BACKGROUND: The efficacy of therapy for cervical cancer is related to the alteration of multiple molecular events and signaling networks during treatment. The aim of this study was to evaluate gene expression alterations in advanced cervical cancers before- and after-trans-uterine arterial chemoembolization- (TUACE).
METHODS: Gene expression patterns in three squamous cell cervical cancers before- and after-TUACE were determined using microarray technique. Changes in AKAP12 and CA9 genes following TUACE were validated by quantitative real-time PCR.
RESULTS: Unsupervised cluster analysis revealed that the after-TUACE samples clustered together, which were separated from the before-TUACE samples. Using a 2-fold threshold, we identified 1131 differentially expressed genes that clearly discriminate after-TUACE tumors from before-TUACE tumors, including 209 up-regulated genes and 922 down-regulated genes. Pathway analysis suggests these genes represent diverse functional categories. Results from real-time PCR confirmed the expression changes detected by microarray.
CONCLUSIONS: Gene expression signature significantly changes during TUACE therapy of cervical cancer. Theses alterations provide useful information for the development of novel treatment strategies for cervical cancers on the molecular level.

Background: Pancreatic cancer is a fatal malignancy with a poor prognosis. The interactions between tumor cells and stromal cells contribute to cancer progression. Pancreatic stellate cells (PSCs) play a key role in tumor-stroma crosstalk of pancreatic cancer. The in-depth exploration for tumor-stroma crosstalk is helpful to develop novel therapeutic strategies. Our aim was to identify the potential core genes and pathways in tumor-stroma crosstalk.
Methods: 3 microarray datasets were from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were screened through bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network were used to obtain the biological roles of DEGs. The top 15 DEGs were explored by principal component analysis. We validated the top 15 DEGs expression in the tumor-stroma crosstalk model in which PSCs were treated with the mixture of Aspc-1 and Panc-1 supernatant.
Results: A total of 221 genes were filtered as DEGs for tumor-stroma crosstalk. The results of principal component analysis for the top 15 DEGs can distinguish three groups. According to the KEGG enrichment, there were 8, 7, and 7 DEGs enriched in cancer related pathway, PI3K-Akt signaling pathway, and microRNAs, respectively. In the tumor-stroma crosstalk model, significant differences can be validated in the AKAP12, CLDN1, CP, FKBP1A, LAMB3, LSM4, MTMR3, PRKARIA, YWHAZ, and JUND expressions.
Conclusions: These results identified the potential core genes and pathways in pancreatic cancer for tumor-stroma crosstalk, which could provide potential targets for the treatment of pancreatic cancer.

A-kinase anchor protein 12 (AKAP12) also known as Gravin and SSeCKS, is a novel potent scaffold protein for many key signaling factors, such as protein kinase C (PKC), PKA, cyclins as well as F-actin. AKAP12 expression is known to be suppressed in several human malignancies including breast, prostate, gastric and colon cancers. In this study, we evaluated the role of AKAP12 in the migration of breast cancer cells, an important cellular process in cancer progression. AKAP12 gene expression was analyzed in human breast cancer tissues using the Gene expression-based Outcome for Breast cancer Online (GOBO) database and TissueScan array, followed by relapse free survival (RFS) analysis with the Kaplan-Meier Plotter. AKAP12 protein was then analyzed in normal MCF10A breast cell line and six different breast cancer cell lines (AU565, Hs578T, MCF7, MDA-MB-231, T47D and ZR751). After which, siRNA-mediated knockdown of AKAP12 was carried out in MCF10A, MDA-MB-231 and Hs578T cells, followed by phenotypic assays. AKAP12 was observed to be reduced in breast cancer tissues as analyzed by GOBO and TissueScan array. Kaplan Meier survival analysis revealed that patients with AKAP12 gene expression had a higher RFS survival. There was also decreased AKAP12 protein expression in breast cancer cell lines compared to MCF10A normal epithelial breast cell line. Knockdown of AKAP12 in both MCF10A cells and Hs578T cells induced cell migration but did not alter cell proliferation. Moreover, siAKAP12 in aggressive MDA-MB-231 breast cancer cells led to an increase in cell migration. Immunofluorescence analysis of AKAP12 depleted MCF10A cells also revealed formation of thick stress fibers which could affect cell migration. Hence, the findings in this study suggest that AKAP12 is a potential metastasis suppressor in breast cancer.

