serum vitamin D levels were measured by ultra-performance liquid chromatography and mass spectrometry. Hematoxylin and eosin stained tissue sections from colonic biopsies were classified histologically as normal or colitis (active/inactive), and scored for the degree of inflammation present (0-3, inactive/absent to severe). Tissue sections from colonic biopsies were also stained by immunohistochemistry for VDR, for which representative diagnostic areas were photographed and scored for staining intensity using a 4-point scale. RESULTS: The IBD cohort was significantly younger (40.40 ± 5.27, P < 0.05) than the non-IBD cohort (56.70 ± 1.64) with a higher prevalence of vitamin D deficiency (40% vs 20%, respectively) and insufficiency (70% vs 50%, respectively). Histologic inflammation was significantly higher in visually diseased mucosa from IBD patients (1.95 ± 0.25) than in normal-appearing mucosa from control patients (0.25 ± 0.08, P < 0.01) and from IBD patients (0.65 ± 0.36, P < 0.05) and correlated inversely with VDR expression in visually diseased colonic tissue from IBD patients (r = -0.44, P < 0.05) and from IBD patients with Crohn's disease (r = -0.69, P < 0.05), but not in normal-appearing colonic tissue from control patients or IBD patients. Control and IBD patient serum vitamin D levels correlated positively with VDR expression in normal colon from control and IBD patients (r = 0.38, P < 0.05) and with patient age (r = 0.54, P < 0.01). CONCLUSION: Levels of serum vitamin D correlate positively with colonic VDR expression in visually normal mucosa whereas inflammation correlates negatively with colonic VDR expression in visually diseased mucosa in Puerto Rican patients.
Asunto(s)
Colitis Ulcerosa/sangre , Colon/química , Enfermedad de Crohn/sangre , Mucosa Intestinal/química , Receptores de Calcitriol/análisis , Deficiencia de Vitamina D/sangre , Vitamina D/análogos & derivados , Adulto , Anciano , Biomarcadores/sangre , Biopsia , Estudios de Casos y Controles , Cromatografía Liquida , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/epidemiología , Colon/patología , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/epidemiología , Femenino , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Prevalencia , Puerto Rico/epidemiología , Vitamina D/sangre , Deficiencia de Vitamina D/diagnóstico , Deficiencia de Vitamina D/epidemiología , Adulto JovenRESUMEN
AIM: To determine the expression of neurokinin-1 receptor (NK-1R), phosphorylated epidermal growth factor receptor (pEGFR), cyclooxygenase-2 (Cox-2), and vitamin D receptor (VDR) in normal, inflammatory bowel disease (IBD), and colorectal neoplasia tissues from Puerto Ricans. METHODS: Tissues from patients with IBD, colitis-associated colorectal cancer (CAC), sporadic dysplasia, and sporadic colorectal cancer (CRC), as well as normal controls, were identified at several centers in Puerto Rico. Archival formalin-fixed, paraffin-embedded tissues were de-identified and processed by immunohistochemistry for NK-1R, pEGFR, Cox-2, and VDR. Pictures of representative areas of each tissues diagnosis were taken and scored by three observers using a 4-point scale that assessed intensity of staining. Tissues with CAC were further analyzed by photographing representative areas of IBD and the different grades of dysplasia, in addition to the areas of cancer, within each tissue. Differences in the average age between the five patient groups were assessed with one-way analysis of variance and Tukey-Kramer multiple comparisons test. The mean scores for normal tissues and tissues with IBD, dysplasia, CRC, and CAC were calculated and statistically compared using one-way analysis of variance and Dunnett's multiple comparisons test. Correlations between protein expression patterns were analyzed with the Pearson's product-moment correlation coefficient. Data are presented as mean ± SE. RESULTS: On average, patients with IBD were younger (34.60 ± 5.81) than normal (63.20 ± 6.13, P < 0.01), sporadic dysplasia (68.80 ± 