RESUMEN
Brother of Regulator of Imprinted Sites (BORIS) or CCCTC-binding factor like (CTCFL) is a nucleotide-binding protein, aberrantly expressed in various malignancies. Expression of BORIS has been found to be associated with the expression of oncogenes which regulate the reactive oxygen species (ROS) biogenesis, DNA double-strand break repair, regulation of stemness, and induction of cellular senescence. In the present study, we have analyzed the effects of knockdown of BORIS, a potential oncogene, on the induction of senescence and tumor suppression. Loss of BORIS downregulated the expression of critical oncogenes such as BMI1, Akt, MYCN, and STAT3, whereas overexpression increased their respective expression levels in MYCN-amplified neuroblastoma cells. BORIS knockdown exhibited high levels of ROS biogenesis, indicating an upregulated mitochondrial superoxide production and thereby induction of senescence. Our study also showed that the loss of BORIS facilitated cellular senescence through the disruption of telomere integrity via altering the expression of various proteins required for telomere capping (POT1, TRF2, and TIN1). In addition to affecting ROS production and DNA damage, BORIS knockdown sensitized the cells toward chemotherapeutic drugs and induced apoptosis. Tumor induction studies on in vivo xenograft mouse models showed that cells with loss of BORIS/CTCFL failed to induce tumors. From our study, we conclude that silencing BORIS/CTCFL influences tumor growth and proliferation by regulating key oncogenes. The results also indicated that the BORIS knockdown can cause cellular senescence and upon a combinatorial treatment with chemotherapeutic drugs can induce enhanced drug sensitivity in MYCN-amplified neuroblastoma cells.
Asunto(s)
Senescencia Celular , Proteínas de Unión al ADN , Neuroblastoma , Animales , Línea Celular Tumoral , Senescencia Celular/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/patología , Especies Reactivas de OxígenoRESUMEN
The cancer-testis antigen CTCFL/BORIS (Brother of Regulator of Imprinted Sites) also known, as a paralog of CTCF -the "master weaver of the genome" is a key transcriptional regulator. Both CTCF and BORIS can bind to the same promoter sequence and recruit diverse proteins. BORIS is also known to be associated with actively translating ribosomes suggesting new roles of BORIS in gene expression. Various studies have attempted to elucidate the role of BORIS in different cell types for the development of targeted therapy depending on molecular signatures and genetic aberrations associated with the disease type. The current study is focused on its role in neuroblastoma. Here, we have deciphered the role of BORIS on TGFß1 pathway which is highly affected by embryonic CTCFL expression. BORIS stabilized the SMAD3 and SMAD4 transcripts leading to prolonged TGFß activation. Further, loss of BORIS abrogated both the canonical and non-canonical TGFß signaling suggesting the dependency of TGFß on BORIS. The effect on the metabolic profile of the neuroblastoma cells were analyzed with change in BORIS expression levels. Also, ectopic expression of BORIS leads to Drp1 phosphorylation (Ser616) enhancing mitochondrial fission followed by a switch in cellular metabolism towards glycolysis. This cellular metabolism switch was in turn supported with a reduction in oxygen consumption rate upon BORIS expression. Interestingly methylome analysis revealed patterns of global histone methylation, a mechanism that regulate important signaling pathways in neuroblastoma. This study analyzes the consequence of BORIS expression in neuroblastoma cells and thereby elucidate its downstream targets, which could help in designing effective therapeutic for treating neuroblastoma. Similar results were obtained in both MYCN amplified and non-MYCN neuroblastoma cell lines, indicating a common mechanism of BORIS/CTCFL action in neuroblastoma.
