9590

AKAP12

A kinase (PRKA) anchor protein 12;AKAP12;A kinase (PRKA) anchor protein 12

A-kinase anchor protein 12|A-kinase anchor protein, 250kDa|AKAP 250|Src-Suppressed C Kinase Substrate|kinase scaffold protein gravin|myasthenia gravis autoantigen gravin

6q24-q25

protein-coding

BACKGROUND: SSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity. METHODS: We use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src. RESULTS: SSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1alpha, Cdc20a and Pdgfr-beta, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7. CONCLUSION: Our data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity.

BACKGROUND: SSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity. METHODS: We use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src. RESULTS: SSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1alpha, Cdc20a and Pdgfr-beta, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7. CONCLUSION: Our data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity.

BACKGROUND: SSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity. METHODS: We use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src. RESULTS: SSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1alpha, Cdc20a and Pdgfr-beta, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7. CONCLUSION: Our data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity.

The ability of SSeCKS/Gravin/AKAP12 (SSeCKS) to negatively regulate cell cycle progression is thought to relate to its spatiotemporal scaffolding activity for key signaling molecules such as protein kinase A and C, calmodulin and cyclins. SSeCKS is downregulated upon progression to malignancy in many cancer types, including melanoma and nonmelanoma skin cancer. The forced re-expression of SSeCKS is especially potent in suppressing metastasis through the inhibition of VEGF-mediated neovascularization. We have previously shown that SSeCKS-null (KO) mice exhibit hyperplasia and focal dysplasia in the prostate marked by activated Akt. To address whether KO mice exhibit increased skin carcinogenesis, WT and KO C57BL/6 mice were treated topically with 12-O-tetradecanoylphorbol-13-acetate and 7,12-dimethylbenzanthracene. Compared to WT mice, KO mice developed squamous papillomas more rapidly and in greater numbers and also exhibited significantly increased progression to squamous cell carcinoma. Untreated KO epidermal layers were thicker than those in age-matched WT mice and exhibited significantly increased levels of FAK and phospho-ERK1/2, known mediators of carcinogen-induced squamous papilloma progression to carcinoma. Compared to protein levels in WT mouse embryo fibroblasts (MEF), SSeCKS levels were increased in FAK-null cells, whereas FAK levels were increased in SSeCKS-null cells. RNAi studies in WT MEF cells suggest that SSeCKS and FAK attenuate each other's expression. Our study implicates a role for SSeCKS in preventing of skin cancer progression possibly through negatively regulating FAK expression.

BACKGROUND: Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. METHODS: metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. RESULTS: In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05). CONCLUSIONS: AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.

BACKGROUND: SSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity. METHODS: We use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src. RESULTS: SSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1alpha, Cdc20a and Pdgfr-beta, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7. CONCLUSION: Our data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity.

SSeCKS, a Src-suppressed protein kinase C substrate with metastasis suppressor activity, is the rodent orthologue of human gravin/AKAP12, a scaffolding protein for protein kinase A and protein kinase C. We show here that the tetracycline-regulated reexpression of SSeCKS in MatLyLu (MLL) prostate cancer cells suppressed formation of macroscopic lung metastases in both spontaneous and experimental models of in vivo metastasis while having minimal inhibitory effects on the growth of primary-site s.c. tumors. SSeCKS decreased angiogenesis in vitro and in vivo by suppressing vascular endothelial growth factor (VEGF) expression in MLL tumor cells as well as in stromal cells. The forced reexpression of VEGF(165) and VEGF(121) isoforms was sufficient to reverse aspects of SSeCKS metastasis-suppressor activity in both the experimental and spontaneous models. SSeCKS reexpression in MLL cells resulted in the down-regulation of proangiogenic genes, such as osteopontin, tenascin C, KGF, angiopoietin, HIF-1alpha, and PDGFRbeta, and the up-regulation of antiangiogenic genes, such as vasostatin and collagen 18a1, a precursor of endostatin. These results suggest that SSeCKS suppresses formation of metastatic lesions by inhibiting VEGF expression and by inducing soluble antiangiogenic factors.

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.

The ability of SSeCKS/Gravin/AKAP12 (SSeCKS) to negatively regulate cell cycle progression is thought to relate to its spatiotemporal scaffolding activity for key signaling molecules such as protein kinase A and C, calmodulin and cyclins. SSeCKS is downregulated upon progression to malignancy in many cancer types, including melanoma and nonmelanoma skin cancer. The forced re-expression of SSeCKS is especially potent in suppressing metastasis through the inhibition of VEGF-mediated neovascularization. We have previously shown that SSeCKS-null (KO) mice exhibit hyperplasia and focal dysplasia in the prostate marked by activated Akt. To address whether KO mice exhibit increased skin carcinogenesis, WT and KO C57BL/6 mice were treated topically with 12-O-tetradecanoylphorbol-13-acetate and 7,12-dimethylbenzanthracene. Compared to WT mice, KO mice developed squamous papillomas more rapidly and in greater numbers and also exhibited significantly increased progression to squamous cell carcinoma. Untreated KO epidermal layers were thicker than those in age-matched WT mice and exhibited significantly increased levels of FAK and phospho-ERK1/2, known mediators of carcinogen-induced squamous papilloma progression to carcinoma. Compared to protein levels in WT mouse embryo fibroblasts (MEF), SSeCKS levels were increased in FAK-null cells, whereas FAK levels were increased in SSeCKS-null cells. RNAi studies in WT MEF cells suggest that SSeCKS and FAK attenuate each other's expression. Our study implicates a role for SSeCKS in preventing of skin cancer progression possibly through negatively regulating FAK expression.