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Irwin H Gelman - One of the best experts on this subject based on the ideXlab platform.
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SSeCKS/AKAP12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.
Oncotarget, 2018Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Shin Akakura 2 , Lingqiu Gao 3 , Jennifer Peresie 3 , Benjamin Balderman 3 and Irwin H. Gelman 3 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA 3 Department of Cancer Genetics and Genomics, Roswell Park Comprehensive Cancer Center, Buffalo 14263, NY, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12; pre-metastatic niche; melanoma; lung endothelial cells; adhesion Received: July 25, 2018 Accepted: August 20, 2018 Published: September 11, 2018 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[ Braf V600E ] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.
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ssecks AKAP12 suppresses metastatic melanoma lung colonization by attenuating src mediated pre metastatic niche crosstalk
Oncotarget, 2018Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Shin Akakura 2 , Lingqiu Gao 3 , Jennifer Peresie 3 , Benjamin Balderman 3 and Irwin H. Gelman 3 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA 3 Department of Cancer Genetics and Genomics, Roswell Park Comprehensive Cancer Center, Buffalo 14263, NY, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12; pre-metastatic niche; melanoma; lung endothelial cells; adhesion Received: July 25, 2018 Accepted: August 20, 2018 Published: September 11, 2018 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[ Braf V600E ] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.
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SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts
Oncotarget, 2017Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Lingqiu Gao 2 , Jennifer Peresie 2 , Benjamin Balderman 2 , Shin Akakura 3 and Irwin H. Gelman 2 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo 14263, NY, USA 3 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12, metastasis, senescent secretome, CXCL9/10, CXCR3 Received: June 09, 2017 Accepted: July 26, 2017 Published: August 09, 2017 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [ 1 ]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[ Braf V600E ] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naive WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naive KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-β-galactosidase, p21 waf1 and p16 ink4a , and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.
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ssecks AKAP12 scaffolding functions suppress b16f10 induced peritoneal metastasis by attenuating cxcl9 10 secretion by resident fibroblasts
Oncotarget, 2017Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Lingqiu Gao 2 , Jennifer Peresie 2 , Benjamin Balderman 2 , Shin Akakura 3 and Irwin H. Gelman 2 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo 14263, NY, USA 3 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12, metastasis, senescent secretome, CXCL9/10, CXCR3 Received: June 09, 2017 Accepted: July 26, 2017 Published: August 09, 2017 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [ 1 ]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[ Braf V600E ] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naive WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naive KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-β-galactosidase, p21 waf1 and p16 ink4a , and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.
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structural environment built by AKAP12 colon mesenchymal cells drives m2 macrophages during inflammation recovery
Scientific Reports, 2017Co-Authors: Junmo Yang, Irwin H Gelman, Jihyeon Park, Eng H LoAbstract:Macrophages exhibit phenotypic plasticity, as they have the ability to switch their functional phenotypes during inflammation and recovery. Simultaneously, the mechanical environment actively changes. However, how these dynamic alterations affect the macrophage phenotype is unknown. Here, we observed that the extracellular matrix (ECM) constructed by AKAP12+ colon mesenchymal cells (CMCs) generated M2 macrophages by regulating their shape during recovery. Notably, rounded macrophages were present in the linear and loose ECM of inflamed colons and polarized to the M1 phenotype. In contrast, ramified macrophages emerged in the contracted ECM of recovering colons and mainly expressed M2 macrophage markers. These contracted structures were not observed in the inflamed colons of AKAP12 knockout (KO) mice. Consequently, the proportion of M2 macrophages in inflamed colons was lower in AKAP12 KO mice than in WT mice. In addition, clinical symptoms and histological damage were more severe in AKAP12 KO mice than in WT mice. In experimentally remodeled collagen gels, WT CMCs drove the formation of a more compacted structure than AKAP12 KO CMCs, which promoted the polarization of macrophages toward an M2 phenotype. These results demonstrated that tissue contraction during recovery provides macrophages with the physical cues that drive M2 polarization.
Bing Su - One of the best experts on this subject based on the ideXlab platform.