Asunto(s)
Proteínas de Unión al ADN , Dinaminas , Neuroblastoma , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Dinámicas Mitocondriales , Neuroblastoma/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The global burden of cervical cancer from low and middle-income groups is increasing at alarming rates with more than half a million women being diagnosed every year. Although the disease is largely preventable when screened and diagnosed in earlier stages, the development of resistance and relapse had resulted in a poor prognosis. Therefore, a comprehensive approach needs to be put forward to understand and develop new preventive and therapeutic strategies to effectively combat cancer. Recently, much attention has been diverted to plant-derivatives for the treatment as they exhibit potent anti-cancer properties and side-effects caused by chemotherapeutic agents can also be prevented. Oleanolic acid and Esculetin are natural compounds known for their anti-cancer properties. Hence, the present study investigates the effect and mechanism of these compounds on cervical carcinoma, using HeLa cells. Posttreatment, it was observed that these compounds inhibited proliferation by both arresting the cells in the sub G1 phase and inducing senescence. Also, a marked reduction in the migration and cell survival was observed, as evidenced by results obtained from wound healing assay and Annexin V-FITC/PI staining. Furthermore, studies on the expression pattern of genes involved in major signaling pathways demonstrated a profound effect of these compounds. Taken together, the results of our study suggest that both Oleanolic acid and esculetin serve as a plausible therapeutic agent.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Redes Reguladoras de Genes/efectos de los fármacos , Ácido Oleanólico/farmacología , Umbeliferonas/farmacología , Neoplasias del Cuello Uterino/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Transducción de Señal/efectos de los fármacos , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
INTRODUCTION: Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder, characterized by the overproduction of myeloid cells in all stages of maturation. It is usually defined by three sequential stages (Chronic, Accelerated and Blast-crisis) where the transition from chronic to accelerated to blast phases is presumed to occur due to secondary genetic changes, viz. accumulation of mutations, activation of downstream pathways and failure of apoptosis. Caspase 9 is the initiator caspase involved in mitochondrial-mediated apoptotic pathway. Polymorphisms in the promoter (-1263 A>G, -712C>T, -293 del) and coding (Ex5 +32G>A) regions of CASP9 gene are found to influence the expression levels by either impairing the activation or loss of expression of CASP9 or insufficient formation of apoptosome. METHODS: The present case-control study was carried out on 999 individuals, comprised of 485CML cases reported at Nizams Institute of Medical Sciences (NIMS), Hyderabad and 514 age and gender-matched healthy individuals from local population. DNA was isolated by non-enzymatic/salting-out method and was genotyped using RFLP technique. RESULTS: It was observed that the presence of G allele of CASP9 -1263A>G polymorphism enhanced the risk for CML while CASP9 -712C>T and CASP9 -293del SNPs conferred protection against development of CML. Haplotype analysis of promoter and exonic polymorphisms had revealed increased risk associated with two haplotypes G_C_del (+)_G (OR-1.61, 95% CI-0.97-2.65, p-0.06#) and G_C_del (-)_G (OR-2.09, 95% CI-0.94-4.66, p-0.07#) suggesting the role of G allele of CASP9 -1263A>G in conferring risk independent of other SNPs. Pairwise LD analysis performed for all the four SNPs revealed the presence of LD among the SNPs. CONCLUSION: The results of the present study therefore concludes the role of CASP9 -1263A>G polymorphism in increasing the risk for the development and progression while CASP9 -712C>T and CASP9 -293del SNPs conferred protection and CASP9 Ex5 +32G>A was involved in conferring resistance which could be in combination with other SNPs or factors affecting them.
RESUMEN
Chronic myeloid leukemia (CML) is a monoclonal myeloproliferative disorder of hematopoietic stem cells (HSCs), characterized by reciprocal translocation, leading to the formation of BCR-ABL oncogene with constitutive tyrosine kinase (TK) activity. This oncogene is known to deregulate different downstream pathways which ultimately lead to cell proliferation, defective DNA repair, and inhibition of apoptosis. Fas (Fas cell surface death receptor) is a member of tumor necrosis factor (TNF) superfamily which interacts with its ligand, FasL, to initiate apoptosis. Promoter polymorphisms in Fas-FasL genes are known to influence the apoptotic signaling. Hence, the present study has been aimed to find out the association of the promoter polymorphisms in Fas and FasL genes with the development and progression of CML. Blood samples from 772 subjects (386 controls and 386 cases) were collected and genotyped for Fas-FasL gene polymorphisms through PCR-RFLP method. The association between SNPs and clinical outcome was analyzed using statistical softwares like SPSS version 20, SNPSTATs, and Haploview 2.1. The study revealed a significant association of Fas -670 G>A and FasL -844 T>C polymorphisms with the development of CML while Fas -670 AG was associated with accelerated phase. Combined risk analysis by taking the risk genotypes in cases and controls revealed a significant increase in CML risk with increase in number of risk genotypes (one risk genotype-OR 1.99 (1.44-2.76), p < 0.0001; two risk genotypes-OR 3.33 (1.91-5.81), p < 0.0001). Kaplan-Meier survival analysis of Fas -670 A>G and FasL -844 T>C showed reduced event-free survival in patients carrying the variant genotypes, Fas -670 GG, 32.363 ± 6.33, and FasL -844 CC, 33.489 ± 5.83, respectively. Our findings revealed a significant association of Fas -670 GG, FasL -844 TC, and CC genotypes with increased risk of CML.
Asunto(s)
Proteína Ligando Fas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Regiones Promotoras Genéticas , Receptor fas/genética , Adulto , Apoptosis , Supervivencia sin Enfermedad , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
LCN2 (Lipocalin 2) is a 25 KD secreted acute phase protein, reported to be a novel regulator of angiogenesis in breast cancer. Up regulation of LCN2 had been observed in multiple cancers including breast cancer, pancreatic cancer and ovarian cancer. However, the role of LCN2 promoter methylation in the formation of microvessels is poorly understood. The aim of this study was to analyze the association of LCN 2 promoter methylation with microvessel formation and tumor cell proliferation in breast cancer patients. The LCN2 promoter methylation status was studied in 64 breast cancer tumors by methylation specific PCR (MSP). Evaluation of microvessel density (MVD) and Ki67 cell proliferation index was achieved by immunohistochemical staining using CD34 and MIB-1 antibodies, respectively. LCN2 promoter unmethylation status was observed in 43 (67.2%) of breast cancer patients whereas LCN2 methylation status was seen in 21 (32.8%). Further, LCN2 promoter unmethylation status was associated with aggressive tumor phenotype and elevated mean MVD in breast cancer patients.
