Genome-wide methylation analyses identifies Non-coding RNA genes dysregulated in breast tumours that metastasise to the brain.

Brain metastases comprise 40% of all metastatic tumours and breast tumours are among the tumours that most commonly metastasise to the brain, the role that epigenetic gene dysregulation plays in this process is not well understood. We carried out 450 K methylation array analysis to investigate epigenetically dysregulated genes in breast to brain metastases (BBM) compared to normal breast tissues (BN) and primary breast tumours (BP). For this, we referenced 450 K methylation data for BBM tumours prepared in our laboratory with BN and BP from The Cancer Genome Atlas. Experimental validation on our initially identified genes, in an independent cohort of BP and in BBM and their originating primary breast tumours using Combined Bisulphite and Restriction Analysis (CoBRA) and Methylation Specific PCR identified three genes (RP11-713P17.4, MIR124-2, NUS1P3) that are hypermethylated and three genes (MIR3193, CTD-2023M8.1 and MTND6P4) that are hypomethylated in breast to brain metastases. In addition, methylation differences in candidate genes between BBM tumours and originating primary tumours shows dysregulation of DNA methylation occurs either at an early stage of tumour evolution (in the primary tumour) or at a later evolutionary stage (where the epigenetic change is only observed in the brain metastasis). Epigentic changes identified could also be found when analysing tumour free circulating DNA (tfcDNA) in patient's serum taken during BBM biopsies. Epigenetic dysregulation of RP11-713P17.4, MIR3193, MTND6P4 are early events suggesting a potential use for these genes as prognostic markers.

Cyclic nucleotide phosphodiesterase-1C (PDE1C) drives cell proliferation, migration and invasion in glioblastoma multiforme cells in vitro.

Cyclic nucleotides (cAMP & cGMP) are critical intracellular second messengers involved in the transduction of a diverse array of stimuli and their catabolism is mediated by phosphodiesterases (PDEs). We previously detected focal genomic amplification of PDE1C in >90 glioblastoma multiforme (GBM) cells suggesting a potential as a novel therapeutic target in these cells. In this report, we show that genomic gain of PDE1C was associated with increased expression in low passage GBM-derived cell cultures. We demonstrate that PDE1C is essential in driving cell proliferation, migration and invasion in GBM cultures since silencing of this gene significantly mitigates these functions. We also define the mechanistic basis of this functional effect through whole genome expression analysis by identifying down-stream gene effectors of PDE1C which are involved in cell cycle and cell adhesion regulation. In addition, we also demonstrate that Vinpocetine, a general PDE1 inhibitor, can also attenuate proliferation with no effect on invasion/migration. Up-regulation of at least one of this gene set (IL8, CXCL2, FOSB, NFE2L3, SUB1, SORBS2, WNT5A, and MMP1) in TCGA GBM cohorts is associated with worse outcome and PDE1C silencing down-regulated their expression, thus also indicating potential to influence patient survival. Therefore we conclude that proliferation, migration, and invasion of GBM cells could also be regulated downstream of PDE1C.

The GALNT9, BNC1 and CCDC8 genes are frequently epigenetically dysregulated in breast tumours that metastasise to the brain.

BACKGROUND: Tumour metastasis to the brain is a common and deadly development in certain cancers; 18-30 % of breast tumours metastasise to the brain. The contribution that gene silencing through epigenetic mechanisms plays in these metastatic tumours is not well understood. RESULTS: We have carried out a bioinformatic screen of genome-wide breast tumour methylation data available at The Cancer Genome Atlas (TCGA) and a broad literature review to identify candidate genes that may contribute to breast to brain metastasis (BBM). This analysis identified 82 candidates. We investigated the methylation status of these genes using Combined Bisulfite and Restriction Analysis (CoBRA) and identified 21 genes frequently methylated in BBM. We have identified three genes, GALNT9, CCDC8 and BNC1, that were frequently methylated (55, 73 and 71 %, respectively) and silenced in BBM and infrequently methylated in primary breast tumours. CCDC8 was commonly methylated in brain metastases and their associated primary tumours whereas GALNT9 and BNC1 were methylated and silenced only in brain metastases, but not in the associated primary breast tumours from individual patients. This suggests differing roles for these genes in the evolution of metastatic tumours; CCDC8 methylation occurs at an early stage of metastatic evolution whereas methylation of GANLT9 and BNC1 occurs at a later stage of tumour evolution. Knockdown of these genes by RNAi resulted in a significant increase in the migratory and invasive potential of breast cancer cell lines. CONCLUSIONS: These findings indicate that GALNT9 (an initiator of O-glycosylation), CCDC8 (a regulator of microtubule dynamics) and BNC1 (a transcription factor with a broad range of targets) may play a role in the progression of primary breast tumours to brain metastases. These genes may be useful as prognostic markers and their products may provide novel therapeutic targets.

Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo.

Breast cancer stem cells (BCSCs) are pan-resistant to different anticancer agents and responsible for cancer relapse. Disulfiram (DS), an antialcoholism drug, targets CSCs and reverses pan-chemoresistance. The anticancer application of DS is limited by its very short half-life in the bloodstream. This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo. The relationship between hypoxia and CSCs was examined by in vitro comparison of BC cells cultured in spheroid and hypoxic conditions. To determine the importance of NFκB activation in bridging hypoxia and CSC-related pan-resistance, the CSC characters and drug sensitivity in BC cell lines were observed in NFκB p65 transfected cell lines. The effect of Lipo-DS on the NFκB pathway, CSCs and chemosensitivity was investigated in vitro and in vivo. The spheroid cultured BC cells manifested CSC characteristics and pan-resistance to anticancer drugs. This was related to the hypoxic condition in the spheres. Hypoxia induced activation of NFκB and chemoresistance. Transfection of BC cells with NFκB p65 also induced CSC characters and pan-resistance. Lipo-DS blocked NFκB activation and specifically targeted CSCs in vitro. Lipo-DS also targeted the CSC population in vivo and showed very strong anticancer efficacy. Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed. Hypoxia induced NFκB activation is responsible for stemness and chemoresistance in BCSCs. Lipo-DS targets NFκB pathway and CSCs. Further study may translate DS into cancer therapeutics.

Epigenetic genome-wide analysis identifies BEX1 as a candidate tumour suppressor gene in paediatric intracranial ependymoma.

Promoter hypermethylation and transcriptional silencing is a common epigenetic mechanism of gene inactivation in cancer. To identify targets of epigenetic silencing in paediatric intracranial ependymoma, we used a pharmacological unmasking approach through treatment of 3 ependymoma short-term cell cultures with the demethylating agent 5-Aza-2'-deoxycytidine followed by global expression microarray analysis. We identified 55 candidate epigenetically silenced genes, which are involved in the regulation of apoptosis, Wnt signalling, p53 and cell differentiation. The methylation status of 26 of these genes was further determined by combined bisulfite restriction analysis (COBRA) and genomic sequencing in a cohort of 40 ependymoma samples. The most frequently methylated genes were BEX1 (27/40 cases), BAI2 (20/40), CCND2 (18/40), and CDKN2A (14/40). A high correlation between promoter hypermethylation and decreased gene expression levels was established by real-time quantitative PCR, suggesting the involvement of these genes in ependymoma tumourigenesis. Furthermore, ectopic expression of brain-expressed X-linked 1 (BEX1) in paediatric ependymoma short-term cell cultures significantly suppressed cell proliferation and colony formation. These data suggest that promoter hypermethylation contributes to silencing of target genes in paediatric intracranial ependymoma. Epigenetic inactivation of BEX1 supports its role as a candidate tumour suppressor gene in intracranial ependymoma, and a potential target for novel therapies for ependymoma in children.

Cytogenetic analysis of paediatric astrocytoma using comparative genomic hybridisation and fluorescence in-situ hybridisation.

Little is known about the cytogenetic and molecular genetic events that lead to the formation of paediatric astrocytoma. We have analysed 57 paediatric astrocytoma (WHO grades I-IV) using comparative genomic hybridisation in order to identify common regions of abnormality. Large regions of copy number alterations were infrequent with 71% of tumours demonstrating no genomic imbalance. Furthermore, the most frequent aberrations (including gain of 6q, 2q, and 7q, and loss of 16 and 12q) occurred in only a subset of cases. High-copy number amplification was seen in five tumours at 12 different regions. The presence of copy number alterations was significantly associated with increasing grade of malignancy, and gain of 12q and the presence of high-copy number amplification were associated with a poor outcome in patients with malignant astrocytoma (P = 0.0039 and 0.0085, respectively). FISH analysis confirmed loss of 1p36 identified by CGH. There was no evidence of amplification of EGFR, CDK4, MET, CDK6, c-myc, or MDM2.

Triptolide simultaneously induces reactive oxygen species, inhibits NF-kappaB activity and sensitizes 5-fluorouracil in colorectal cancer cell lines.