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ssecks AKAP12 induces repulsion between human prostate cancer and microvessel endothelial cells through the activation of semaphorin 3f
Biochemical and Biophysical Research Communications, 2017Co-Authors: Wei Su, Lijuan Zhang, Qingkun Shang, Bing SuAbstract:Abstract Metastasis remains the primary cause of prostate cancer related death. Cancer cells need to contact endothelial cells and disrupt endothelial junctions to cross the endothelium for invasion and metastasis. The suppression of heterotypic repulsion between cancer and endothelial cells allows cancer cells to invade into the surrounding tissue. Here, we demonstrate that SSeCKS/AKAP12 induced repulsion between human prostate cancer and microvessel endothelial cells, which was mediated by an angiogenesis inhibitor Semaphorin 3F. Moreover, we examined AKAP12 and Semaphorin 3F mRNA expression in 42 prostate cancer and 30 benign prostatic hyperplasia tissue samples, and found that the expression of AKAP12 and Semaphorin 3F mRNA was inversely associated with the degree of aggressiveness of prostate cancer cells and tissues. An ordinal logistic regression analysis indicates that there is a positive association between the expression of AKAP12 and Semaphorin 3F in prostate cancer, suggesting that the activation of Semaphorin 3F by SSeCKS/AKAP12 may be involved in prostate cancer progression and metastasis.
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SSeCKS/AKAP12 induces repulsion between human prostate cancer and microvessel endothelial cells through the activation of Semaphorin 3F
Biochemical and Biophysical Research Communications, 2017Co-Authors: Wei Su, Lijuan Zhang, Qingkun Shang, Bing SuAbstract:Abstract Metastasis remains the primary cause of prostate cancer related death. Cancer cells need to contact endothelial cells and disrupt endothelial junctions to cross the endothelium for invasion and metastasis. The suppression of heterotypic repulsion between cancer and endothelial cells allows cancer cells to invade into the surrounding tissue. Here, we demonstrate that SSeCKS/AKAP12 induced repulsion between human prostate cancer and microvessel endothelial cells, which was mediated by an angiogenesis inhibitor Semaphorin 3F. Moreover, we examined AKAP12 and Semaphorin 3F mRNA expression in 42 prostate cancer and 30 benign prostatic hyperplasia tissue samples, and found that the expression of AKAP12 and Semaphorin 3F mRNA was inversely associated with the degree of aggressiveness of prostate cancer cells and tissues. An ordinal logistic regression analysis indicates that there is a positive association between the expression of AKAP12 and Semaphorin 3F in prostate cancer, suggesting that the activation of Semaphorin 3F by SSeCKS/AKAP12 may be involved in prostate cancer progression and metastasis.
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rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation sensitive high resolution melting ms hrm analysis application in colorectal cancer
Clinica Chimica Acta, 2010Co-Authors: Ming Guan, Bing Su, Ji Li, Min Li, Yuan LuAbstract:Abstract Background Colorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers. Methods We applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines. Results In this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p = 0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation. Conclusions We demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer.
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quantitative assessment of AKAP12 promoter methylation in colorectal cancer using methylation sensitive high resolution melting correlation with duke s stage
Cancer Biology & Therapy, 2010Co-Authors: Ming Guan, Bing Su, Chuanzhong Ye, Ji Li, Xinju Zhang, Min Li, Yuan LuAbstract:BACKGROUND: The expression of AKAP12 (A Kinase anchoring protein 12) is markedly reduced in a variety of cancers. The purpose of this study was to establish a methylation-sensitive high resolution melting (MS-HRM) assay for the quantitative detection of AKAP12 promoter methylation and expression and the association with clinicopathological variables in human colorectal cancer. We also assessed the effect of AKAP12 re-expression on cell growth and colony formation. RESULTS: Downregulation or loss of AKAP12 mRNA expression was detected in 31 of 45 tissue samples (68.9%). No significant correlation was observed between the reduced expression levels and patient age, gender, Duke's stage or tumor differentiation. Methylation (>1%) of the AKAP12 promoter region was present in 35 of 45 (77.8%) carcinoma tissue samples and 6 of 45 (13.3%) adjacent tissue samples. AKAP12 methylation was significantly higher in the colorectal cancer tissues exhibiting advanced Duke's stages. Treatment of the three colorectal carcinoma cell lines (LoVo, COLO320 and SW480) with completely methylated AKAP12 with inhibitors of DNA methyltransferase (5-Aza-2'-deoxycytidine) markedly increased expression of AKAP12 and decreased methylation levels. Ectopic expression of AKAP12 in the LoVo cell line suppressed cell growth and inhibited colony formation. METHODS: The AKAP12 gene was examined by quantitative RT-PCR, MS-HRM analysis and bisulfite sequencing in 45 paired tissue samples obtained from primary colorectal carcinomas and the corresponding adjacent tissues. In addition, five colorectal carcinoma cell lines (LoVo, COLO205, SW480, LS174T and COLO320) were investigated and western blot analysis was used to investigate changes in protein expression. A proliferation assay and soft agar assay were performed after overexpression of AKAP12. CONCLUSION: Our study demonstrated that MS-HRM is a robust, fast and sensitive method for AKAP12 methylation analysis. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of this cancer.