Breast carcinoma is one of the most common types of malignant neoplasms, and is associated with high rates of morbidity and mortality. Altered gene expression is critical in the development of breast cancer. To identify the important differentially expressed genes and microRNAs in breast carcinoma, mRNA (GSE26910, GSE42568, and GSE89116) and microRNA (GSE35412) microarray datasets were downloaded from the Gene Expression Omnibus database. The differentially expressed microRNA expression data were extracted with GEO2R online software. The DAVID online database was used to perform a function and pathway enrichment analysis of the key identified differentially expressed genes. A protein-protein interaction (PPI) network was constructed using the STRING online database, and visualized in Cytoscape software. The effect of the expression level of the key identified genes on overall survival (OS) time was analyzed by using the Kaplan-Meier Plotter online database. Furthermore, the online miRNA databases TargetScan, microT-CDS, and TarBase were used to identify the target genes of the differentially expressed miRNAs. A total of 254 differentially expressed genes were identified, which were enriched in cell adhesion, polysaccharide binding, extracellular region part and ECM-receptor interactions. The PPI network contained 250 nodes and 375 edges. Five differentially expressed genes were found to be significantly negatively correlated with the differentially expressed miRNAs, which were potentially also target genes for miRNAs. Four of the five genes, including AKAP12, SOPB, TCF7L2, COL12A1 and TXNIP were downregulated, and were associated with the OS of patients with breast carcinoma. In addition, a total of 130 differentially expressed miRNAs were identified. In conclusion, these results constitute a novel model for miRNA-mRNA differential expression patterns, and further studies may provide potential targets for diagnosing and understanding the mechanisms of breast carcinoma.

BACKGROUND: Dysregulation of microRNA (miRNA) expression is associated with hallmarks of aggressive tumor phenotypes, e.g., enhanced cell growth, proliferation, invasion, and anchorage independent growth in prostate cancer (PCa).
METHODS: Serum-based miRNA profiling involved 15 men diagnosed with non-metastatic (stage I, III) and metastatic (stage IV) PCa and five age-matched disease-free men using miRNA arrays with select targets confirmed by quantitative real-time PCR (qRT-PCR). The effect of miR-186-5p inhibition or ectopic expression on cellular behavior of PCa cells (i.e., PC-3, MDA-PCa-2b, and LNCaP) involved the use bromodeoxyuridine (BrdU) incorporation, invasion, and colony formation assays. Assessment of the impact of miR-186-5p inhibition or overexpression on selected targets entailed microarray analysis, qRT-PCR, and/or western blots. Statistical evaluation used the modified t-test and ANOVA analysis.
RESULTS: MiR-186-5p was upregulated in serum from PCa patients and metastatic PCa cell lines (i.e., PC-3, MDA-PCa-2b, LNCaP) compared to serum from disease-free individuals or a normal prostate epithelial cell line (RWPE1), respectively. Inhibition of miR-186-5p reduced cell proliferation, invasion, and anchorage-independent growth of PC-3 and/or MDA-PCa-2b PCa cells. AKAP12, a tumor suppressor target of miR-186-5p, was upregulated in PC-3 and MDA-PCa-2b cells transfected with a miR-186-5p inhibitor. Conversely, ectopic miR-186-5p expression in HEK 293 T cells decreased AKAP12 expression by 30%. Both pAKT and β-catenin levels were down-regulated in miR-186-5p inhibited PCa cells.
CONCLUSIONS: Our findings suggest miR-186-5p plays an oncogenic role in PCa. Inhibition of miR-186-5p reduced PCa cell proliferation and invasion as well as increased AKAP12 expression. Future studies should explore whether miR-186-5p may serve as a candidate prognostic indicator and a therapeutic target for the treatment of aggressive prostate cancer.

