The brain is a common site of metastatic disease in patients with breast cancer, which has few therapeutic options and dismal outcomes. The purpose of our study was to identify common and rare events that underlie breast cancer brain metastasis. We performed deep genomic profiling, which integrated gene copy number, gene expression and DNA methylation datasets on a collection of breast brain metastases. We identified frequent large chromosomal gains in 1q, 5p, 8q, 11q, and 20q and frequent broad-level deletions involving 8p, 17p, 21p and Xq. Frequently amplified and overexpressed genes included ATAD2, BRAF, DERL1, DNMTRB and NEK2A. The ATM, CRYAB and HSPB2 genes were commonly deleted and underexpressed. Knowledge mining revealed enrichment in cell cycle and G2/M transition pathways, which contained AURKA, AURKB and FOXM1. Using the PAM50 breast cancer intrinsic classifier, Luminal B, Her2+/ER negative, and basal-like tumors were identified as the most commonly represented breast cancer subtypes in our brain metastasis cohort. While overall methylation levels were increased in breast cancer brain metastasis, basal-like brain metastases were associated with significantly lower levels of methylation. Integrating DNA methylation data with gene expression revealed defects in cell migration and adhesion due to hypermethylation and downregulation of PENK, EDN3, and ITGAM. Hypomethylation and upregulation of KRT8 likely affects adhesion and permeability. Genomic and epigenomic profiling of breast brain metastasis has provided insight into the somatic events underlying this disease, which have potential in forming the basis of future therapeutic strategies.

Summary Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2•− and H2O2 are capable of disrupting intracellular iron metabolism thereby selectively sensitizing non-small cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Cancer cells, relative to normal cells, demonstrate significant alterations in metabolism that are proposed to result in increased steady-state levels of mitochondrial-derived reactive oxygen species (ROS) such as O2•−and H2O2. It has also been proposed that cancer cells increase glucose and hydroperoxide metabolism to compensate for increased levels of ROS. Given this theoretical construct, it is reasonable to propose that forcing cancer cells to use mitochondrial oxidative metabolism by feeding ketogenic diets that are high in fats and low in glucose and other carbohydrates, would selectively cause metabolic oxidative stress in cancer versus normal cells. Increased metabolic oxidative stress in cancer cells would in turn be predicted to selectively sensitize cancer cells to conventional radiation and chemotherapies. This review summarizes the evidence supporting the hypothesis that ketogenic diets may be safely used as an adjuvant therapy to conventional radiation and chemotherapies and discusses the proposed mechanisms by which ketogenic diets may enhance cancer cell therapeutic responses.

SUMMARY Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2.− and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Object An increasing incidence of brain cancer has been reported for the last three decades. In this study of brain cancer incidence and patient survival in the US, the authors attempt to update information on trends by examining data provided by the Surveillance, Epidemiology, and End Results (SEER) Program. Methods Population-based data from the SEER Program were used to calculate the incidence of and survival rates for people with brain cancer. The approximate Poisson method was used to calculate relative risks for brain cancer and to determine a 95% confidence interval. Annual age-standardized incidence rates were calculated, and time-trend analysis was conducted using joinpoint regression analysis. The relative risks of brain cancer were 1.48 for men compared with women, 3.18 for elderly persons compared with young adults, 1.86 for Caucasian patients compared with African-American patients, and 1.35 for those in metropolitan counties compared with those in nonmetropolitan counties. The incidence of brain cancer increased until 1987, when the annual percentage of change reversed direction, decreasing from 1.68 to −0.44%. The elderly experienced an increase until 1985, but their rates were stable thereafter. Rising trends were noticed for glioblastoma multiforme (GBM), oligodendroglioma, anaplastic astrocytoma, medulloblastoma, and mixed glioma, and falling trends were observed for astrocytoma not otherwise specified and malignant glioma. The survival rate for patients with GBM has not shown improvement in the last two decades. Conclusions Increased risk of brain cancer is associated with being male, Caucasian, elderly, and residing in a metropolitan county. The incidence rate of brain cancer in the US is gradually declining, but the rising trend of GBM combined with its poor survival rate is disconcerting and needs further exploration.

An additional tumor suppressor gene on chromosome 9p telomeric to the CDKN2A/B locus has long been postulated to exist. Using Affymetrix 250K single nucleotide polymorphism arrays to screen for copy number changes in glioblastoma multiforme (GBM), we detected a high frequency of deletions of the PTPRD gene, which encodes a receptor protein tyrosine phosphatase at chromosome 9p23-24.1. Missense and nonsense mutations of PTPRD were identified in a subset of the samples lacking deletions, including an inherited mutation with somatic loss of the wild-type allele. We then sequenced the gene in melanoma and identified 10 somatic mutations in 7 of 57 tumors (12%). Reconstitution of PTPRD expression in GBM and melanoma cells harboring deletions or mutations led to growth suppression and apoptosis that was alleviated by both the somatic and constitutional mutations. These data implicate PTPRD in the pathogenesis of tumors of neuroectodermal origin and, when taken together with other recent reports of PTPRD mutations in adenocarcinoma of the colon and lung, suggest that PTPRD may be one of a select group of tumor suppressor genes that are inactivated in a wide range of common human tumor types. [Cancer Res 2008; 68(24):10300-6]

Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2 ¶-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis. (Cancer Res 2006; 66(13): 6665-74)

Brain metastasis (BM) can affect ~25% of non-small cell lung cancer (NSCLC) patients during their lifetime. Efforts to characterize patients that will develop BM have been disappointing. microRNAs (miRNAs) regulate the expression of target mRNAs. miRNAs play a role in regulating a variety of targets and, consequently, multiple pathways, which make them a powerful tool for early detection of disease, risk assessment, and prognosis. We investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. miRNA microarray profiling was performed on samples from clinically matched NSCLC from seven patients with BM (BM+) and six without BM (BM−). Using t-test and further qRT-PCR validation, eight miRNAs were confirmed to be significantly differentially-expressed. Of these, expression of miR-328 and miR-330-3p were able to correctly classify BM+ vs. BM− patients. This classifier was used on a validation cohort (n=15) and it correctly classified 12/15 patients. Gene expression analysis comparing A549 parental and A549 cells stably transfected to over-express miR-328 (A549–328) identified several significantly differentially-expressed genes. PRKCA was one of the genes over-expressed in A549–328 cells. Additionally, A549–328 cells had significantly increased cell migration compared to A549 cells, which was significantly reduced upon PRKCA knockdown. In summary, miR-328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA and with further corroboration in additional independent cohorts, these miRNAs may be incorporated into clinical treatment decision making to stratify NSCLC patients at higher risk for developing BM.