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Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer.
Clinica chimica acta; international journal of clinical chemistry, 2010Co-Authors: Ming Guan, Bing Su, Ji Li, Min Li, Weizhe Ma, Yuan LuAbstract:Colorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers. We applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines. In this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p=0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation. We demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer. Copyright 2010 Elsevier B.V. All rights reserved.
Yuan Lu - One of the best experts on this subject based on the ideXlab platform.
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re expression of AKAP12 inhibits progression and metastasis potential of colorectal carcinoma in vivo and in vitro
PLOS ONE, 2011Co-Authors: Ming Guan, Tingting Hu, Xiaoye Gu, Yuan LuAbstract:Background: AKAP12/Gravin (A kinase anchor protein 12) is one of the A-kinase scaffold proteins and a potential tumor suppressor gene in human primary cancers. Our recent study demonstrated the highly recurrent loss of AKAP12 in colorectal cancer and AKAP12 reexpression inhibited proliferation and anchorage-independent growth in colorectal cancer cells, implicating AKAP12 in colorectal cancer pathogenesis. Methods: To evaluate the effect of this gene on the progression and metastasis of colorectal cancer, we examined the impact of overexpressing AKAP12 in the AKAP12-negative human colorectal cancer cell line LoVo, the single clone (LoVoAKAP12) compared to mock-transfected cells (LoVo-CON). Results: pCMV6-AKAP12-mediated AKAP12 re-expression induced apoptosis (3% to 12.7%, p,0.01), migration (89.667.5 cells to 31.064.1 cells, p,0.01) and invasion (82.765.2 cells to 24.763.3 cells, p,0.01) of LoVo cells in vitro compared to control cells. Nude mice injected with LoVo-AKAP12 cells had both significantly reduced tumor volume (p,0.01) and increased apoptosis compared to mice given AKAP12-CON. The quantitative human-specific Alu PCR analysis showed overexpression of AKAP12 suppressed the number of intravasated cells in vivo (p,0.01). Conclusion: These results demonstrate that AKAP12 may play an important role in tumor growth suppression and the survival of human colorectal cancer.
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quantitative assessment of AKAP12 promoter methylation in human prostate cancer using methylation sensitive high resolution melting correlation with gleason score
Urology, 2011Co-Authors: Jian Gong, Ming Guan, Tingting Hu, Jinghui Hu, Junhua Du, Haowen Jiang, Yuan LuAbstract:Objectives To quantitatively investigate the A kinase anchoring protein 12 ( AKAP12 ) gene promoter methylation and its association with clinicopathologic variables in human prostate cancer (PCa). The AKAP12 gene has shown reduced expression and marked hypermethylation in a variety of cancers. Methods The percentage levels of DNA methylation were measured in 78 PCa, 22 benign prostatic hyperplasia, and 22 normal adjacent tissue samples using an AKAP12 methylation-sensitive high-resolution melting assay. AKAP12 gene expression was also examined in 4 human prostate carcinoma cell lines, PC-3, DU145, LNCaP, and 22RV1, using quantitative reverse transcriptase-polymerase chain reaction and methylation-sensitive high-resolution melting analysis and after DNA methyltransferase inhibition with 5-aza-2′-deoxycytidine. Results Methylation (>1%) of the AKAP12 promoter region was present in 47 (60.2%) of the 78 PCa, 5 (22.7%) of the 22 benign prostatic hyperplasia, and 2 (9.1%) of the 22 adjacent normal tissue samples. AKAP12 methylation was significantly greater in the PCa than in the benign prostatic hyperplasia or adjacent tissue samples ( P AKAP12 methylation was significantly greater in the PCa samples with higher Gleason scores ( P = .03); however, no correlation was found with age, pT category, or serum prostate-specific antigen level. Reverse transcriptase-polymerase chain reaction demonstrated that PC-3 and DU-145 cells expressed AKAP12 RNA and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in the LNCaP and 22RV1 cells. Treatment of LNCaP cells with 5-aza-2′-deoxycytidine markedly decreased the methylation levels and increased the expression of AKAP12 . Conclusions The results of the present study have demonstrated that AKAP12 promoter methylation is a frequent event in human PCa. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of PCa.