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.

There is a need to better understand meningioma oncogenesis for biomarker discovery and development of targeted therapies. Histological or genetic criteria do not accurately predict aggressiveness. Post-translational studies in meningioma progression are lacking. In the present work, we introduce a combination of mass spectrometry-based phosphoproteomics and peptide array kinomics to profile atypical and anaplastic (high-grade) meningiomas. In the discovery set of fresh-frozen tissue specimens (14), the A-kinase anchor protein 12 (AKAP12) protein was found downregulated across the grades. AKAP12 knockdown in benign meningioma cells SF4433 increases proliferation, cell cycle, migration, invasion, and confers an anaplastic profile. Differentially regulated pathways were characteristic of high-grade meningiomas. Low AKAP12 expression in a larger cohort of patients (75) characterized tumor invasiveness, recurrence, and progression, indicating its potential as a prognostic biomarker. These results demonstrate AKAP12 as a central regulator of meningioma aggressiveness with a possible role in progression.

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.

There is a growing interest in the use of naturally occurring agents in cancer prevention. This study investigated the garlic extract affects in bladder cancer (BC) prevention. The effect of garlic extract in cancer prevention was evaluated using the T24 BC BALB/C-nude mouse xenograft model. Microarray analysis of tissues was performed to identify differences in gene expression between garlic extract intake and control diet, and gene network analysis was performed to assess candidate mechanisms of action. Furthermore, we investigated the expression value of selected genes in the data of 165 BC patients. Compared to the control group, significant differences in tumor volume and tumor weight were observed in the groups fed 20 mg/kg (p<0.05), 200 mg/kg, and 1000 mg/kg of garlic extract (p<0.01). Genes (645) were identified as cancer prevention-related genes (fold change >2 and p<0.05) by tissue microarray analysis. A gene network analysis of 279 of these genes (p<0.01) was performed using Cytoscape/ClueGo software: 36 genes and 37 gene ontologies were mapped to gene networks. Protein kinase A (PKA) signaling pathway including AKAP12, RDX, and RAB13 genes were identified as potential mechanisms for the activity of garlic extract in cancer prevention. In BC patients, AKAP12 and RDX were decreased but, RAB13 was increased. Oral garlic extract has strong cancer prevention activity in vivo and an acceptable safety profile. PKA signaling process, especially increasing AKAP12 and RDX and decreasing RAB13, are candidate pathways that may mediate this prevention effect.

Increased glucocorticoid receptor (GR) expression and activity following androgen blockade can contribute to castration-resistant prostate cancer (CRPC) progression. Therefore, we hypothesized that GR antagonism will have therapeutic benefit in CRPC. However, the FDA-approved nonselective, steroidal GR antagonist, mifepristone, lacks GR specificity, reducing its therapeutic potential. Here, we report that two novel nonsteroidal and highly selective GR modulators (SGRM), CORT118335 and CORT108297, have the ability to block GR activity in prostate cancer and slow CRPC progression. In contrast to mifepristone, these novel SGRMs did not affect androgen receptor (AR) signaling, but potently inhibited GR transcriptional activity. Importantly, SGRMs decreased GR-mediated tumor cell viability following AR blockade.