Asunto(s)
Proteínas de Fase Aguda/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Metilación de ADN/genética , Lipocalinas/genética , Microvasos/patología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Anticuerpos Antinucleares/genética , Anticuerpos Monoclonales/genética , Antígenos CD34/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Antígeno Ki-67/genética , Lipocalina 2 , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Fenotipo , Regulación hacia Arriba/genéticaRESUMEN
B-cell lymphoma 2 (BCL2) and BCL2-associated X protein (BAX) proteins are anti-apoptotic and pro-apoptotic determinants of mitochondrial-mediated apoptosis, and their relative expression determines the cell fate. The promoter polymorphisms in these genes were shown to alter the protein function or expression and exert an impact on apoptosis regulation. Deregulation in the expression of any of these genes leads to disruption of cellular homeostasis and malignant transformation. The present study was aimed to determine the association of BCL2-938C>A and BAX-248G>A promoter polymorphisms with origin and progression of acute myeloid leukemia (AML). We also have performed combined genotype analysis to evaluate the cumulative effect of risk genotypes in the AML development. These polymorphisms were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 221 AML patients and 305 age- and sex-matched healthy controls. Our study revealed that BCL2-938CA (p = 0.018) and BAX-248GG (0.043) genotypes were significantly associated with increased risk for AML occurrence. BAX-248A allele had shown decreased risk for AML. The combined analysis had shown that BCL2-938CA+AA-BAX-248GG group had a 1.63-fold (95 % CI: 1.08-2.45, p = 0.02) increased risk for AML. None of the clinical variables had shown any significant association with both polymorphisms. With respect to complete remission (CR) rate, BAX-248GG genotype (p = 0.002) and G allele (p = 0.009) had conferred significant risk for complete remission failure. Although the log rank test was not significant, survival analysis had shown a trend where BCL2-938CA genotype, and BAX-248GG had reduced median disease-free survival (DFS) of 9 and 10 months, respectively. In conclusion, BCL2-938C>A and BAX-248G>A gene polymorphisms might contribute to the origin of AML. Moreover, influence of BAX-248GG genotype on CR and DFS rate suggests that the BAX-248G>A polymorphism can serve as marker for poor prognosis in AML.
Asunto(s)
Biomarcadores de Tumor/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2/genética , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Niño , Preescolar , ADN/análisis , ADN/genética , Femenino , Estudios de Seguimiento , Humanos , India , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Pronóstico , Tasa de Supervivencia , Adulto JovenRESUMEN
Abnormal apoptosis is one of the hallmarks of cancers including acute myeloid leukemia (AML), as it plays a pivotal role in precisely maintaining self-renewal, proliferation, and differentiation properties of hematopoietic stem cells (HSCs). Caspase9 (CASP9), an initiator caspase activated by mitochondrial-mediated apoptotic pathway (intrinsic pathway), triggers cascade of effector caspases and executes apoptosis. Functional SNPs in CASP9 might influence the gene expression leading to altered apoptosis which confer the risk to AML. To test this hypothesis, we have analyzed four CASP9 gene polymorphisms [CASP9 - 1263A > G (rs4645978), CASP9 - 712C > T (rs4645981), CASP9 - 293_275del CGTGAGGTC AGTGCGGGGA (-293del) (rs4645982), and CASP9 Ex5 + 32G > A (rs1052576)] in 180 AML cases and 304 age- and sex-matched healthy controls. We performed various statistical analyses to determine the potential interactions between these SNPs and AML. The study revealed that presence of G allele at CASP9 - 1263 position elevates the risk of AML 1.53-fold and CT/TT genotype at CASP9 - 712 position by 2.60-fold under dominant model of inheritance. Two CASP9 haplotypes, G-del(+)-C-A and G-del(+)-T-A, were found to be significantly associated with increased AML risk by 2.19- (95 % confidence interval (CI), 1.09-4.39; p = 0.028) and 11.75-fold (95 % CI, 1.01-136.57; p = 0.05), respectively. Further, multidimensionality reduction (MDR) analysis had revealed single locus CASP9 - 712C > T SNP and four loci CASP9 - 1263A > G, CASP9 - 293del, CASP9 - 712C > T, and CASP9 Ex5 + 32G > A SNPs as highest predicting models for AML development. Our results revealed a significant association of two SNPs in CASP9 (-1263A > G and -712C > T) and two haplotypes of the four SNP combinations with AML susceptibility.