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rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation sensitive high resolution melting ms hrm analysis application in colorectal cancer
Clinica Chimica Acta, 2010Co-Authors: Ming Guan, Bing Su, Ji Li, Min Li, Yuan LuAbstract:Abstract Background Colorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers. Methods We applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines. Results In this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p = 0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation. Conclusions We demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer.
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quantitative assessment of AKAP12 promoter methylation in colorectal cancer using methylation sensitive high resolution melting correlation with duke s stage
Cancer Biology & Therapy, 2010Co-Authors: Ming Guan, Bing Su, Chuanzhong Ye, Ji Li, Xinju Zhang, Min Li, Yuan LuAbstract:BACKGROUND: The expression of AKAP12 (A Kinase anchoring protein 12) is markedly reduced in a variety of cancers. The purpose of this study was to establish a methylation-sensitive high resolution melting (MS-HRM) assay for the quantitative detection of AKAP12 promoter methylation and expression and the association with clinicopathological variables in human colorectal cancer. We also assessed the effect of AKAP12 re-expression on cell growth and colony formation. RESULTS: Downregulation or loss of AKAP12 mRNA expression was detected in 31 of 45 tissue samples (68.9%). No significant correlation was observed between the reduced expression levels and patient age, gender, Duke's stage or tumor differentiation. Methylation (>1%) of the AKAP12 promoter region was present in 35 of 45 (77.8%) carcinoma tissue samples and 6 of 45 (13.3%) adjacent tissue samples. AKAP12 methylation was significantly higher in the colorectal cancer tissues exhibiting advanced Duke's stages. Treatment of the three colorectal carcinoma cell lines (LoVo, COLO320 and SW480) with completely methylated AKAP12 with inhibitors of DNA methyltransferase (5-Aza-2'-deoxycytidine) markedly increased expression of AKAP12 and decreased methylation levels. Ectopic expression of AKAP12 in the LoVo cell line suppressed cell growth and inhibited colony formation. METHODS: The AKAP12 gene was examined by quantitative RT-PCR, MS-HRM analysis and bisulfite sequencing in 45 paired tissue samples obtained from primary colorectal carcinomas and the corresponding adjacent tissues. In addition, five colorectal carcinoma cell lines (LoVo, COLO205, SW480, LS174T and COLO320) were investigated and western blot analysis was used to investigate changes in protein expression. A proliferation assay and soft agar assay were performed after overexpression of AKAP12. CONCLUSION: Our study demonstrated that MS-HRM is a robust, fast and sensitive method for AKAP12 methylation analysis. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of this cancer.
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Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer.
Clinica chimica acta; international journal of clinical chemistry, 2010Co-Authors: Ming Guan, Bing Su, Ji Li, Min Li, Weizhe Ma, Yuan LuAbstract:Colorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers. We applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines. In this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p=0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation. We demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer. Copyright 2010 Elsevier B.V. All rights reserved.
Shin Akakura - One of the best experts on this subject based on the ideXlab platform.
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SSeCKS/AKAP12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.
Oncotarget, 2018Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Shin Akakura 2 , Lingqiu Gao 3 , Jennifer Peresie 3 , Benjamin Balderman 3 and Irwin H. Gelman 3 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA 3 Department of Cancer Genetics and Genomics, Roswell Park Comprehensive Cancer Center, Buffalo 14263, NY, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12; pre-metastatic niche; melanoma; lung endothelial cells; adhesion Received: July 25, 2018 Accepted: August 20, 2018 Published: September 11, 2018 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[ Braf V600E ] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.