OBJECTIVE: To screen the gene signature for distinguishing patients with high risks from those with low-risks for colon cancer recurrence and predicting their prognosis.
METHODS: Five microarray datasets of colon cancer samples were collected from Gene Expression Omnibus database and one was obtained from The Cancer Genome Atlas (TCGA). After preprocessing, data in GSE17537 were analyzed using the Linear Models for Microarray data (LIMMA) method to identify the differentially expressed genes (DEGs). The DEGs further underwent PPI network-based neighborhood scoring and support vector machine (SVM) analyses to screen the feature genes associated with recurrence and prognosis, which were then validated by four datasets GSE38832, GSE17538, GSE28814 and TCGA using SVM and Cox regression analyses.
RESULTS: A total of 1207 genes were identified as DEGs between recurrence and no-recurrence samples, including 726 downregulated and 481 upregulated genes. Using SVM analysis and five gene expression profile data confirmation, a 15-gene signature (HES5, ZNF417, GLRA2, OR8D2, HOXA7, FABP6, MUSK, HTR6, GRIP2, KLRK1, VEGFA, AKAP12, RHEB, NCRNA00152 and PMEPA1) were identified as a predictor of recurrence risk and prognosis for colon cancer patients.
CONCLUSION: Our identified 15-gene signature may be useful to classify colon cancer patients with different prognosis and some genes in this signature may represent new therapeutic targets.

Non-small cell lung cancer (NSCLC) remains one of the leading causes of death worldwide, and thus new molecular targets need to be identified to improve treatment efficacy. Although epidermal growth factor receptor (EGFR)/KRAS mutation-driven lung tumorigenesis is well understood, the mechanism of EGFR/KRAS-independent signal activation remains elusive. Enhanced TFAP2C (transcription factor activating enhancer-binding protein 2C) expression is associated with poor prognosis in some types of cancer patients, but little is known of its relation with the pathogenesis of lung cancer. In the present study, we found that TFAP2C overexpression was associated with cell cycle activation and NSCLC cell tumorigenesis. Interestingly, TFAP2C blocked AKAP12-mediated cyclin D1 inhibition by inducing the overexpression of oncogenic microRNA (miRNA)-183 and simultaneously activated cyclin-dependent kinase 6-mediated cell cycle progression by downregulating tumor-suppressive miRNA-33a. In a mouse xenograft model, TFAP2C promoted lung tumorigenesis and disease aggressiveness via the miR-183 and miR-33a pathways. The study provides a mechanism of mitogenic and oncogenic signaling via two functionally opposed miRNAs and suggests that TFAP2C-induced cell cycle hyperactivation contributes to lung tumorigenesis.

Cutaneous squamous cell carcinomas (cSCCs) are common human carcinomas. Despite having metastasizing capacities, they usually show less aggressive progression compared to squamous cell carcinoma (SCC) of other organs. Metastasis suppressor proteins (MSPs) are a group of proteins that control and slow-down the metastatic process. In this study, we established the importance of seven well-defined MSPs including NDRG1, NM23-H1, RhoGDI2, E-cadherin, CD82/KAI1, MKK4, and AKAP12 in cSCCs. Protein expression levels of the selected MSPs were detected in 32 cSCCs, 6 in situ SCCs, and two skin cell lines (HaCaT, A-431) by immunohistochemistry. The results were evaluated semi-quantitatively using the HSCORE system. In addition, mRNA expression levels were detected by qRT-PCR in the cell lines. The HSCOREs of NM23-H1 were similar in cSCCs and normal skin tissues, while RGHOGDI2, E-cadherin and AKAP12 were significantly downregulated in cSCCs compared to normal skin. The levels of MKK4, NDRG1 and CD82 were partially conserved in cSCCs. In stage I SCCs, nuclear staining of NM23-H1 (NM23-H1nuc) was significantly lower than in stage II/III SCCs. Only nuclear staining of MKK4 (MKK4nuc) showed significantly higher scores in in situ carcinomas compared to invasive SCCs. In conclusion, similar to other human tumors, we have demonstrated complex differential expression patterns for the MSPs in in-situ and invasive cSCCs. This complex MSP signature warrants further biological and experimental pathway research.

A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of AKAP12 has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., α, β, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12α expression. Promoter methylation of AKAP12α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates AKAP12α expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis.