The in vitro chemosensitizing effect of triptolide (PG490) on the cytotoxicity of 5-fluorouracil (5-FU) was determined in three colorectal cancer (CRC) cell lines. PG490 alone was toxic to CRC cell lines (IC(50)s: 1.39-5.51nM). The cytotoxicity of 5-FU to CRC cell lines was 4-15-fold sensitized by combining use of low concentration of PG490. CI-isobologram indicated that the effect of PG490 plus 5-FU was synergistic. PG490 and 5-FU treatment induced activated caspase 3 and Bax expression and inhibited Bcl-2 expression. PG490 simultaneously induced generation of reactive oxygen species (ROS) and inhibited NF-kappaB transcriptional activity.

Disulfiram/copper complex inhibiting NFkappaB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines.

Most of the gemcitabine (dFdC) resistant cell lines manifested high NFkappaB activity. The NFkappaB activity can be induced by dFdC and 5-FU exposure. The chemosensitizing effect of disulfiram (DS), an anti-alcoholism drug and NFkappaB inhibitor, and copper (Cu) on the chemoresistant cell lines was examined. The DS/Cu complex significantly enhanced the cytotoxicity of dFdC (resistant cells: 12.2-1085-fold) and completely reversed the dFdC resistance in the resitant cell lines. The dFdC-induced NFkappaB activity was markedly inhibited by DS/Cu complex. The data from this study indicated that DS may be used in clinic to improve the therapeutic effect of dFdC in breast and colon cancer patients.

Vascular endothelial growth factor in astroglioma stem cell biology and response to therapy.

Malignant astrogliomas are among the most aggressive, highly vascular and infiltrating tumours bearing a dismal prognosis, mainly due to their resistance to current radiation treatment and chemotherapy. Efforts to identify and target the mechanisms that underlie astroglioma resistance have recently focused on candidate cancer stem cells, their biological properties, interplay with their local microenvironment or 'niche', and their role in tumour progression and recurrence. Both paracrine and autocrine regulation of astroglioma cell behaviour by locally produced cytokines such as the vascular endothelial growth factor (VEGF) are emerging as key factors that determine astroglioma cell fate. Here, we review these recent rapid advances in astroglioma research, with emphasis on the significance of VEGF in astroglioma stem-like cell biology. Furthermore, we highlight the unique DNA damage checkpoint properties of the CD133-marker-positive astroglioma stem-like cells, discuss their potential involvement in astroglioma radioresistance, and consider the implications of this new knowledge for designing combinatorial, more efficient therapeutic strategies.

Microarray analysis of pediatric ependymoma identifies a cluster of 112 candidate genes including four transcripts at 22q12.1-q13.3.

Ependymomas are glial cell-derived tumors characterized by varying degrees of chromosomal abnormalities and variability in clinical behavior. Cytogenetic analysis of pediatric ependymoma has failed to identify consistent patterns of abnormalities, with the exception of monosomy of 22 or structural abnormalities of 22q. In this study, a total of 19 pediatric ependymoma samples were used in a series of expression profiling, quantitative real-time PCR (Q-PCR), and loss of heterozygosity experiments to identify candidate genes involved in the development of this type of pediatric malignancy. Of the 12,627 genes analyzed, a subset of 112 genes emerged as being abnormally expressed when compared to three normal brain controls. Genes with increased expression included the oncogene WNT5A; the p53 homologue p63; and several cell cycle, cell adhesion, and proliferation genes. Underexpressed genes comprised the NF2 interacting gene SCHIP-1 and the adenomatous polyposis coli (APC)-associated gene EB1 among others. We validated the abnormal expression of six of these genes by Q-PCR. The subset of differentially expressed genes also included four underexpressed transcripts mapping to 22q12.313.3. By Q-PCR we show that one of these genes, 7 CBX7(22q13.1), was deleted in 55% of cases. Other genes mapping to cytogenetic hot spots included two overexpressed and three underexpressed genes mapping to 1q31-41 and 6q21-q24.3, respectively. These genes represent candidate genes involved in ependymoma tumorigenesis. To the authors' knowledge, this is the first time microarray analysis and Q-PCR have been linked to identify heterozygous/homozygous deletions.

Adenovirus vector-mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane.

Carboxypeptidase G2 (CPG2) is a powerful prodrug-converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug-activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol (GPI) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI-anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir (GCV), a suicide gene therapy approach already in the clinic. In a short-term human glioma culture (IN1760) resistant to the clinical chemotherapeutic drug CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), thymidine kinase/GCV effected no cell killing compared to 70% cell killing with our system.