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ssecks AKAP12 suppresses metastatic melanoma lung colonization by attenuating src mediated pre metastatic niche crosstalk
Oncotarget, 2018Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Shin Akakura 2 , Lingqiu Gao 3 , Jennifer Peresie 3 , Benjamin Balderman 3 and Irwin H. Gelman 3 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA 3 Department of Cancer Genetics and Genomics, Roswell Park Comprehensive Cancer Center, Buffalo 14263, NY, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12; pre-metastatic niche; melanoma; lung endothelial cells; adhesion Received: July 25, 2018 Accepted: August 20, 2018 Published: September 11, 2018 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[ Braf V600E ] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.
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SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts
Oncotarget, 2017Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Lingqiu Gao 2 , Jennifer Peresie 2 , Benjamin Balderman 2 , Shin Akakura 3 and Irwin H. Gelman 2 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo 14263, NY, USA 3 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12, metastasis, senescent secretome, CXCL9/10, CXCR3 Received: June 09, 2017 Accepted: July 26, 2017 Published: August 09, 2017 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [ 1 ]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[ Braf V600E ] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naive WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naive KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-β-galactosidase, p21 waf1 and p16 ink4a , and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.
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ssecks AKAP12 scaffolding functions suppress b16f10 induced peritoneal metastasis by attenuating cxcl9 10 secretion by resident fibroblasts
Oncotarget, 2017Co-Authors: Masashi Muramatsu, Shin Akakura, Jennifer Peresie, Benjamin Balderman, Irwin H GelmanAbstract:// Masashi Muramatsu 1 , Lingqiu Gao 2 , Jennifer Peresie 2 , Benjamin Balderman 2 , Shin Akakura 3 and Irwin H. Gelman 2 1 Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan 2 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo 14263, NY, USA 3 Frontiers in Bioscience Research Institute in Aging and Cancer, Irvine 92618, CA, USA Correspondence to: Irwin H. Gelman, email: Irwin.gelman@roswellpark.org Keywords: SSeCKS/AKAP12, metastasis, senescent secretome, CXCL9/10, CXCR3 Received: June 09, 2017 Accepted: July 26, 2017 Published: August 09, 2017 ABSTRACT SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [ 1 ]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[ Braf V600E ] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naive WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naive KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-β-galactosidase, p21 waf1 and p16 ink4a , and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.
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A transgenic mouse model for early prostate metastasis to lymph nodes
Cancer Research, 2014Co-Authors: Hyun Kyung Ko, Shin Akakura, Jennifer Peresie, David W. Goodrich, Barbara A. Foster, Irwin H GelmanAbstract:The emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event. The individual genetic loss of the metastasis suppressor, SSeCKS/Gravin/AKAP12 or Rb , genes that are downregulated or deleted in human prostate cancer, results in prostatic hyperplasia. Here, we show that the combined loss of AKAP12 and Rb results in prostatic intraepithelial neoplasia (PIN) that fails to progress to malignancy after 18 months. Strikingly, 83% of mice with PIN lesions exhibited metastases to draining lymph nodes, marked by relatively differentiated tumor cells expressing markers of basal (p63, cytokeratin 14) and luminal (cytokeratin 8 and androgen receptor) epithelial cells, although none expressed the basal marker, cytokeratin 5. The finding that PIN lesions contain increased numbers of p63/AR-positive, cytokeratin 5-negative basal cells compared with WT or AKAP12 −/− prostate lobes suggests that these transitional cells may be the source of the lymph node metastases. Taken together, these data suggest that in the context of Rb loss, AKAP12 suppresses the oncogenic proliferation and early metastatic spread of basal-luminal prostate tumor cells. Cancer Res; 74(3); 945–53. ©2014 AACR .
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AKAP12 Endogenous Transcripts Suppress The Proliferation, Migration And Invasion Of Colorectal Cancer Cells By Directly Targeting oncomiR-183-5p.