AKAP12/Gravin (A kinase anchor protein 12) belongs to the group of A-kinase scaffold proteins and functions as a tumor suppressor in some human primary cancers. While AKAP12 is found consistently downregulated in hepatocellular carcinoma (HCC), its involvement in hepatocarcinogenesis has not been fully elucidated. We identified targeting sites for miR-103 in the 3'-untranslated region (3'-UTR) of AKAP12 by bioinformatic analysis and confirm their function by a luciferase reporter gene assay. We reveal miR-103 expression to be inversely correlated with AKAP12 in HCC tissue samples and show that overexpressed miR-103 promotes cell proliferation and inhibits apoptosis by downregulating AKAP12 expression in HCC cell lines. On the other hand, repression of miR-103 suppresses proliferation and promotes apoptosis in HCC cells by increasing AKAP12. In xenografted HCC tumors, overexpression of AKAP12 suppresses tumor growth whereas overexpression of miR-103 enhances tumor growth while repressing AKAP12. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we investigated whether AKAP12 expression affected telomerase activity in HCC cells. Both AKAP12 overexpression and protein kinase Cα (PKCα) inhibition prevent nuclear translocation and phosphorylation of TERT and reduce telomerase activity in HCC cells. These findings indicate that miR-103 potentially acts as an oncogene in HCC by inhibiting AKAP12 expression and raise the possibility that miR-103 increases telomerase activity by increasing PKCα activity. Thus, miR-103 may represent a new potential diagnostic and therapeutic target for HCC treatment.

In a genome-wide association study conducted among Chinese women, we identified the single nucleotide polymorphism (SNP) rs2046210 at 6q25.1 for breast cancer risk. To explore a potential regulatory role for this risk locus, we measured expression levels of nine genes at the locus in breast cancer tissue and adjacent normal tissue samples obtained from 67 patients recruited in the Shanghai Breast Cancer Study. We found that rs2046210 had a statistically significant association with the expression levels of the AKAP12 and ESR1 genes in adjacent normal breast tissues. Women who carry the AA/AG risk genotypes had higher expressions of these two genes compared to those who carry G/G genotypes (P = 0.02 and 0.04 for the AKAP12 and ESR1, respectively). However, no significant differences of SNP rs2046210 with gene expression levels were found in tumor tissues. In The Cancer Genome Atlas samples, the AA/AG risk genotypes of SNP rs2046210 were associated with a significantly higher expression level of the AKAP12 gene and a lower level of the ESR1 gene in tumor tissue. Functional analysis using ENCODE data revealed that SNP rs7763637, which is in strong linkage disequilibrium with SNP rs2046210, is likely a potential functional variant, regulating the AKAP12 gene. Taken together, these results from our study suggest that the association between the 6q25.1 locus and breast cancer risk may be mediated through SNPs that regulate expressions of the AKAP12 gene.

Scaffold proteins are critical hubs within cells that have the ability to modulate upstream signaling molecules and their downstream effectors to fine-tune biological responses. Although they can serve as focal points for association of signaling molecules and downstream pathways that regulate tumorigenesis, little is known about how the tumor microenvironment affects the expression and activity of scaffold proteins. This study demonstrates that hypoxia, a common element of solid tumors harboring low oxygen levels, regulates expression of a specific variant of the scaffold protein AKAP12 (A-kinase anchor protein 12), AKAP12v2, in metastatic melanoma. In turn, through a kinome-wide phosphoproteomic and MS study, we demonstrate that this scaffolding protein regulates a shift in protein kinase A (PKA)-mediated phosphorylation events under hypoxia, causing alterations in tumor cell invasion and migration in vitro, as well as metastasis in an in vivo orthotopic model of melanoma. Mechanistically, the shift in AKAP12-dependent PKA-mediated phosphorylations under hypoxia is due to changes in AKAP12 localization vs. structural differences between its two variants. Importantly, our work defines a mechanism through which a scaffold protein can be regulated by the tumor microenvironment and further explains how a tumor cell can coordinate many critical signaling pathways that are essential for tumor growth through one individual scaffolding protein.