4.42, P < 0.01), sporadic cancer (74.80 ± 4.91, P < 0.001), and CAC (57.50 ± 5.11, P < 0.05) patients. NK-1R in cancer tissue (sporadic CRC, 1.73 ± 0.34; CAC, 1.57 ± 0.53) and sporadic dysplasia (2.00 ± 0.45) were higher than in normal tissues (0.73 ± 0.19). pEGFR was significantly increased in sporadic CRC (1.53 ± 0.43) and CAC (2.25 ± 0.47) when compared to normal tissue (0.07 ± 0.25, P < 0.05, P < 0.001, respectively). Cox-2 was significantly increased in sporadic colorectal cancer (2.20 ± 0.23 vs 0.80 ± 0.37 for normal tissues, P < 0.05). In comparison to normal (2.80 ± 0.13) and CAC (2.50 ± 0.33) tissues, VDR was significantly decreased in sporadic dysplasia (0.00 ± 0.00, P < 0.001 vs normal, P < 0.001 vs CAC) and sporadic CRC (0.47 ± 0.23, P < 0.001 vs normal, P < 0.001 vs CAC). VDR levels negatively correlated with NK-1R (r = -0.48) and pEGFR (r = -0.56) in normal, IBD, sporadic dysplasia and sporadic CRC tissue, but not in CAC. CONCLUSION: Immunohistochemical NK-1R and pEGFR positivity with VDR negativity can be used to identify areas of sporadic colorectal neoplasia. VDR immunoreactivity can distinguish CAC from sporadic cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/química , Receptores ErbB/análisis , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/metabolismo , Receptores de Calcitriol/análisis , Receptores de Neuroquinina-1/análisis , Adulto , Anciano , Biopsia , Estudios de Casos y Controles , Neoplasias Colorrectales/etnología , Neoplasias Colorrectales/patología , Ciclooxigenasa 2/análisis , Femenino , Hispánicos o Latinos , Humanos , Enfermedades Inflamatorias del Intestino/etnología , Enfermedades Inflamatorias del Intestino/patología , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Fosforilación , Puerto Rico/epidemiologíaRESUMEN
While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging.
Asunto(s)
Envejecimiento/fisiología , Daño del ADN/fisiología , ADN Mitocondrial , Mitocondrias Hepáticas/genética , Estrés Oxidativo/fisiología , Envejecimiento/metabolismo , Animales , Catalasa/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Glutatión Peroxidasa/metabolismo , Leucocitos Mononucleares/metabolismo , Peroxidación de Lípido/fisiología , Hígado/anatomía & histología , Hígado/metabolismo , Macaca mulatta , Masculino , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/fisiología , Carbonilación Proteica/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Inflammatory bowel disease (IBD) is a gastrointestinal disorder of unknown etiology or cure. One complication of IBD is an increased risk for development of colon cancer. The aims of this study were to use a previously established rat model of colitis to develop a new model of colitis-associated colon cancer and ascertain the involvement of three cancer-related genes: K-ras, adenomatous polyposis coli (APC), and p53. Four groups of rats were used: reactivated 1,2-dimethylhydrazine [DMH; trinitrobenzene sulfonic acid (TNBS) was used to induce colitis followed by a weekly s.c. dose of DMH], prolonged reactivation (inflammation was induced with TNBS, then maintained twice a week), saline-DMH (animals received saline instead of TNBS followed by a weekly dose of DMH), and normal (received no treatment). Animals were sacrificed at 5, 10, or 15 weeks, and colon samples were taken for pathologic analysis and gene mutation detection. No dysplasia was found in the normal group. The highest incidences of dysplasia were as follows: prolonged reactivation group at 5 weeks (60%), reactivated DMH group at 10 weeks (83%), and saline-DMH group at 15 weeks (67%). Carcinoma was found in both the prolonged reactivation and saline-DMH groups. No mutations were found in the K-ras oncogene; however 62% of the APC samples (exon 15 at nucleotide 2778) and 76% of p53 (exon 6 at nucleotide 1327) showed substitutions. The prolonged reactivation group may be considered a new model of colitis-associated colon cancer, offering the potential to study cancer prevention strategies for patients with IBD.