OncoTargets and Therapy, 2019Co-Authors: Tingting Hu, Ping He, Ke Li, Xuan Wu, Yuan Li, Zhiyuan Wu, Ming GuanAbstract:Purpose: Restoring lost function to suppressor gene products has captured the interest of the research community in the field of gene therapy. AKAP12, also known as Gravin/AKAP250, is a tumor suppressor gene, and its deregulation may be responsible for cancer progression. The aim of this study was to investigate whether AKAP12 mRNA has an anti-cancer function by regulating onco-miRNA expression in colorectal cancer (CRC) cells. Methods: miRNAs targeting AKAP12 were predicted by bioinformatics analysis and further confirmed by dual-luciferase reporter assays and RT-qPCR. The altered expression of microRNA was validated in early-stage CRC tumor tissues by miRseq. Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. Cell invasion and migration were detected by transwell and wound healing assays, respectively. In vivo experiments were conducted to confirm the in vitro findings. Results: Among all miRNAs, reversed correlation between AKAP12 expression and miRNA-183-5p expression was most significant. Luciferase assays revealed that AKAP12 directly targeted miR-183-5p. The miRseq data showed that miR-183 was also dysregulated at the early stage of tumor development and upregulated in late sub-stage II CRC patients (P
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AKAP12 endogenous transcripts suppress the proliferation migration and invasion of colorectal cancer cells by directly targeting oncomir 183 5p
OncoTargets and Therapy, 2019Co-Authors: Tingting Hu, Ping He, Ke Li, Xuan Wu, Yuan Li, Zhiyuan Wu, Ming GuanAbstract:Purpose: Restoring lost function to suppressor gene products has captured the interest of the research community in the field of gene therapy. AKAP12, also known as Gravin/AKAP250, is a tumor suppressor gene, and its deregulation may be responsible for cancer progression. The aim of this study was to investigate whether AKAP12 mRNA has an anti-cancer function by regulating onco-miRNA expression in colorectal cancer (CRC) cells. Methods: miRNAs targeting AKAP12 were predicted by bioinformatics analysis and further confirmed by dual-luciferase reporter assays and RT-qPCR. The altered expression of microRNA was validated in early-stage CRC tumor tissues by miRseq. Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. Cell invasion and migration were detected by transwell and wound healing assays, respectively. In vivo experiments were conducted to confirm the in vitro findings. Results: Among all miRNAs, reversed correlation between AKAP12 expression and miRNA-183-5p expression was most significant. Luciferase assays revealed that AKAP12 directly targeted miR-183-5p. The miRseq data showed that miR-183 was also dysregulated at the early stage of tumor development and upregulated in late sub-stage II CRC patients (P<0.01). Mechanistic analysis both in vitro and in vivo demonstrated that anti-miR-183-5p depressed cell proliferation, migration, and invasion in CRC cells while miR-183-5p overexpression resulted in opposite effects. Conclusion: Our findings suggested that oncomiR-183-5p promoted the proliferation, migration, and invasion of CRC cells. AKAP12 miRNA-binding elements (MREs) suppressed miRNA-183-5p activities. Any change in expression of AKAP12 thus affected miRNA-183-5p. This may be another anti-tumor mechanism in addition to protein-mediation that regulates tumor suppressor genes.
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upregulation of AKAP12 with hdac3 depletion suppresses the progression and migration of colorectal cancer
International Journal of Oncology, 2018Co-Authors: Ping He, Tingting Hu, Ke Li, Shibao Li, Ming GuanAbstract:: A-kinase anchor protein 12 (AKAP12; also known as Gravin) functions as a tumor suppressor in several human primary cancers. However, the potential correlation between histone deacetylase 3 (HDAC3) and AKAP12 and the underlying mechanisms remain unclear. Thus, in this study, in an aim to shed light into this matter, the expression levels of HDAC3 and AKAP12 in 96 colorectal cancer (CRC) and adjacent non-cancerous tissues, as well as in SW480 cells were examined by immunohistochemical, RT-qPCR and western blot analyses. The effects of HDAC3 and AKAP12 on the proliferation, apoptosis and metastasis of CRC cells were examined by cell counting kit-8 (CCK-8) assay, colony formation assays, flow cytometry, cell cycle analysis and Transwell assays. The results revealed that the reduction or loss of AKAP12 expression was detected in 69 (71.8%) of the 96 tissue specimens, whereas HDAC3 was upregulated in 50 (52.1%) of the 96 tumor tissue specimens. AKAP12 expression was markedly increased upon treatment with the HDAC3 inhibitors, trichostatin A (TSA) and RGFP966, at both the mRNA and protein level. Mechanistically, the direct binding of HDAC3 within the intron-1 region of AKAP12 was identified to be indispensable for the inhibition of AKAP12 expression. Moreover, the proliferation, colony-forming ability, cell cycle progression and the migration of the CRC cells were found to be promoted in response to AKAP12 silencing or AKAP12/HDAC3 co-silencing, whereas transfection with si-HDAC3 yielded opposite effects. Apart from the elevated expression of the anti-apoptotic protein, Bcl-2, after AKAP12 knockdown, the increased activity of PI3K/AKT signaling was found to be indispensable for AKAP12-mediated colony formation and migration. On the whole, these findings indicate that AKAP12 may be a potential prognostic predictor and therapeutic target for the treatment of CRC in combination with HDAC3.