A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient. Associations between the identified candidate proteins and patient outcome were assessed in a discovery cohort of 545 patients and two validation cohorts totaling 795 independent SOC patients. Among the 81 differentially abundant proteins identified (q < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated in all models of paclitaxel-resistant HGSOC. Furthermore, elevated AKAP12 transcript expression was associated with worse progression-free and overall survival. Associations with outcome were observed in three independent cohorts and remained significant after adjusted multivariate modeling. We further provide evidence to support that differential gene methylation status is associated with elevated expression of AKAP12 in taxol-resistant ovarian cancer cells and ovarian cancer patient subsets. Elevated expression and shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic and predictive marker for progression-free and overall survival in SOC patients.

BACKGROUND: Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs.
METHODS: A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile.
RESULTS: NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs.
CONCLUSION: The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Metastasis suppressor genes (MSGs) have contributed to an understanding of regulatory pathways unique to the lethal metastatic process. When re-expressed in experimental models, MSGs block cancer spread to, and colonization of distant sites without affecting primary tumor formation. Genes have been identified with expression patterns inverse to a single MSG, and found to encode functional, druggable signaling pathways. We now hypothesize that common signaling pathways mediate the effects of multiple MSGs. By gene expression profiling of human MCF7 breast carcinoma cells expressing a scrambled siRNA, or siRNAs to each of 19 validated MSGs (NME1, BRMS1, CD82, CDH1, CDH2, CDH11, CASP8, MAP2K4, MAP2K6, MAP2K7, MAPK14, GSN, ARHGDIB, AKAP12, DRG1, CD44, PEBP1, RRM1, KISS1), we identified genes whose expression was significantly opposite to at least five MSGs. Five genes were selected for further analysis: PDE5A, UGT1A, IL11RA, DNM3 and OAS1. After stable downregulation of each candidate gene in the aggressive human breast cancer cell line MDA-MB-231T, in vitro motility was significantly inhibited. Two stable clones downregulating PDE5A (phosphodiesterase 5A), an enzyme involved in the regulation of cGMP-specific signaling, exhibited no difference in cell proliferation, but reduced motility by 47 and 66 % compared to the empty vector-expressing cells (p = 0.01 and p = 0.005). In an experimental metastasis assay, two shPDE5A-MDA-MB-231T clones produced 47-62 % fewer lung metastases than shRNA-scramble expressing cells (p = 0.045 and p = 0.009 respectively). This study demonstrates that previously unrecognized genes are inversely related to the expression of multiple MSGs, contribute to aspects of metastasis, and may stand as novel therapeutic targets.

Lung cancer is the top cause of cancer-related deceases. One of the reasons is the development of resistance to the chemotherapy treatment. In particular, cancer stem cells (CSCs), can escape treatment and regenerate the bulk of the tumor. In this article, we describe a comparison between cancer cells resistant to cisplatin and CSCs, both derived from the non-small-cell lung cancer cell lines H460 and A549. Cisplatin-resistant cells were obtained after a single treatment with the drug. CSCs were isolated by culture in defined media, under nonadherent conditions. The isolated CSCs were clonogenic, could be differentiated into adherent cells and were less sensitive to cisplatin than the original cells. Cisplatin resistant and CSCs were able to generate primary tumors and to metastasize when injected into immunodeficient Nu/Nu mice, although they formed smaller tumors with a larger latency than untreated cells. Notably, under appropriated proportions, CSCs synergized with differentiated cells to form larger tumors. CSCs also showed increased capacity to induce angiogenesis in Nu/Nu mice. Conversely, H460 cisplatin-resistant cells showed increased tendency to develop bone metastasis. Gene expression analysis showed that several genes involved in tumor development and metastasis (EGR1, COX2, MALAT1, AKAP12, ADM) were similarly induced in CSC and cisplatin-resistant H460 cells, in agreement with a close similarity between these two cell populations. Cells with the characteristic growth properties of CSCs were also isolated from surgical samples of 18 out of 44 lung cancer patients. A significant correlation (P = 0.028) was found between the absence of CSCs and cisplatin sensitivity.

