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Methylation-mediated downregulation of the B-cell translocation gene 3 (BTG3) in breast cancer cells.

ABSTRACT: The incidence of solid tumors is low in individuals with Down syndrome (trisomy 21), suggesting the presence of one or more tumor suppressor genes on chromosome 21. Consistent with this finding, previous work has demonstrated frequent loss of heterozygosity (LOH) of a small (< 5 Mb) region of chromosome 21, particularly in breast cancer, indicating that a tumor suppressor gene(s) may be located in this region. We investigated the expression of BTG3, a gene in the LOH region on chromosome 21, in breast cancer cell lines. BTG3 has been shown to be a negative regulator of SRC tyrosine kinase, and BTG3 is a target of p53 and inhibits the activity of the E2F1 transcription factor. Here we demonstrate that in a wide variety of human breast cancer cell lines, BTG3 expression is markedly reduced in the absence of detectable mutations in the BTG3 promoter and coding region. In these cell lines, the promoter region of the BTG3 gene is hypermethylated when compared to normal breast cell lines. BTG3 gene expression can be restored by treatment with 5'-aza-deoxycytidine, an inhibitor of DNA methylation. These data support the hypothesis that BTG3 may act to suppress tumorigenesis and that hypermethylation is an important mechanism for inactivation of BTG3 and perhaps other tumor suppressor genes. The findings are consistent with a role for an additional copy of BTG3 in the reduced incidence of breast cancer in individuals with Down syndrome.

SUBMITTER: Yu J

PROVIDER: S-EPMC6042006 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Methylation-mediated downregulation of the B-cell translocation gene 3 (BTG3) in breast cancer cells.

Yu Jingwei J Zhang Yingsha Y Qi Zhongxia Z Kurtycz Daniel D Vacano Guido G Patterson David D

Gene expression 20080101 3

The incidence of solid tumors is low in individuals with Down syndrome (trisomy 21), suggesting the presence of one or more tumor suppressor genes on chromosome 21. Consistent with this finding, previous work has demonstrated frequent loss of heterozygosity (LOH) of a small (< 5 Mb) region of chromosome 21, particularly in breast cancer, indicating that a tumor suppressor gene(s) may be located in this region. We investigated the expression of BTG3, a gene in the LOH region on chromosome 21, in ...[more]

PMID: 18590053

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Project description:Twist1, a key transcription factor regulating epithelial-mesenchymal transition and cancer metastasis, is highly expressed in invasive cancers in contrast to the loss of BTG2/TIS21 expression. Based on our observation that forced expression of BTG2/TIS21 downregulated Twist1 protein expression without altering mRNA level, we investigated molecular mechanisms of the BTG2/TIS21-inhibited Twist1 translation in the triple negative breast cancer (TNBC) cells and in vivo BTG2/TIS21-knockout (KO) mice and human breast cancer tissues. (1) C-terminal domain of Twist1 and Box B of BTG2/TIS21 interacted with each other, which abrogated Twist1 activity. (2) BTG2/TIS21 inhibited translational initiation by depleting eIF4E availability via inhibiting 4EBP1 phosphorylation. (3) Expression of BTG2/TIS21 maintained p-eIF2? that downregulates initiation of protein translation, confirmed by eIF2?-AA mutant expression and BTG2/TIS21 knockdown in MEF cells. (4) cDNA microarray analysis revealed significantly higher expression of initiation factors-eIF2A, eIF3A, and eIF4G2-in the BTG2/TIS21-KO mouse than that in the wild type. (5) BTG2/TIS21-inhibited translation initiation lead to the collapse of polysome formation and the huge peak of 80s monomer in the BTG2/TIS21 expresser, but not in the control. (6) mRNAs and protein expressions of elongation factors were also downregulated by BTG2/TIS21 expression in TNBC cells, but much higher in both TIS21-KO mice and lymph node-positive human breast cancers. (7) BTG2/TIS21-mediated Twist1 loss was not due to the protein degradation by ubiquitination and autophagy activation. (8) Twist1 protein level was significantly higher in various organs of TIS21-KO mice compared with that in the control, indicating the in vivo role of BTG2/TIS21 gene in the regulation of Twist1 protein level. Altogether, the present study support our hypothesis that BTG2/TIS21 is a promising target to combat with metastatic cancers with high level of Twist1 without BTG2/TIS21 expression.