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re expression of AKAP12 inhibits progression and metastasis potential of colorectal carcinoma in vivo and in vitro
PLOS ONE, 2011Co-Authors: Ming Guan, Tingting Hu, Xiaoye Gu, Yuan LuAbstract:Background: AKAP12/Gravin (A kinase anchor protein 12) is one of the A-kinase scaffold proteins and a potential tumor suppressor gene in human primary cancers. Our recent study demonstrated the highly recurrent loss of AKAP12 in colorectal cancer and AKAP12 reexpression inhibited proliferation and anchorage-independent growth in colorectal cancer cells, implicating AKAP12 in colorectal cancer pathogenesis. Methods: To evaluate the effect of this gene on the progression and metastasis of colorectal cancer, we examined the impact of overexpressing AKAP12 in the AKAP12-negative human colorectal cancer cell line LoVo, the single clone (LoVoAKAP12) compared to mock-transfected cells (LoVo-CON). Results: pCMV6-AKAP12-mediated AKAP12 re-expression induced apoptosis (3% to 12.7%, p,0.01), migration (89.667.5 cells to 31.064.1 cells, p,0.01) and invasion (82.765.2 cells to 24.763.3 cells, p,0.01) of LoVo cells in vitro compared to control cells. Nude mice injected with LoVo-AKAP12 cells had both significantly reduced tumor volume (p,0.01) and increased apoptosis compared to mice given AKAP12-CON. The quantitative human-specific Alu PCR analysis showed overexpression of AKAP12 suppressed the number of intravasated cells in vivo (p,0.01). Conclusion: These results demonstrate that AKAP12 may play an important role in tumor growth suppression and the survival of human colorectal cancer.
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quantitative assessment of AKAP12 promoter methylation in human prostate cancer using methylation sensitive high resolution melting correlation with gleason score
Urology, 2011Co-Authors: Jian Gong, Ming Guan, Tingting Hu, Jinghui Hu, Junhua Du, Haowen Jiang, Yuan LuAbstract:Objectives To quantitatively investigate the A kinase anchoring protein 12 ( AKAP12 ) gene promoter methylation and its association with clinicopathologic variables in human prostate cancer (PCa). The AKAP12 gene has shown reduced expression and marked hypermethylation in a variety of cancers. Methods The percentage levels of DNA methylation were measured in 78 PCa, 22 benign prostatic hyperplasia, and 22 normal adjacent tissue samples using an AKAP12 methylation-sensitive high-resolution melting assay. AKAP12 gene expression was also examined in 4 human prostate carcinoma cell lines, PC-3, DU145, LNCaP, and 22RV1, using quantitative reverse transcriptase-polymerase chain reaction and methylation-sensitive high-resolution melting analysis and after DNA methyltransferase inhibition with 5-aza-2′-deoxycytidine. Results Methylation (>1%) of the AKAP12 promoter region was present in 47 (60.2%) of the 78 PCa, 5 (22.7%) of the 22 benign prostatic hyperplasia, and 2 (9.1%) of the 22 adjacent normal tissue samples. AKAP12 methylation was significantly greater in the PCa than in the benign prostatic hyperplasia or adjacent tissue samples ( P AKAP12 methylation was significantly greater in the PCa samples with higher Gleason scores ( P = .03); however, no correlation was found with age, pT category, or serum prostate-specific antigen level. Reverse transcriptase-polymerase chain reaction demonstrated that PC-3 and DU-145 cells expressed AKAP12 RNA and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in the LNCaP and 22RV1 cells. Treatment of LNCaP cells with 5-aza-2′-deoxycytidine markedly decreased the methylation levels and increased the expression of AKAP12 . Conclusions The results of the present study have demonstrated that AKAP12 promoter methylation is a frequent event in human PCa. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of PCa.