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.

We describe a novel bioinformatic and translational pathology approach, gene Signature Finder Algorithm (gSFA) to identify biomarkers associated with Colorectal Cancer (CRC) survival. Here a robust set of CRC markers is selected by an ensemble method. By using a dataset of 232 gene expression profiles, gSFA discovers 16 highly significant small gene signatures. Analysis of dichotomies generated by the signatures results in a set of 133 samples stably classified in good prognosis group and 56 samples in poor prognosis group, whereas 43 remain unreliably classified. AKAP12, DCBLD2, NT5E and SPON1 are particularly represented in the signatures and selected for validation in vivo on two independent patients cohorts comprising 140 tumor tissues and 60 matched normal tissues. Their expression and regulatory programs are investigated in vitro. We show that the coupled expression of NT5E and DCBLD2 robustly stratifies our patients in two groups (one of which with 100% survival at five years). We show that NT5E is a target of the TNF-α signaling in vitro; the tumor suppressor PPARγ acts as a novel NT5E antagonist that positively and concomitantly regulates DCBLD2 in a cancer cell context-dependent manner.

CONTEXT: Colorectal cancer is the second leading cause of cancer deaths worldwide. DNA microarray-based technologies allow simultaneous analysis of expression of thousands of genes.
AIM: To search for important molecular markers and pathways that hold great promise for further treatment of patients with colorectal cancer.
MATERIALS AND METHODS: Here, we performed a comprehensive gene-level assessment of colorectal cancer using 35 colorectal cancer and 24 normal samples.
RESULTS: It was shown that AURKA, MT1G, and AKAP12 had a high degree of response in colorectal cancer. Besides, we further explored the underlying molecular mechanism within these different genes.
CONCLUSIONS: The results indicated calcium signaling pathway and vascular smooth muscle contraction pathway were the two significant pathways, giving hope to provide insights into the development of novel therapeutic targets and pathways.

PURPOSE: : Colorectal cancer (CRC) is a common and potentially lethal disease. A number of genetic aberrations is known to take place in colorectal carcinogenesis, which leads to progressive alteration of normal mechanisms controlling cell growth. A-kinase-anchoring protein 12 (AKAP12) plays a role in cell proliferation, angiogenesis and cytoskeletal remodeling. The purpose of this study was to demonstrate the role of the AKAP12 gene expression in CRC patients and to determine its relationship (if any) with prognosis.
METHODS: AKAP12 gene expression was investigated by immunohistochemistry.
RESULTS: A total of 55 patients (63.6% males, 36.4% females) with histologically confirmed CRC were studied. Normal intestinal epithelium showed weak basal staining, dysplastic areas were stained mildly, whereas all of the cancer cells were stained completely with AKAP12.
CONCLUSION: AKAP12 gene seems to play a role in colorectal carcinogenesis.

The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined.

AIMS: This study was to investigate whether the clinically acceptable colchicine concentrations had anti-cancer effects on hepatocellular carcinoma (HCC) and their anti-cancer mechanisms.
MAIN METHODS: Two human HCC cell lines (HCC24/KMUH, HCC38/KMUH) and two human cancer-associated fibroblast (CAF) cell lines (F28/KMUH, F59/KMUH) were investigated by proliferative assay, microarray, quantitative reverse transcriptase-polymerase chain reaction, and nude mouse study using clinically acceptable colchicine concentrations.
KEY FINDINGS: Both 2 and 6ng/mL colchicine significantly inhibited the cellular proliferation of all cell lines tested (P<0.05). The anti-proliferative effects of colchicine on F28/KMUH, HCC24/KMUH and HCC38/KMUH cells were dose-dependent. The anti-proliferative effects of 6ng/mL colchicine on both HCC cell lines were similar to the effects of 1μg/mL epirubicin. The anti-proliferative effects of colchicine on HCC cells could be partially explained by dose-dependent up-regulations of 2 anti-proliferative genes (AKAP12, TGFB2) in these cells. TGFB2 was also up-regulated in CAFs but was not dose-dependent. Up-regulation of MX1 which can accelerate cell death was a common effect of 6ng/mL colchicine on both CAF cell lines, but 2ng/mL colchicine down-regulated MX1 in F28/KMUH cells. Nude mouse (BALB/c-nu) experiment showed that colchicine-treated mice (0.07mgcolchicine/kg/day×14days) had lower increased tumor volume ratios, slower tumor growth rates and larger percentages of tumor necrotic areas than control mice (all P<0.05).
SIGNIFICANCE: Clinically acceptable colchicine concentrations have anti-cancer effects on HCC. This drug has potential for the palliative treatment of HCC.

