Moon-Chang Choi

AKAP12/Gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity.

AKAP12/Gravin, one of the A-kinase anchoring proteins (AKAPs), functions as a kinase scaffold protein and as a dynamic regulator of the β2-adrenergic receptor complex. However, the biological role of AKAP12 in cancer development is not well understood. The AKAP12 gene encodes two major isoforms of 305 and 287 kDa (designated AKAP12A and AKAP12B, respectively, in this report). We found that these two isoforms are independently expressed and that they are probably under the control of two different promoters. Moreover, both isoforms were absent from the majority of human gastric cancer cells. The results from methylation-specific PCR (MSP) and bisulfite sequencing revealed that the 5′ CpG islands of both AKAP12A and AKAP12B are frequently hypermethylated in gastric cancer cells. Treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor efficiently restored the expression of AKAP12 isoforms, confirming that DNA methylation is directly involved in the transcriptional silencing of AKAP12 in gastric cancer cells. Hypermethylation of AKAP12A CpG island was also detected in 56% (10 of 18) of primary gastric tumors. The restoration of AKAP12A in AKAP12-nonexpressing cells reduced colony formation and induced apoptotic cell death. In conclusion, our results suggest that AKAP12A may function as an important negative regulator of the survival pathway in human gastric cancer.

Class II histone deacetylases play pivotal roles in heat shock protein 90-mediated proteasomal degradation of vascular endothelial growth factor receptors

Vascular endothelial growth factor receptors (VEGFRs) perform pivotal roles in both tumor growth and angiogenesis. In this study, we report that histone deacetylase inhibitors (HDIs) induce a reduction in VEGFR1 and VEGFR2 protein expression via the inhibition of class II histone deacetylases (HDACs) in human cancer cell lines. After HDI treatment, VEGFR1 and VEGFR2 were shown to be downregulated in a proteasome-dependent manner. HDI treatment induced a reduction in the binding of heat shock protein (Hsp) 90 to VEGFR1 or VEGFR2, followed by an increase of the binding of Hsp70 to VEGFR1 or VEGFR2. However, we noted no remarkable changes in the binding of Hsp90/Hsp70 to VEGFR3. HDI treatment effectively inhibited the activities of HDAC6 and HDAC10. Furthermore, the knock-down of HDAC6 or HDAC10, which was accomplished via the siRNA transfection, induced depletion of VEGFR1 or VEGFR2 proteins. Overall, these results indicate that HDAC6 and HDAC10 play important roles in Hsp-mediated VEGFR regulation.

Inhibition of histone deacetylase 10 induces thioredoxin-interacting protein and causes accumulation of reactive oxygen species in SNU-620 human gastric cancer cells

Histone deacetylase (HDAC)10, a novel class IIb histone deacetylase, is the most similar to HDAC6, since both contain a unique second catalytic domain. Unlike HDAC6, which is located in the cytoplasm, HDAC10 resides in both the nucleus and cytoplasm. The transcriptional targets of HDAC10 that are associated with HDAC10 gene regulation have not been identified. In the present study, we found that knockdown of HDAC10 significantly increased the mRNA expression levels of thioredoxin-interacting protein (TXNIP) in SNU-620 human gastric cancer cells; whereas inhibition of HDAC1, HDAC2, and HDAC6 did not affect TXNIP expression. TXNIP is the endogenous inhibitor of thioredoxin (TRX), which acts as a cellular antioxidant. Real-time PCR and immunoblot analysis confirmed that inhibition of HDAC10 induced TXNIP expression. Compared to class I only HDAC inhibitors, inhibitors targeting both class I and II upregulated TXNIP, indicating that TXNIP is regulated by class II HDACs such as HDAC10. We further verified that inhibition of HDAC10 induced release of cytochrome c and activated apoptotic signaling molecules through accumulation of reactive oxygen species (ROS). Taken together, our results demonstrate that HDAC10 is involved in transcriptional downregulation of TXNIP, leading to altered ROS signaling in human gastric cancer cells. How TXNIP is preferentially regulated by HDAC10 needs further investigation.

Aberrant methylation of integrin alpha4 gene in human gastric cancer cells.

Integrins are adhesion receptors that mediate both cell–extracellular matrix and cell–cell interactions. It has also been reported that the loss of integrin α4 expression might be associated with metastasis in several cancers. However, the molecular mechanism for loss of their expression in cancers has not been explored. In the present study, we found that the integrin α4 expression is lost in human gastric cancer cell lines and that this is recovered by treatment with DNA methyltransferase inhibitor, implying transcriptional silencing by DNA methylation. Methylation-specific PCR (MSP) and bisulfite genomic DNA sequencing demonstrated the CpG methylation-dependent silencing of integrin α4 expression in eight of nine (88.8%) gastric cancer cell lines and in 84.7% of 46 primary tumors. We also investigated whether the restoration of integrin α4 in integrin α4-inactivated cells affects their ability to invade extracellular matrix, using matrigel assays. Interestingly, integrin α4-stable transfectants had markedly less invasive ability than the parental cells. Taken together, these results suggest that the transcriptional repression of the integrin α4 gene is caused by aberrant DNA methylation, and that this may play an important role in human gastric carcinogenesis.