Project description:BackgroundYAP, a potent oncogene and major downstream effector of the mammalian Hippo tumor suppressor pathway can act as either oncogene or tumor suppressor gene based on the type of tissue involved. Despite various studies, the role and mechanism through which YAP mediates its tumor suppressor or oncogenic effects are not yet fully understood. Therefore in the present study we aimed to investigate YAP at DNA, mRNA and protein level and also attempted to correlate our molecular findings with various clinicopathological variables of the patients.MethodsThe study comprised of a total 137 genetically unrelated women with sporadic breast cancer cases and normal adjacent tissues not infiltrated with tumor. Mutation of YAP gene was analyzed by automated DNA sequencing. YAP promoter methylation was studied using MS-PCR. Expression at mRNA and protein level was studied using qPCR and IHC respectively.ResultsIn our study YAP mRNA expression was found to be 8.65 ± 6.17 fold downregulated in 67.15% cases. The expression of YAP when analyzed at the protein level by IHC was found to be absent in 78.83% cases. Results from MS-PCR analysis showed that YAP promoter methylation plays an important role in declining the expression of YAP protein. The absence of YAP protein coincided with 86.60% methylated cases thereby showing a very strong correlation (p = 0.001). We also investigated YAP mutation at the major check point sites in the Hippo pathway and observed no mutation. A significant association was observed on correlating mRNA expression with clinical stages (p = 0.038) and protein expression with ER status (p = 0.018) among Indian breast cancer patients.ConclusionThe expression of YAP was found to be downregulated in response to aberrant promoter methylation. The downregulation of YAP are consistent with previous studies suggesting it to have a tumor suppressive role in breast cancer. We did not observe any mutation at the major check point sites in the Hippo pathway.

Project description:E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion, and its loss has been associated with metastasis development. Although E-cadherin downregulation was previously reported in canine prostate cancer (PC), the mechanism involved in this process is unclear. It is well established that dogs, besides humans, spontaneously develop PC with high frequency; therefore, canine PC is an interesting model to study human PC. In human PC, CDH1 methylation has been associated with E-cadherin downregulation. However, no previous studies have described the methylation pattern of CDH1 promoter in canine PC. Herein, we evaluated the E-cadherin protein and gene expression in canine PC compared to normal tissues. DNA methylation pattern was investigated as a regulatory mechanism of CDH1 silencing. Our cohort is composed of 20 normal prostates, 20 proliferative inflammatory atrophy (PIA) lesions, 20 PC, and 11 metastases from 60 dogs. The E-cadherin protein expression was assessed by immunohistochemistry and western blotting and gene expression by qPCR. Bisulfite- pyrosequencing assay was performed to investigate the CDH1 promoter methylation pattern. Membranous E-cadherin expression was observed in all prostatic tissues. A higher number of E-cadherin negative cells was detected more frequently in PC compared to normal and PIA samples. High-grade PC showed a diffuse membranous positive immunostaining. Furthermore, PC patients with a higher number of E-cadherin negative cells presented shorter survival time and higher Gleason scores. Western blotting and qPCR assays confirmed the immunohistochemical results, showing lower E-cadherin protein and gene expression levels in PC compared to normal samples. We identified CDH1 promoter hypermethylation in PIA and PC samples. An in vitro assay with two canine prostate cancer cells (PC1 and PC2 cell lines) was performed to confirm the methylation as a regulatory mechanism of E-cadherin expression. PC1 cell line presented CDH1 hypermethylation and after 5-Aza-dC treatment, a decreased CDH1 methylation and increased gene expression levels were observed. Positive E-cadherin cells were massively found in metastases (mean of 90.6%). In conclusion, low levels of E-cadherin protein, gene downregulation and CDH1 hypermethylation was detected in canine PC. However, in metastatic foci occur E-cadherin re-expression confirming its relevance in these processes.

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Methylation-mediated downregulation of the B-cell translocation gene 3 (BTG3) in breast cancer cells.

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Methylation-mediated downregulation of the B-cell translocation gene 3 (BTG3) in breast cancer cells.

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