Acute leukemias are caused by genetic and epigenetic mechanisms involving tumor suppressor genes and oncogenes. Aberrant DNA methylation patterns are the most frequent molecular alterations detected in acute myeloid leukemia (AML). Gravin is down-regulated in several solid tumors and is implicated in tumorigenesis. To explore its role in the molecular pathogenesis and its possible prognostic importance in AML, we have evaluated the expression levels of the gravin gene in 83 acute myeloid leukemia patients as compared with controls using quantitative real-time polymerase chain reaction (qRT-PCR). Mean gravin expression was 0.53 ± 1.34 and 8.81 ± 11.6 for patients and controls, respectively, and was found to be about 16-fold lower than controls. Gravin gene expression was lower than controls in 83.1 % (69/83) and was similar to controls in 16.9 % (14/83) of cases (p < 0.0001). It was found that there was no significant correlation between gravin expression and laboratory prognostic markers (p > 0.05). Gravin expression was highest in complete remission (1.065 ± 1.79) and lowest in relapse (0.019 ± 0.03) with a statistical difference (p = 0.004). Patients with gravin expression below median level had higher risk to develop relapse (OR = 8.689, 95 % CI = 2.464-30.638; p < 0.0001). No statistical correlation was reported between gravin expression and survival times (OS, DFS) (p = 0.482, 0.409, respectively), and this was confirmed in multivariate analysis. Gravin gene expression was found to be decreased in acute myeloid leukemia, and the degree of its decreased expression has been found to be correlated with poor prognosis.

BACKGROUND: Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genome-wide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies.
PRINCIPAL FINDINGS: The cell lines showed complex patterns of DNA copy number changes, where genomic copy number gains were significantly associated with gene-rich regions and losses with gene-poor regions. By integrating the datasets, 350 genes were identified as having two types of aberrations (gain/over-expression, hypo-methylation/over-expression, loss/under-expression or hyper-methylation/under-expression) using a recurrence threshold of 6/19 (>30%) cell lines. The genes showed in general alterations in either DNA copy number or DNA methylation, both within individual samples and across the sample panel. These 350 genes are involved in embryonic skeletal system development and morphogenesis, as well as remodelling of extracellular matrix. The aberrations of three selected genes, CXCL5, DLX5 and RUNX2, were validated in five cell lines and five tumour samples using PCR techniques. Several genes were hyper-methylated and under-expressed compared to normal osteoblasts, and expression could be reactivated by demethylation using 5-Aza-2'-deoxycytidine treatment for four genes tested; AKAP12, CXCL5, EFEMP1 and IL11RA. Globally, there was as expected a significant positive association between gain and over-expression, loss and under-expression as well as hyper-methylation and under-expression, but gain was also associated with hyper-methylation and under-expression, suggesting that hyper-methylation may oppose the effects of increased copy number for detrimental genes.
CONCLUSIONS: Integrative analysis of genome-wide genetic and epigenetic alterations identified dependencies and relationships between DNA copy number, DNA methylation and mRNA expression in osteosarcomas, contributing to better understanding of osteosarcoma biology.