Aberrant methylation of integrin α 4 gene in human gastric cancer cells

Integrins are adhesion receptors that mediate both cell–extracellular matrix and cell–cell interactions. It has also been reported that the loss of integrin α4 expression might be associated with metastasis in several cancers. However, the molecular mechanism for loss of their expression in cancers has not been explored. In the present study, we found that the integrin α4 expression is lost in human gastric cancer cell lines and that this is recovered by treatment with DNA methyltransferase inhibitor, implying transcriptional silencing by DNA methylation. Methylation-specific PCR (MSP) and bisulfite genomic DNA sequencing demonstrated the CpG methylation-dependent silencing of integrin α4 expression in eight of nine (88.8%) gastric cancer cell lines and in 84.7% of 46 primary tumors. We also investigated whether the restoration of integrin α4 in integrin α4-inactivated cells affects their ability to invade extracellular matrix, using matrigel assays. Interestingly, integrin α4-stable transfectants had markedly less invasive ability than the parental cells. Taken together, these results suggest that the transcriptional repression of the integrin α4 gene is caused by aberrant DNA methylation, and that this may play an important role in human gastric carcinogenesis.

DNA methyltransferase 3B acts as a co-repressor of the human polycomb protein hPc2 to repress fibroblast growth factor receptor 3 transcription.

DNA methyltransferase 3B has been demonstrated to mediate gene silencing. The mechanisms how DNA methyltransferase 3B is targeted to specific regions and represses gene transcription, however, are not well understood. Here we show that by using yeast two-hybrid screening, DNA methyltransferase 3B interacts with the human polycomb protein, hPc2. This interaction was verified via co-immunoprecipitation and GST pull-down assay. Sequential deletion analysis showed that the region of DNA methyltransferase 3B responsible for interaction is mapped to the N-terminal regulatory domain. By performing a cDNA microarray analysis in HCT 116 cells, we identified that the expression of fibroblast growth factor receptor 3 is significantly increased upon the small interference RNA-mediated knockdown of hPc2, suggesting fibroblast growth factor receptor 3 as a potential target of hPc2. We further found that DNA methyltransferase 3B enhances hPc2-mediated transcriptional repression of fibroblast growth factor receptor 3, which does not require its de novo methyltransferase activity. Taken together, these results suggest that DNA methyltransferase 3B functions as a co-repressor of polycomb protein in inducing transcriptional repression independent of DNA methylation.

Aberrant methylation of integrin α4 gene in human gastric cancer cells

Integrins are adhesion receptors that mediate both cell–extracellular matrix and cell–cell interactions. It has also been reported that the loss of integrin α4 expression might be associated with metastasis in several cancers. However, the molecular mechanism for loss of their expression in cancers has not been explored. In the present study, we found that the integrin α4 expression is lost in human gastric cancer cell lines and that this is recovered by treatment with DNA methyltransferase inhibitor, implying transcriptional silencing by DNA methylation. Methylation-specific PCR (MSP) and bisulfite genomic DNA sequencing demonstrated the CpG methylation-dependent silencing of integrin α4 expression in eight of nine (88.8%) gastric cancer cell lines and in 84.7% of 46 primary tumors. We also investigated whether the restoration of integrin α4 in integrin α4-inactivated cells affects their ability to invade extracellular matrix, using matrigel assays. Interestingly, integrin α4-stable transfectants had markedly less invasive ability than the parental cells. Taken together, these results suggest that the transcriptional repression of the integrin α4 gene is caused by aberrant DNA methylation, and that this may play an important role in human gastric carcinogenesis.

A-kinase anchoring protein 12 regulates the completion of cytokinesis

A-kinase anchoring protein 12 (AKAP12) gene is frequently inactivated in human gastric cancer and in several other cancers due to promoter hypermethylation. However, the biological function of AKAP12 in tumorigenesis remains to be identified. Aneuploidy, a hallmark of cancer cells, is often caused by abnormal cell division. In the present study, AKAP12 was found to localize to the cell periphery during interphase and to the actomyosin contractile ring during cytokinesis. Furthermore, AKAP12 depletion using small interfering RNA increased the number of multinucleated cells, and disrupted the completion of cytokinesis. Interestingly, the inhibition of myosin light chain kinase (MLCK), a key regulator of actomyosin contractility, removed AKAP12 from the cell periphery during interphase and from the contractile ring during cytokinesis, suggesting that AKAP12 might be a downstream effector of MLCK. Our findings implicate AKAP12 in the regulation of cytokinesis progression, and suggest a novel role for AKAP12 tumor suppressor.

Overexpression of A-kinase anchoring protein 12A activates sterol regulatory element binding protein-2 and enhances cholesterol efflux in hepatic cells

A-kinase anchoring protein 12 (AKAP12) is known to function as a scaffold protein and as a putative tumor suppressor. However, little is known about the biological role of AKAP12 in hepatic cells. In this study, we performed micro-array analysis to identify the downstream pathway of AKAP12A, and found that AKAP12A overexpression up-regulates the expressions of several cholesterol-associated genes including HMG-CoA reductase and LDL receptor, which have been reported to be controlled by sterol regulatory element binding protein-2 (SREBP-2). It was found that AKAP12A activates SREBP-2 in hepatic cells, as demonstrated by the presence of its cleavage product, whereas the activation of sterol regulatory element binding protein-1 was not remarkably changed. Moreover, AKAP12A-induced SREBP-2 activation was found to depend on SREBP cleavage-activating protein (SCAP), as inhibition of SCAP by RNAi or sterols blocked SREBP-2 activation in response to AKAP12A overexpression. Interestingly, the hydrophobic amine U18666A caused dramatic movement of AKAP12A from the plasma membrane to cytosol and lysosomal membranes. Moreover, cholesterol depletion from the plasma membrane (using methyl-beta-cyclodextrin) caused a shift of AKAP12A from the plasma membrane to the cytoplasm. Cholesterol binding assay revealed that the N-terminal region of AKAP12A binds directly to cholesterol in vitro. Furthermore, AKAP12A overexpression enhanced [3H]-cholesterol efflux to extracellular acceptors, suggesting that AKAP12A may activate SREBP-2 by increasing cholesterol efflux. In conclusion, the present study suggests that AKAP12A is a novel regulator of cellular cholesterol metabolism.